Bumetanide Derivatives for the Therapy of Stroke and Other Neurological Diseases/Disorders Involving NKCCs
20210163406 · 2021-06-03
Inventors
Cpc classification
A61P25/28
HUMAN NECESSITIES
C07C311/37
CHEMISTRY; METALLURGY
C07C311/39
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
International classification
C07C311/39
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates to bumetanide derivatives of formula (I) as well as pharmaceutical compositions comprising these compounds for use in the treatment or prevention of neurological diseases/disorders involving Na+-K+-20Γ-cotransporters (NKCCs), such as stroke, traumatic brain injury (TBI), spinal cord injury (SCI), peripheral nerve injury (PNI), brain edema, or glioma, and particularly for use in the treatment or prevention of stroke. The invention likewise relates to a method of treating or preventing a neurological disease or disorder involving an NKCC, such as stroke, TBI, SCI, PNI, brain edema, or glioma, the method comprising administering a compound of formula (I) to a subject in need thereof.
##STR00001##
Claims
1. A compound of formula (I) ##STR00015## wherein: R.sup.1 is selected from —(C.sub.1-4 alkylene)-NH—(C.sub.1-4 alkylene)-R.sup.11, —COO—(C.sub.1-4 alkylene)-R.sup.11, —O—CO—(C.sub.1-4 alkylene)-R.sup.11, —CO—(C.sub.1-4 alkylene)-R.sup.11, —CO—NH—(C.sub.1-4 alkylene)-R.sup.11, —CO—N(C.sub.1-4 alkyl)-(C.sub.1-4 alkylene)-R.sup.11, —NH—CO—(C.sub.1-4 alkylene)-R.sup.11 and —N(C.sub.1-4 alkyl)-CO—(C.sub.1-4 alkylene)-R.sup.11; R.sup.11 is independently selected from —CF.sub.3, —CN and halogen; R.sup.2 is selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S (C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, -O—(C.sub.1-6 haloalkyl), —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)-CO—(C.sub.1-6 alkyl), —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—SO.sub.2—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-SO.sub.2—(C.sub.1-6 alkyl); R.sup.3 is selected from —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —SO.sub.2—N═(C.sub.1-6 alkylidene) and —SO.sub.2-halogen, wherein the alkyl moiety of said —SO.sub.2—NH(C.sub.1-6 alkyl), one or both of the alkyl moieties of said —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), and the alkylidene moiety of said —SO.sub.2—N═(C.sub.1-6 alkylidene) are each optionally substituted with one or more groups independently selected from halogen, —CF.sub.3, —CN, —NO.sub.2, —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —OH, —O(C.sub.1-6 alkyl), —SH and —S(C.sub.1-6 alkyl); R.sup.4 is selected from —O—R.sup.41, —S—R.sup.41, —NH—R.sup.41, —N(C.sub.1-6 alkyl)-R.sup.41, halogen, hydrogen, carbocyclyl and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups R.sup.42; R.sup.41 is selected from —(C.sub.0-4 alkylene)-carbocyclyl, —(C.sub.0-4 alkylene)-heterocyclyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl, wherein the carbocyclyl moiety of said —(C.sub.0-4 alkylene)-carbocyclyl and the heterocyclyl moiety of said —(C.sub.0-4 alkylene)-heterocyclyl are each optionally substituted with one or more groups R.sup.42, and wherein said C.sub.1-6 alkyl, said C.sub.2-6 alkenyl, said C.sub.2-6 alkynyl, the alkylene moiety of said —(C.sub.0-4 alkylene)-carbocyclyl, and the alkylene moiety of said —(C.sub.0-4 alkylene)-heterocyclyl are each optionally substituted with one or more groups R.sup.43; each R.sup.42 is independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl), —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)-CO—(C.sub.1-6 alkyl), —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—SO.sub.2—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-SO.sub.2—(C.sub.1-6 alkyl); each R.sup.43 is independently selected from —OH, —O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, —CF.sub.3, —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-CO—(C.sub.1-6 alkyl); R.sup.5 is selected from —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NO.sub.2 and hydrogen, wherein the alkyl moiety of said —NH(C.sub.1-6 alkyl) and one or both of the alkyl moieties of said —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl) are each optionally substituted with one or more groups independently selected from halogen, —CF.sub.3, —CN, —NO.sub.2, —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —OH, —O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), carbocyclyl and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups R.sup.51; each R.sup.51 is independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S (C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl), —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)-CO—(C.sub.1-6 alkyl), —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—SO.sub.2—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-SO.sub.2—(C.sub.1-6 alkyl); and R.sup.6 is selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl), —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)-CO—(C.sub.1-6 alkyl), —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—SO.sub.2—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-SO.sub.2—(C.sub.1-6 alkyl); or a pharmaceutically acceptable salt or solvate thereof.
2. The compound according to claim 1, wherein R.sup.1 is —(C.sub.1-4 alkylene)-NH—(C.sub.1-4 alkylene)-CF.sub.3.
3. The compound according to claim 1, wherein R.sup.2 is hydrogen.
4. The compound according to claim 1, wherein R.sup.3 is selected from —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-4 alkyl), —SO.sub.2—N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and —SO.sub.2—N═(C.sub.1-4 alkylidene), and further wherein the alkyl moiety of said —SO.sub.2—NH(C.sub.1-4 alkyl), one or both of the alkyl moieties of said —SO.sub.2—N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and the alkylidene moiety of said —SO.sub.2—N═(C.sub.1-4 alkylidene) are each optionally substituted with one group selected from —NH.sub.2, —NH(C.sub.1-4 alkyl) and —N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl).
5. The compound according to claim 1, wherein R.sup.3 is —SO.sub.2—NH.sub.2.
6. The compound according to claim 1, wherein R.sup.4 is selected from —O-aryl, —O-heteroaryl, —S-aryl, —S-heteroaryl, —NH-aryl, —NH-heteroaryl, —N(C.sub.1-4 alkyl)-aryl, —N(C.sub.1-4 alkyl)-heteroaryl, aryl and heteroaryl, and wherein aryl and heteroaryl moiety are each optionally substituted with one or more groups independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl and —CN.
7. The compound according to cliam 1, wherein R.sup.4 is —O-phenyl.
8. The compound according to claim 1, wherein R.sup.5 is selected from —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), and —NO.sub.2.
9. The compound according to claim 1, wherein R.sup.5 is —NH—CH.sub.2CH.sub.2CH.sub.2CH.sub.3.
10. The compound according to claim 1, wherein R.sup.6 is hydrogen.
11. The compound according to claim 1, wherein the compound of formula (I) is selected from the group consisting of: ##STR00016## ##STR00017## and pharmaceutically acceptable salts or solvents thereof.
12. The compound according to claim 1, wherein said compounds is ##STR00018## or pharmaceutically acceptable salts or solvate thereof.
13. A pharmaceutical composition comprising a compound according to claim 1 and at least one pharmaceutically acceptable excipient.
14. (canceled)
15. A method of treating or preventing a neurological disease or disorder involving a Na.sup.+—K.sup.+-2Cl.sup.−-cotransporter (NKCC), the method comprising administering a compound according to claim 1 to a subject in need thereof.
16. The method according to claim 15, wherein the neurological disease or disorder involving an NKCC is selected from the group consisting of stroke, traumatic brain injury, spinal cord injury, peripheral nerve injury, brain edema, glioma, an autism spectrum disorder, Alzheimer's disease, schizophrenia, and Down syndrome.
17. The method according to claim 15, wherein the neurological disease or disorder involving an NKCC is selected from the group consisting of stroke, traumatic brain injury, spinal cord injury, peripheral nerve injury, brain edema, and glioma.
18. The method according to claim 15, wherein the neurological disease or disorder involving an NKCC is stroke.
19. The method according to claim 15, wherein the subject is a human.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0047]
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
DETAILED DESCRIPTION OF THE INVENTION
[0055] As described above, the present invention relates to the treatment or prevention of a neurological disease or disorder involving (or mediated by) an NKCC, particularly a neurological disease or disorder involving (or mediated by) NKCC1, such as, e.g., stroke (particularly ischemic stroke; including also stroke in subjects/patients with hypertension), traumatic brain injury, spinal cord injury, peripheral nerve injury, brain edema, or glioma, using a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.
[0056] The neurological disease or disorder to be treated or prevented in accordance with the present invention, specifically the neurological disease or disorder involving an NKCC (preferably NKCC1), is not particularly limited, and is preferably selected from stroke (e.g., ischemic stroke; including, in particular, the use of the compounds according to the invention in promoting recovery after stroke, or the use of said compounds in reducing brain damage and/or neurological deficits after stroke), traumatic brain injury, spinal cord injury (including also chronic pain caused by spinal cord injury), peripheral nerve injury, brain edema, glioma (e.g., oligodendroglioma, ependymoma, subependymoma, choroid plexus papilloma, choroid plexus carcinoma, glioblastoma multiforme, astrocytoma, oligoastrocytoma, gliomatosis cerebri, or gliosarcoma), an autism spectrum disorder (e.g., autism, Asperger syndrome, childhood disintegrative disorder, or a pervasive developmental disorder as part of an autism spectrum disorder), Alzheimer's disease, schizophrenia, or Down syndrome (particularly mental disability in patients with Down syndrome). It is particularly preferred that the disease or disorder to be treated or prevented in accordance with the invention is stroke (particularly ischemic stroke), traumatic brain injury, spinal cord injury, peripheral nerve injury, brain edema, or glioma. The present invention particularly relates to the treatment or prevention of stroke (preferably in a human subject/patient, who may be male or female, and particularly in a male human subject/patient).
[0057] The compound of formula (I) as well as the pharmaceutically acceptable salt or solvate thereof will be described in more detail in the following:
##STR00003##
[0058] In formula (I), R.sup.1 is selected from —(C.sub.1-4 alkylene)-NH—(C.sub.1-4 alkylene)-R.sup.11 (e.g., —CH.sub.2—NH—CH.sub.2—R.sup.11), —COO—(C.sub.1-4 alkylene)-R.sup.11 (e.g., —COO—CH.sub.2—R.sup.11), —O—CO—(C.sub.1-4 alkylene)-R.sup.11, —CO—(C.sub.1-4 alkylene)-R.sup.11, —CO—NH—(C.sub.1-4 alkylene)-R.sup.11 (e.g., —CO—NH—CH.sub.2—R.sup.11), —CO—N(C.sub.1-4 alkyl)-(C.sub.1-4 alkylene)-R.sup.11, —NH—CO—(C.sub.1-4 alkylene)-R.sup.11 and —N(C.sub.1-4 alkyl)-CO—(C.sub.1-4 alkylene)-R.sup.11, wherein R.sup.11 is independently selected from —CF.sub.3, —CN and halogen (e.g., —F, —Cl, —Br or —I). Preferably, R.sup.11 is independently selected from —CF.sub.3 and —CN; more preferably, R.sup.11 is —CF.sub.3. Specific examples of such R.sup.1 groups include the corresponding groups R.sup.1 of the compounds described in the examples section. It is particularly preferred that R.sup.1 is —(C.sub.1-4 alkylene)-NH—(C.sub.1-4 alkylene)-CF.sub.3, and even more preferably R.sup.1 is —CH.sub.2—NH—CH.sub.2—CF.sub.3.
[0059] R.sup.2 is selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, haloalkyl), —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)—CO—(C.sub.1-6 alkyl), —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—SO.sub.2—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-SO.sub.2—(C.sub.1-6 alkyl). Preferably, R.sup.2 is selected from hydrogen, C.sub.1-6 alkyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl) and —CN. More preferably, R.sup.2 is hydrogen or C.sub.1-.sub.4 alkyl. Even more preferably, R.sup.2 is hydrogen.
[0060] R.sup.3 is selected from —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —SO.sub.2N═(C.sub.1-6 alkylidene) and —SO.sub.2-halogen, wherein the alkyl moiety of said —SO.sub.2—NH(C.sub.1-6 alkyl), one or both of the alkyl moieties of said —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), and the alkylidene moiety of said —SO.sub.2—N═(C.sub.1-6 alkylidene) are each optionally substituted with one or more (e.g., one, two or three) groups independently selected from halogen, —CF.sub.3, —CN, —NO.sub.2, —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —OH, —O(C.sub.1-6 alkyl), —SH and —S(C.sub.1-6 alkyl). Preferably, R.sup.3 is selected from —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-4 alkyl), —SO.sub.2—N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and —SO.sub.2—N═(C.sub.1-4 alkylidene), wherein the alkyl moiety of said —SO.sub.2—NH(C.sub.1-4 alkyl), one or both of the alkyl moieties of said —SO.sub.2—N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and the alkylidene moiety of said —SO.sub.2—N═(C.sub.1-4 alkylidene) are each optionally substituted with one or more groups (particularly one group) independently selected from halogen, —CF.sub.3, —CN, —NO.sub.2, —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —OH, —O(C.sub.1-6 alkyl), —SH and —S(C.sub.1-6 alkyl). More preferably, R.sup.3 is selected from —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-4 alkyl), —SO.sub.2—N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and —SO.sub.2—N═(C.sub.1-4 alkylidene), wherein the alkyl moiety of said —SO.sub.2—NH(C.sub.1-4 alkyl), one or both of the alkyl moieties of said —SO.sub.2—N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and the alkylidene moiety of said —SO.sub.2—N═(C.sub.1-4 alkylidene) are each optionally substituted with one group selected from —NH.sub.2, —NH(C.sub.1-4 alkyl) and —N(C.sub.14 alkyl)(C.sub.1-4 alkyl). Even more preferably, R.sup.3 is selected from —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-4 alkyl), —SO.sub.2—N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), —SO.sub.2—NH—(C.sub.1-4 alkylene)-NH.sub.2, —SO.sub.2—NH—(C.sub.1-4 alkylene)-NH(C.sub.1-4 alkyl), —SO.sub.2—NH—(C.sub.1-4 alkylene)-N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), —SO.sub.2—N═(C.sub.1-4 alkylidene)-NH.sub.2, —SO.sub.2—N═(C.sub.1-4 alkylidene)-NH(C.sub.1-4 alkyl) and —SO.sub.2—N═(C.sub.1-4 alkylidene)-N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl). Yet even more preferably, R.sup.3 is selected from —SO.sub.2—NH.sub.2, —SO.sub.2—NH—CH.sub.3, —SO.sub.2—N(CH.sub.3).sub.2, —SO.sub.2—NH—(C.sub.1-4 alkylene)-NH.sub.2, —SO.sub.2—NH—(C.sub.1-4 alkylene)-NH—CH.sub.3, —SO.sub.2—NH—(C.sub.1-4 alkylene)-N(CH.sub.3).sub.2 (e.g., —SO.sub.2—NH—CH.sub.2CH.sub.2—N(CH.sub.3).sub.2), —SO.sub.2—N═(C.sub.1-4 alkylidene)-NH.sub.2, —SO.sub.2—N═(C.sub.1-4 alkylidene)-NH—CH.sub.3 and —SO.sub.2—N═(C.sub.1-4 alkylidene)-N(CH.sub.3).sub.2 (e.g., —SO.sub.2—N═CH—N(CH.sub.3).sub.2). Still more preferably, R.sup.3 is —SO.sub.2—NH.sub.2.
[0061] R.sup.4 is selected from —O—R.sup.41, —S—R.sup.41, —NH—R.sup.41, —N(C.sub.1-6 alkyl)-R.sup.41, halogen (e.g., —Cl), hydrogen, carbocyclyl and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.42. Preferably, R.sup.4 is selected from —O—R.sup.41, —S—R.sup.41, —NH—R.sup.41, —N(C.sub.1-6 alkyl)-R.sup.41, halogen, carbocyclyl and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups R.sup.42. More preferably, R.sup.4 is selected from —O—R.sup.41, —S—R.sup.41, —NH—R.sup.41, —N(C.sub.1-6 alkyl)-R.sup.41, carbocyclyl (e.g., aryl, cycloalkyl, or cycloalkenyl) and heterocyclyl (e.g., heteroaryl, heterocycloalkyl, or heterocycloalkenyl), wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups R.sup.42. Even more preferably, R.sup.4 is selected from —O—R.sup.41, —S—R.sup.41, —NH—R.sup.41, —N(C.sub.1-4 alkyl)-R.sup.41, aryl and heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R.sup.42.
[0062] R.sup.41 is selected from —(C.sub.0-4 alkylene)-carbocyclyl, —(C.sub.0-4 alkylene)-heterocyclyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl, wherein the carbocyclyl moiety of said —(C.sub.0-4 alkylene)-carbocyclyl and the heterocyclyl moiety of said —(C.sub.0-4 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.42, and wherein said C.sub.1-6 alkyl, said C.sub.2-6 alkenyl, said C.sub.2-6 alkynyl, the alkylene moiety of said —(C.sub.0-4 alkylene)-carbocyclyl, and the alkylene moiety of said —(C.sub.0-4 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.43. Preferably, R.sup.41 is selected from —(C.sub.0-4 alkylene)-carbocyclyl, —(C.sub.0-4 alkylene)-heterocyclyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl, wherein the carbocyclyl moiety of said —(C.sub.0-4 alkylene)-carbocyclyl is selected from cycloalkyl, cycloalkenyl and aryl, wherein the heterocyclyl moiety of said —(C.sub.0-4 alkylene)-heterocyclyl is selected from heterocycloalkyl, heterocycloalkenyl and heteroaryl, wherein the carbocyclyl moiety of said —(C.sub.0-4 alkylene)-carbocyclyl and the heterocyclyl moiety of said —(C.sub.0-4 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.42, and further wherein said C.sub.1-6 alkyl, said C.sub.2-6 alkenyl, said C.sub.2-6 alkynyl, the alkylene moiety of said —(C.sub.0.4 alkylene)-carbocyclyl, and the alkylene moiety of said —(C.sub.0-4 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.43. More preferably, R.sup.41 is selected from —(C.sub.0-4 alkylene)-aryl and —(C.sub.0-4 alkylene)-heteroaryl, wherein the aryl moiety of said —(C.sub.0-4 alkylene)-aryl and the heteroaryl moiety of said —(C.sub.0-4 alkylene)-heteroaryl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.42, and further wherein the alkylene moiety of said —(C.sub.0-4 alkylene)-aryl and the alkylene moiety of said —(C.sub.0-4 alkylene)-heteroaryl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.43. Even more preferably, R.sup.41 is selected from —(C.sub.0-4 alkylene)-aryl and —(C.sub.0-4 alkylene)-heteroaryl, wherein the aryl moiety of said —(C.sub.0-4 alkylene)-aryl and the heteroaryl moiety of said —(C.sub.0-4 alkylene)-heteroaryl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.42. A preferred example of the aryl moiety of said —(C.sub.0-4 alkylene)-aryl is phenyl. A preferred example of the heteroaryl moiety of said —(C.sub.0-4 alkylene)-heteroaryl is a 5- or 6-membered monocyclic heteroaryl having 1 or 2 ring heteroatoms independently selected from oxygen, nitrogen and sulfur (wherein the remaining ring atoms are carbon atoms), such as, e.g., imidazolyl, thiophenyl, or pyrimidinyl. Still more preferably, R.sup.41 is selected from phenyl and heteroaryl, wherein said heteroaryl is a 5- or 6-membered monocyclic heteroaryl having 1 or 2 ring heteroatoms independently selected from oxygen, nitrogen and sulfur (the remaining ring atoms of the monocyclic heteroaryl are carbon atoms), and further wherein said phenyl or said heteroaryl is optionally substituted with one or more (e.g., one, two or three) groups R.sup.42.
[0063] Each R.sup.42 is independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—CO—(C.sub.1-6 haloalkyl), —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)-CO—(C.sub.1-6 alkyl), —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—SO.sub.2—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-SO.sub.2—(C.sub.1-6 alkyl). Preferably, each R.sup.42 is independently selected from C.sub.1-6 alkyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl) and —CN.
[0064] Each R.sup.43 is independently selected from —OH, —O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, —CF.sub.3, —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-CO—(C.sub.1-6 alkyl). Preferably, each R.sup.43 is independently selected from —OH, —O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, —CF.sub.3 and —CN.
[0065] In accordance with the above definitions, it is particularly preferred that R.sup.4 is selected from —O—(C.sub.0-4 alkylene)-aryl, —O—(C.sub.0-4 alkylene)-heteroaryl, —S—(C.sub.0-4 alkylene)-aryl, —S—(C.sub.0-4 alkylene)-heteroaryl, —NH—(C.sub.0-4 alkylene)-aryl, —NH—(C.sub.0-4 alkylene)-heteroaryl, —N(C.sub.1-4 alkyl)-(C.sub.0-4 alkylene)-aryl, —N(C.sub.1-4 alkyl)-(C.sub.0-4 alkylene)-heteroaryl, aryl and heteroaryl, wherein the aryl moiety of any of the aforementioned groups, the heteroaryl moiety of any of the aforementioned groups, said aryl and said heteroaryl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.42. Even more preferably, R.sup.4 is selected from —O-aryl, —O-heteroaryl, —S-aryl, —S-heteroaryl, —NH-aryl, —NH-heteroaryl, —N(C.sub.1-4 alkyl)-aryl, —N(C.sub.1-4 alkyl)-heteroaryl, aryl and heteroaryl, wherein the aryl moiety of any of the aforementioned groups, the heteroaryl moiety of any of the aforementioned groups, said aryl and said heteroaryl are each optionally substituted with one or more groups independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl and —CN. Yet even more preferably, R.sup.4 is selected from —O-phenyl, —O-heteroaryl, —S-phenyl, —S-heteroaryl, —NH-phenyl, —NH-heteroaryl, —N(C.sub.1-4 alkyl)-phenyl, —N(C.sub.1-4 alkyl)-heteroaryl, phenyl and heteroaryl, wherein said heteroaryl or the heteroaryl moiety of any of the aforementioned groups is a 5- or 6-membered monocyclic heteroaryl having 1 or 2 ring heteroatoms independently selected from oxygen, nitrogen and sulfur (the remaining ring atoms of the monocyclic heteroaryl are carbon atoms), and further wherein the phenyl moiety of any of the aforementioned groups, the heteroaryl moiety of any of the aforementioned groups, said phenyl and said heteroaryl are each optionally substituted with one or more groups independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl and —CN. Still more preferably, R.sup.4 is —O-phenyl.
[0066] R.sup.5 is selected from —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NO.sub.2 and hydrogen, wherein the alkyl moiety of said —NH(C.sub.1-6 alkyl) and one or both of the alkyl moieties of said —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl) are each optionally substituted with one or more (e.g., one, two or three) groups independently selected from halogen, —CF.sub.3, —CN, —NO.sub.2, —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —OH, —O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), carbocyclyl and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups R.sup.51. Preferably, R.sup.5 is selected from —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl) and —NO.sub.2, wherein the alkyl moiety of said —NH(C.sub.1-6 alkyl) and one or both of the alkyl moieties of said —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl) are each optionally substituted with one or more (e.g., one, two or three) groups independently selected from halogen, —CF.sub.3, —CN, —NO.sub.2, —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —OH, —O(C.sub.1-6 alkyl), —SH and —S(C.sub.1-6 alkyl). More preferably, R.sup.5 is selected from —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), and —-NO.sub.2. Even more preferably, R.sup.5 is selected from —NH.sub.2, —NH(C.sub.1-6 alkyl), and —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl). Yet even more preferably, R.sup.5 is —NH(C.sub.1-6 alkyl). Still more preferably, R.sup.5 is —-NH—CH.sub.2CH.sub.2CH.sub.2CH.sub.3.
[0067] Each R.sup.51 is independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl), —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)-CO—(C.sub.1-6 alkyl), —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—SO.sub.2—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-SO.sub.2—(C.sub.1-6 alkyl). Preferably, each R.sup.51 is independently selected from C.sub.1-6 alkyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl) and —CN.
[0068] R.sup.6 is selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl), —CN, —NO.sub.2, —CHO, —CO—(C.sub.1-6 alkyl), —COOH, —COO—(C.sub.1-6 alkyl), —O—CO—(C.sub.1-6 alkyl), —CO—NH.sub.2, —CO—NH(C.sub.1-6 alkyl), —CO—N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—CO—(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)—O—(C.sub.1-6 alkyl), —SO.sub.2—NH.sub.2, —SO.sub.2—NH(C.sub.1-6 alkyl), —SO.sub.2-N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), —NH—SO.sub.2—(C.sub.1-6 alkyl) and —N(C.sub.1-6 alkyl)-SO.sub.2—(C.sub.1-6 alkyl). Preferably, R.sup.6 is selected from hydrogen, C.sub.1-6 alkyl, —OH, —O(C.sub.1-6 alkyl), —O(C.sub.1-6 alkylene)-OH, —O(C.sub.1-6 alkylene)-O(C.sub.1-6 alkyl), —SH, —S(C.sub.1-6 alkyl), —NH.sub.2, —NH(C.sub.1-6 alkyl), —N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 haloalkyl) and —CN. More preferably, R.sup.6 is hydrogen or C.sub.1-4 alkyl. Even more preferably, R.sup.6 is hydrogen.
[0069] The compound of formula (I) may be, for example, any one of the specific compounds described in the examples section of this specification, either in non-salt form (e.g., free base/acid form) or as a pharmaceutically acceptable salt or solvate of the respective compound.
[0070] In particular, the compound of formula (I) may be a compound of any one of the following formulae, or a pharmaceutically acceptable salt or solvate thereof:
##STR00004## ##STR00005##
[0071] For a person skilled in the field of synthetic chemistry, various ways for the preparation of the compounds of formula (I) will be readily apparent. For example, the compounds of formula (I) can be prepared in accordance with or in analogy to the synthetic routes described in the examples section.
[0072] The following definitions apply throughout the present specification, unless specifically indicated otherwise.
[0073] The term “hydrocarbon group” refers to a group consisting of carbon atoms and hydrogen atoms.
[0074] The term “alicyclic” is used in connection with cyclic groups and denotes that the corresponding cyclic group is non-aromatic.
[0075] As used herein, the term “alkyl” refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. A “C.sub.1-5 alkyl” denotes an alkyl group having 1 to 6 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless defined otherwise, the term “alkyl” preferably refers to C.sub.1-4 alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.
[0076] As used herein, the term “alkenyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term “C.sub.2-6 alkenyl” denotes an alkenyl group having 2 to 6 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl, or prop-2-en-1-yl), butenyl, butadienyl (e.g., buta-1,3-dien-1-yl or buta-1,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl). Unless defined otherwise, the term “alkenyl” preferably refers to C.sub.2-4 alkenyl.
[0077] As used herein, the term “alkynyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more carbon-to-carbon double bonds. The term “C.sub.2-6 alkynyl” denotes an alkynyl group having 2 to 6 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl. Unless defined otherwise, the term “alkynyl” preferably refers to C.sub.2-4 alkynyl.
[0078] As used herein, the term “alkylene” refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched. A “C.sub.1-15 alkylene” denotes an alkylene group having 1 to 15 carbon atoms, and the term “C.sub.0-15 alkylene” indicates that a covalent bond (corresponding to the option “C.sub.0 alkylene”) or a C.sub.1-16 alkylene is present. Preferred exemplary alkylene groups are methylene (—CH.sub.2—), ethylene (e.g., —CH.sub.2—CH.sub.2— or —CH(—CH.sub.3)—), propylene (e.g., —CH.sub.2—CH.sub.2—CH.sub.2—, —CH(—CH.sub.2—CH.sub.3)—, —CH.sub.2—CH(—CH.sub.3)—, or —CH(—CH.sub.3)—CH.sub.2—), or butylene (e.g., —CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—). Unless defined otherwise, the term “alkylene” preferably refers to C.sub.1-4 alkylene (including, in particular, linear C.sub.1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.
[0079] As used herein, the term “alkylidene” refers to a divalent acyclic hydrocarbon group which may be linear or branched, which is connected to the remainder of the respective compound via a double bond, and which does not comprise any other double bond (i.e., which does not comprise any double bond except for the one that connects the alkylidene group to the remainder of the respective compound) or any triple bond. An alkylidene group may, e.g., be attached to a carbon atom or to a nitrogen atom of the remainder of the respective compound. A “C.sub.1-6 alkylidene” denotes an alkylidene group having 1 to 6 carbon atoms. Preferred exemplary alkylidene groups are methylidene (═CH.sub.2), ethylidene (═CH—CH.sub.3), propylidene (e.g., ═CH—CH.sub.2CH.sub.3 or ═C(—CH.sub.3)—CH.sub.3), or butylidene (e.g., ═CH—CH.sub.2CH.sub.2OH.sub.3, ═C(—CH.sub.3)—CH.sub.2CH.sub.3, or ═CH—CH(—CH.sub.3)—CH.sub.3). Unless defined otherwise, the term “alkylidene” preferably refers to C.sub.1-4 alkylidene.
[0080] As used herein, the term “carbocyclyl” refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, “carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl.
[0081] As used herein, the term “heterocyclyl” refers to a ring group, including monocyclic rings as well as bridged ring, Spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. For example, each heteroatom-containing ring comprised in said ring group may contain one or two O atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. Unless defined otherwise, “heterocyclyl” preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.
[0082] As used herein, the term “aryl” refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). “Aryl” may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1H-indenyl), anthracenyl, phenanthrenyl, 9H-fluorenyl, or azulenyl. Unless defined otherwise, an “aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.
[0083] As used herein, the term “heteroaryl” refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said aromatic ring group may contain one or two O atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. “Heteroaryl” may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1-benzopyranyl or 4H-1-benzopyranyl), isochromenyl (e.g., 1H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolyl (e.g., 3H-indolyl), isoindolyl, indazolyl, indolizinyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (e.g., [1,10]phenanthrolinyl, [1,7]phenanthrolinyl, or [4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (i.e., furazanyl), or 1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, or 1,3,4-thiadiazolyl), phenoxazinyl, pyrazolo[1,5-a]pyrimidinyl (e.g., pyrazolo[1,5-a]pyrimidin-3-yl), 1,2-benzoisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzo[b]thiophenyl (i.e., benzothienyl), triazolyl (e.g., 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, or 4H-1,2,4-triazolyl), benzotriazolyl, 1H-tetrazolyl, 2H-tetrazolyl, triazinyl (e.g., 1,2,3-triazinyl, 1,2,4-triazinyl, or 1,3,5-triazinyl), furo[2,3-c]pyridinyl, dihydrofuropyridinyl (e.g., 2,3-dihydrofuro[2,3-c]pyridinyl or 1,3-dihydrofuro[3,4-c]pyridinyl), imidazopyridinyl (e.g., imidazo[1,2-a]pyridinyl or imidazo[3,2-a]pyridinyl), quinazolinyl, thienopyridinyl, tetrahydrothienopyridinyl (e.g., 4,5,6,7-tetrahydrothieno[3,2-c]pyridinyl), dibenzofuranyl, 1,3-benzodioxolyl, benzodioxanyl (e.g., 1,3-benzodioxanyl or 1,4-benzodioxanyl), or coumarinyl. Unless defined otherwise, the term “heteroaryl” preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a “heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. Moreover, unless defined otherwise, particularly preferred examples of a “heteroaryl” include pyridinyl (e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), imidazolyl, thiazolyl, 1H-tetrazolyl, 2H-tetrazolyl, thienyl (i.e., thiophenyl), or pyrimidinyl.
[0084] As used herein, the term “cycloalkyl” refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). “Cycloalkyl” may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl. Unless defined otherwise, “cycloalkyl” preferably refers to a C.sub.3-11 cycloalkyl, and more preferably refers to a C.sub.3-7 cycloalkyl. A particularly preferred “cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members. Moreover, unless defined otherwise, particularly preferred examples of a “cycloalkyl” include cyclohexyl or cyclopropyl, particularly cyclohexyl.
[0085] As used herein, the term “heterocycloalkyl” refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said saturated ring group may contain one or two O atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. “Heterocycloalkyl” may, e.g., refer to aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, azepanyl, diazepanyl (e.g., 1,4-diazepanyl), oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g., morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, 1,3-dioxolanyl, tetrahydropyranyl, 1,4-dioxanyl, oxepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl (i.e., thiolanyl), 1,3-dithiolanyl, thianyl, thiepanyl, decahydroquinolinyl, decahydroisoquinolinyl, or 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl. Unless defined otherwise, “heterocycloalkyl” preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, “heterocycloalkyl” refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. Moreover, unless defined otherwise, particularly preferred examples of a “heterocycloalkyl” include tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrofuranyl.
[0086] As used herein, the term “cycloalkenyl” refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond. “Cycloalkenyl” may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless defined otherwise, “cycloalkenyl” preferably refers to a C.sub.3-11 cycloalkenyl, and more preferably refers to a C.sub.3-7 cycloalkenyl. A particularly preferred “cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.
[0087] As used herein, the term “heterocycloalkenyl” refers to an unsaturated alicyclic (non-aromatic) ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms. For example, each heteroatom-containing ring comprised in said unsaturated alicyclic ring group may contain one or two O atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. “Heterocycloalkenyl” may, e.g., refer to imidazolinyl (e.g., 2-imidazolinyl (i.e., 4,5-dihydro-1H-imidazolyl), 3-imidazolinyl, or 4-imidazolinyl), tetrahydropyridinyl (e.g., 1,2,3,6-tetrahydropyridinyl), dihydropyridinyl (e.g., 1,2-dihydropyridinyl or 2,3-dihydropyridinyl), pyranyl (e.g., 2H-pyranyl or 4H-pyranyl), thiopyranyl (e.g., 2H-thiopyranyl or 4H-thiopyranyl), dihydropyranyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrazinyl, dihydroisoindolyl, octahydroquinolinyl (e.g., 1,2,3,4,4a,5,6,7-octahydroquinolinyl), or octahydroisoquinolinyl (e.g., 1,2,3,4,5,6,7,8-octahydroisoquinolinyl). Unless defined otherwise, “heterocycloalkenyl” preferably refers to a 3 to 11 membered unsaturated alicyclic ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms; more preferably, “heterocycloalkenyl” refers to a 5 to 7 membered monocyclic unsaturated non-aromatic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms.
[0088] As used herein, the term “halogen” refers to fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (—I).
[0089] As used herein, the term “haloalkyl” refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group. “Haloalkyl” may, e.g., refer to —CF.sub.3, —CHF.sub.2, —CH.sub.2F, —CF.sub.2—CH.sub.3, —CH.sub.2—CF.sub.3, —CH.sub.2—CHF.sub.2, —CH.sub.2—CF.sub.2—CH.sub.3, —CH.sub.2—CF.sub.2—CF.sub.3, or —CH(CF.sub.3).sub.2. A particularly preferred “haloalkyl” group is —CF.sub.3.
[0090] As used herein, the terms “optional”, “optionally” and “may” denote that the indicated feature may be present but can also be absent. Whenever the term “optional”, “optionally” or “may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression “X is optionally substituted with Y” (or “X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be “optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.
[0091] Various groups are referred to as being “optionally substituted” in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless defined otherwise, the “optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.
[0092] A skilled person will appreciate that the substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.
[0093] As used herein, unless explicitly indicated otherwise or contradicted by context, the terms “a”, “an” and “the” are used interchangeably with “one or more” and “at least one”. Thus, for example, a composition comprising “a” compound of formula (I) can be interpreted as referring to a composition comprising “one or more” compounds of formula (I).
[0094] As used herein, the term “about” preferably refers to ±10% of the indicated numerical value, more preferably to ±5% of the indicated numerical value, and in particular to the exact numerical value indicated. If the term “about” is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint −10% of its indicated numerical value to the upper endpoint +10% of its indicated numerical value, more preferably to the range from of the lower endpoint −5% to the upper endpoint +5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint. If the term “about” is used in connection with the endpoint of an open-ended range, it preferably refers to the corresponding range starting from the lower endpoint −10% or from the upper endpoint +10%, more preferably to the range starting from the lower endpoint −5% or from the upper endpoint +5%, and even more preferably to the open-ended range defined by the exact numerical value of the corresponding endpoint. If the term “about” is used in connection with a parameter that is quantified in integers, such as the number of nucleotides in a given nucleic acid, the numbers corresponding to ±10% or ±5% of the indicated numerical value are to be rounded to the nearest integer (using the tie-breaking rule “round half up”).
[0095] As used herein, the term “comprising” (or “comprise”, “comprises”, “contain”, “contains”, or “containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of “containing, inter alia”, i.e., “containing, among further optional elements, . . . ”. In addition thereto, this term also includes the narrower meanings of “consisting essentially of” and “consisting of”. For example, the term “A comprising B and C” has the meaning of “A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., “A containing B, C and D” would also be encompassed), but this term also includes the meaning of “A consisting essentially of B and C” and the meaning of “A consisting of B and C” (i.e., no other components than B and C are comprised in A).
[0096] The scope of the invention embraces all pharmaceutically or physiologically acceptable salt forms of the compounds of formula (I) which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), 2-naphthalenesulfonate (napsylate), 3-phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts. Preferred pharmaceutically/physiologically acceptable salts of the compounds of formula (I) include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt. A particularly preferred pharmaceutically/physiologically acceptable salt of the compound of formula (I) is a hydrochloride salt.
[0097] Moreover, the scope of the invention embraces the compounds of formula (I) in any solvated form, including, e.g., solvates with water (i.e., as a hydrate) or solvates with organic solvents such as, e.g., methanol, ethanol or acetonitrile (i.e., as a methanolate, ethanolate or acetonitrilate), or in any crystalline form (i.e., as any polymorph), or in amorphous form. It is to be understood that such solvates of the compounds of the formula (I) also include solvates of pharmaceutically acceptable salts of the compounds of the formula (I).
[0098] Furthermore, the compounds of formula (I) may exist in the form of different isomers, in particular stereoisomers (including, e.g., geometric isomers (or cis/trans isomers), enantiomers and diastereomers) or tautomers. All such isomers of the compounds of formula (I) are contemplated as being part of the present invention, either in admixture or in pure or substantially pure form. As for stereoisomers, the invention embraces the isolated optical isomers of the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures/racemates). The racemates can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization. The present invention further encompasses any tautomers of the compounds provided herein.
[0099] The scope of the invention also embraces compounds of formula (I), in which one or more atoms are replaced by a specific isotope of the corresponding atom. For example, the invention encompasses compounds of formula (I), in which one or more hydrogen atoms (or, e.g., all hydrogen atoms) are replaced by deuterium atoms (i.e., .sup.2H; also referred to as “D”). Accordingly, the invention also embraces compounds of formula (I) which are enriched in deuterium. Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol-% hydrogen-1 (.sup.1H) and about 0.0156 mol-% deuterium (.sup.2H or D). The content of deuterium in one or more hydrogen positions in the compounds of formula (I) can be increased using deuteration techniques known in the art. For example, a compound of formula (I) or a reactant or precursor to be used in the synthesis of the compound of formula (I) can be subjected to an H/D exchange reaction using, e.g., heavy water (D.sub.2O). Further suitable deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012; William J S et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11-12), 635-644, 2010; Modvig A et al., J Org Chem, 79, 5861-5868, 2014. The content of deuterium can be determined, e.g., using mass spectrometry or NMR spectroscopy. Unless specifically indicated otherwise, it is preferred that the compound of formula (I) is not enriched in deuterium. Accordingly, the presence of naturally occurring hydrogen atoms or .sup.1H hydrogen atoms in the compounds of formula (I) is preferred.
[0100] The present invention also embraces compounds of formula (I), in which one or more atoms are replaced by a positron-emitting isotope of the corresponding atom, such as, e.g., 18.sub.F, .sup.11C, .sup.13N, .sup.15O, .sup.76Br, .sup.77Br, .sup.120I and/or .sup.124I. Such compounds can be used as tracers, trackers or imaging probes in positron emission tomography (PET). The invention thus includes (i) compounds of formula (I), in which one or more fluorine atoms (or, e.g., all fluorine atoms) are replaced by .sup.18F atoms, (ii) compounds of formula (I), in which one or more carbon atoms (or, e.g., all carbon atoms) are replaced by .sup.110 atoms, (iii) compounds of formula (I), in which one or more nitrogen atoms (or, e.g., all nitrogen atoms) are replaced by .sup.13N atoms, (iv) compounds of formula (I), in which one or more oxygen atoms (or, e.g., all oxygen atoms) are replaced by .sup.15O atoms, (v) compounds of formula (I), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by .sup.76Br atoms, (vi) compounds of formula (I), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by .sup.77Br atoms, (vii) compounds of formula (I), in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by .sup.120I atoms, and (viii) compounds of formula (I), in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by .sup.124I atoms. In general, it is preferred that none of the atoms in the compounds of formula (I) are replaced by specific isotopes.
[0101] The compounds provided herein may be administered as compounds per se or may be formulated as medicaments. The medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers.
[0102] The pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., poly(ethylene glycol), including poly(ethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor° HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxyethyl-γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, dihydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, sulfobutylether-γ-cyclodextrin, glucosyl-α-cyclodextrin, glucosyl-β-cyclodextrin, diglucosyl-β-cyclodextrin, maltosyl-α-cyclodextrin, maltosyl-β-cyclodextrin, maltosyl-γ-cyclodextrin, maltotriosyl-β-cyclodextrin, maltotriosyl-γ-cyclodextrin, dimaltosyl-β-cyclodextrin, methyl-β-cyclodextrin, a carboxyalkyl thioether, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, a vinyl acetate copolymer, vinyl pyrrolidone, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, or any combination thereof.
[0103] The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in “Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22.sup.nd edition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration. Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
[0104] The compounds of formula (I) or the above described pharmaceutical compositions comprising a compound of formula (I) may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose), gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic (including intravitreal or intracameral), rectal, or vaginal administration.
[0105] If said compounds or pharmaceutical compositions are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracardially, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and/or by using infusion techniques. For parenteral administration, the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
[0106] Said compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
[0107] The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
[0108] Alternatively, said compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compounds of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch.
[0109] Said compounds or pharmaceutical compositions may also be administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include, e.g., polylactides (see, e.g., U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP133988). Sustained-release pharmaceutical compositions also include liposomally entrapped compounds. Liposomes containing a compound of the present invention can be prepared by methods known in the art, such as, e.g., the methods described in any one of: DE3218121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP0052322; EP0036676; EP088046; EP0143949; EP0142641; JP 83-118008; U.S. Pat. Nos. 4,485,045; 4,544,545; and EP0102324.
[0110] Said compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes, or the ocular route. For ophthalmic use, they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
[0111] It is also envisaged to prepare dry powder formulations of the compounds of formula (I) for pulmonary administration, particularly inhalation. Such dry powders may be prepared by spray drying under conditions which result in a substantially amorphous glassy or a substantially crystalline bioactive powder. Accordingly, dry powders of the compounds of the present invention can be made according to the emulsification/spray drying process disclosed in WO 99/16419 or WO 01/85136. Spray drying of solution formulations of the compounds of the invention can be carried out, e.g., as described generally in the “Spray Drying Handbook”, 5th ed., K. Masters, John Wiley & Sons, Inc., NY (1991), in WO 97/41833, or in WO 03/053411.
[0112] For topical application to the skin, said compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
[0113] The present invention thus relates to the compounds or the pharmaceutical compositions provided herein, wherein the corresponding compound or pharmaceutical composition is to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, infrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route. Particularly preferred routes of administration are topical administration, oral administration or parenteral administration.
[0114] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual subject 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 individual subject undergoing therapy.
[0115] A proposed, yet non-limiting dose of the compounds according to the invention for oral administration to a human (of approximately 70 kg body weight) may be 0.05 to 2000 mg, particularly 0.1 mg to 1000 mg, of the active ingredient per unit dose. The unit dose may be administered, e.g., 1 to 3 times per day. The unit dose may also be administered 1 to 7 times per week, e.g., with not more than one administration per day. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient/subject as well as the severity of the condition to be treated. The precise dose and also the route of administration will ultimately be at the discretion of the attendant physician or veterinarian.
[0116] The compound of formula (I) or a pharmaceutical composition comprising the compound of formula (I) can be administered in monotherapy (e.g., without concomitantly administering any further therapeutic agents, or without concomitantly administering any further therapeutic agents against the same disease that is to be treated or prevented with the compound of formula (I)). However, the compound of formula (I) or a pharmaceutical composition comprising the compound of formula (I) can also be administered in combination with one or more further therapeutic agents, such as, e.g., one or more further therapeutic agents selected from phenobarbital, phenytoin, valproate (or valproic acid), carbamazepine, lamotrigine, levetiracetam, ethosuximide, and pharmaceutically acceptable salts of any of the aforementioned agents. If the compound of formula (I) is used in combination with a second therapeutic agent active against the same disease or condition, the dose of each compound may differ from that when the corresponding compound is used alone, in particular, a lower dose of each compound may be used. The combination of the compound of formula (I) with one or more further therapeutic agents (e.g., one or more of the corresponding exemplary therapeutic agents mentioned above) may comprise the simultaneous/concomitant administration of the compound of formula (I) and the further therapeutic agent(s) (either in a single pharmaceutical formulation or in separate pharmaceutical formulations), or the sequential/separate administration of the compound of formula (I) and the further therapeutic agent(s). If administration is sequential, either the compound of formula (I) according to the invention or the one or more further therapeutic agents may be administered first. If administration is simultaneous, the one or more further therapeutic agents may be included in the same pharmaceutical formulation as the compound of formula (I), or they may be administered in one or more different (separate) pharmaceutical formulations.
[0117] The subject or patient to be treated in accordance with the present invention may be an animal (e.g., a non-human animal). Preferably, the subject/patient is a mammal. More preferably, the subject/patient is a human (e.g., a male human or a female human) or a non-human mammal (such as, e.g., a guinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse, a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, an orangutan, a gibbon, a sheep, cattle, or a pig). Most preferably, the subject/patient to be treated in accordance with the invention is a human.
[0118] The term “treatment” of a disorder or disease as used herein is well known in the art. “Treatment” of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient/subject. A patient/subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease).
[0119] The “treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The “treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease. Accordingly, the “treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above). The treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).
[0120] The term “prevention” of a disorder or disease as used herein is also well known in the art. For example, a patient/subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease. The subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators. It is to be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms). Thus, the term “prevention” comprises the use of a compound of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
[0121] It is to be understood that the present invention specifically relates to each and every combination of features described herein, including any combination of general and/or preferred features. In particular, the invention specifically relates to each combination of meanings (including general and/or preferred meanings) for the various groups and variables comprised in formula (I).
[0122] In this specification, a number of documents including patent applications and scientific literature are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
[0123] The reference in this specification to any prior publication (or information derived therefrom) is not and should not be taken as an acknowledgment or admission or any form of suggestion that the corresponding prior publication (or the information derived therefrom) forms part of the common general knowledge in the technical field to which the present specification relates.
[0124] The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
EXAMPLES
[0125] The compounds described in this section are defined by their chemical formulae and their corresponding chemical names. In case of conflict between any chemical formula and the corresponding chemical name indicated herein, the present invention relates to both the compound defined by the chemical formula and the compound defined by the chemical name, and particularly relates to the compound defined by the chemical formula.
Example 1: Synthesis of Various Compounds According to the Invention
[0126] General Methods
[0127] All chemicals and solvents were purchased from commercial suppliers (Sigma Aldrich, Merck, Apollo Scientific and TCI Europe) at analytical grade. Bumetanide was obtained from OChem Inc., Des Plaines, Ill., US.
[0128] To monitor reactions via thin layer chromatography, silica gel F.sub.254 coated aluminum sheets from Merck were used.
[0129] As a stationary phase for column chromatography silica gel 60 70-230 mesh ASTM from Merck was used.
[0130] Melting points were measured on a ThermoGalen Kofler hot stage microscope.
[0131] .sup.1H- and .sup.13C-NMR spectra were recorded on a Bruker Advance (200 and 50 MHz respectively) and chemical shifts are reported in ppm relatively to the solvent residual line or tetramethylsilane as internal standard.
[0132] Mass spectra were recorded on a Shimadzu (GC-17A; MS-QP5050A) spectrometer. The peak intensity is specified in per cent relative to the biggest signal in the spectrum.
[0133] Elemental analysis were performed by Mag. Johannes Theiner at the University of Vienna and all reported values are within +/−0.4% of the calculated values.
3-(Butylamino)-5-(chloromethyl)-2-phenoxy-benzenesulfonamide (TEPS 76; Reference)
[0134] ##STR00006##
[0135] 1 mmol (0.35 g) of 3-(butylamino)-5-(hydroxymethyl)-2-phenoxy-benzenesulfonamide (Toeliner K et al., Annals of Neurology (2014), 75(4), 550-562) was dissolved in 5 mL of thionyl chloride and heated to 80° C. for three hours. The thionyl chloride was evaporated under reduced pressure and the substance was vacuum-dried for one hour. The product was purified by recrystallization from 70% MeOH, yielding 0.34 g of brown crystals (92% yield). .sup.1H NMR (200 MHz, chloroform-d) δ 7.43-7-27 (m, 3H), δ 7.08 (t, J=7.3 Hz, 1H), δ 7.02-6.79 (m, 3H), δ 4.88 (s, 2H) δ 4.57 (s, 2H), δ 3.07 (t, J=6.9 Hz, 2H), δ 1.54-1.33 (m, 2H), δ 1.28-1.08 (m, 2H), δ 0.83, J=7.1 Hz (t, 3H). MS m/z: 368/370 M.sup.+
3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide (STS66)
[0136] ##STR00007##
[0137] General Procedure A:
[0138] 1 mmol (369 mg) of 3-(butylamino)-5-(chloromethyl)-2-phenoxy-benzenesulfonamide (TEPS 76) was dissolved in 3 mL dimethylformamide (DMF). To this 2 mmol (157 μl) of 2,2,2-trifluoroethylamine were added and the mixture was stirred at room temperature overnight. After the reaction was completed, which was verified by thin layer chromatography, the fluid was evaporated under reduced pressure, yielding a white crude product. This crude product was purified by column chromatography (ethyl acetate/petroleum ether 6+4) and recrystallization from 70% MeOH, yielding 130 mg of white crystals (30% yield). .sup.1H NMR (200 MHz, Methanol-d4) δ 7.34-7.18 (m, 3H), δ 7.09-6.96 (m, 2H), δ 6.94-6.83 (m, 2H), δ 3.90 (s, 2H), δ 3.29-3.17 (m, 2H), δ 3.09 (t, J=6.8 Hz, 2H), δ 1.49-1.32 (m, 2H), δ 1.26-1.06 (m, 2H), δ 0.81 (t, J=7.2 Hz, 3H). MS m/z: 431 M.sup.+
3-(Butylamino)-5-[(cyanomethylamino)methyl]-2-phenoxy-benzenesulfonamide (TEPS 13)
[0139] ##STR00008##
[0140] TEPS 13 was prepared according to general procedure A, but instead of 2,2,2-trifluoroethylamine, 1.2 mmol (167 μl) triethylamine and 1.2 mmol (71 μl) of aminoacetonitrile were added. The crude product was purified by column chromatography (EtOAc/petroleum ether 1+1) and recrystallization from 70% EtOH yielding 180 mg of beige powder (46% yield). .sup.1H NMR (200 MHz, chloroform-d) δ 7.40-7.22 (m, 3H), δ 7.16-6.85 (m, 4H), δ 5.02 (s, 2H), δ 3.91 (s, 2H), δ 3.88-3.78 (m, 1 H), δ 3.60 (s, 2H), δ 3.20-3.00 (m, 2H), δ 2.99-2.83 (d, 1H), δ 1.49-1.34 (m, 2H), δ 1.26-1.12 (m, 2H), δ 0.90-0.74 (m, 3H). MS m/z: 388 M.sup.+
3-(Butylamino)-4-phenoxy-5-sulfamoyl-N-(2,2,2-trifluoroethyl)benzamide (TEPS 23)
[0141] ##STR00009##
[0142] To a solution of 1 mmol (364 mg) of Bumetanide in 5 mL dry tetrahydrofuran 1.2 mmol (194 mg) of 1,1-carbonyldiimidazole (CDI) were added and the mixture was stirred for two hours. Once TLC did not show any bumetanide remaining, 2 mmol (157 μl) of trifluoroethylamine were added and the mixture was stirred at room temperature overnight. Once the reaction was completed it was poured into 20 ml of 5% NaHCO.sub.3 and extracted with ethyl acetate. The organic phase was then dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure. The crude product was then purified by recrystallization from EtOH to yield 159 mg of white powder (36% yield). .sup.1H NMR (200 MHz, Methanol-d.sub.4) δ 7.73 (d, J=2.1 Hz, 1H), 7.44 (d, J=2.1 Hz, 1H), 7.38-7.20 (m, 2H), 7.14-6.98 (m, 1H), 6.98-6.86 (m, 2H), 4.10 (q, J=9.3 Hz, 2H), 3.13 (t, J=6.8 Hz, 2H), 1.56-1.34 (m, 2H), 1.30-1.03 (m, 3H), 0.82 (t, J=7.2 Hz, 3H). MS m/z: 445 M.sup.+
3-[(2,2,2-Trifluoroethylamino)methyl]benzenesulfonamide
[0143] ##STR00010##
[0144] 5 mmol (1.41 g) of 2,2,2-trifluoro-N-[(4-sulfamoylphenyl)methyl]acetamide (Augurusa, A., et al., 2016) were dissolved in 10 mL dry tetrahydrofuran (THF). The mixture was cooled at 0-4° C. and flooded with argon gas. 25 mmol (12.5 mL) of LiAIH.sub.4 (2.0 M in THF) were added carefully in three portions every 30 minutes, then the solution was heated to 60° C. for 3 hours. The mixture was stirred overnight at room temperature. Again, the mixture was cooled at 0-4° C. and the reaction was quenched with 5% aqueous NH.sub.4Cl. 2 N HCl was added until the mixture was completely clear and extracted two times with ethyl acetate. The aqueous phase was neutralized by adding 2 M NaOH and again extracted two times with ethyl acetate. The second organic phase was dried over sodium sulfate and evaporated under reduced pressure. Afterwards the product was recrystallized from isopropanol. The resulting product yielded 387 mg of white crystals (28.9% yield). .sup.1H NMR (200 MHz, DMSO-d.sub.6) δ 7.79 (A-part of AB system, J.sub.AB=8.3 Hz, 2H), 7.52 (B-part of AB system, J.sub.AB=8.3 Hz, 2H), 7.31 (s, 2H), 3.86 (d, J=5.7 Hz, 2H), 3.32-3.11 (m, 2H), 3.09-2.96 (m, 1H). .sup.13C NMR (50 MHz, DMSO-d.sub.6) δ 144.3, 142.6, 126.2 (q, J=279.3 Hz), 128.1, 125.6, 51.8, 48.7 (q, J=30.3 Hz). MS m/z: 269 M.sup.+
3-(Benzylamino)-2-(4-fluorophenoxy)-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide (TEPS 88)
[0145] ##STR00011##
Step 1: 4-(4-fluorophenoxy)-3-nitro-5-sulfamoyl-benzoic acid (TEPS 84)
[0146] To a suspension of 20 mmol (5.61 g) of 4-chloro-3-nitro-5-sulfamoyl-benzoic acid (561 mg) (WO 2012/018635) in 30 mL water, 80 mmol NaHCO.sub.3 (6.8 g) were added cautiously followed by 40 mmol (4.77 g) 4-fluorophenol. This solution was stirred at 85° for 16 hours. After cooling to room temperature, the precipitate was filtered off and dissolved in 10 mL of hot water. Then 6N HCI was added and the resulting precipitate was filtered off and dried to yield 4.35 g of a yellow solid (61% yield). .sup.1H NMR (200 MHz, DMSO) δ 14.01 (brs, 1H), 8.83-8.54 (m, 2H), 7.88 (s, 2H), 7.15 (t, J=8.8 Hz, 2H), 7.05-6.86 (m, 3H). .sup.13C NMR (50 MHz, DMSO) δ 164.7, 158.56 (d, J=239.5 Hz), 153.25 (d, J=2.3 Hz), 148.2, 143.4, 140.3, 133.6, 130.9, 128.6, 118.24 (d, J=8.5 Hz), 116.56 (d, J=23.7 Hz). MS m/z 356
Step 2: 3-amino-4-(4-fluorophenoxy)-5-sulfamoyl-benzoic acid (TEPS 85)
[0147] To an aqueous solution of LiOH (adjusted to pH 11) 10 mmol (3.56 g) TEPS84 and 350 mg palladium on activated charcoal (5% Pd/C) were added. The resulting mixture was hydrogenated at room temperature. When the H.sub.2 uptake became negligible, the mixture was filtered and the filtrate was acidified with 6N HCl and extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and dried under reduced pressure to yield 2.15 g of a brown solid (66% yield). .sup.1H NMR (200 MHz, DMSO) δ 7.78-7.47 (m, 2H), 7.30 (s, 2H), 7.19-7.00 (m, 2H), 6.99-6.76 (m, 2H), 5.32 (s, 2H). .sup.13C NMR (50 MHz, DMSO) δ 166.9, 157.8 (d, J=236.9 Hz), 152.8 (d, J=2.0 Hz), 143.2, 139.3, 138.3, 128.3, 120.7, 117.3 (d, J=8.4 Hz), 116.1, 115.7. MS m/z 326
Step 3: Methyl 3-(benzylamino)-4-(4-fluorophenoxy)-5-sulfamoyl-benzoate (TEPS 86)
[0148] To a suspension of 2 mmol (652 mg) TEPS85 in 10 mL MeOH 5 mmol (0.6 mL) benzylbromide were added. The mixture was then refluxed for 16 hour to form a solution. After the reaction was completed, MeOH was removed under reduced pressure and 20 mL 5% NaHCO.sub.3 were added. This mixture was extracted three times with ethyl acetate and the combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (ethyl acetate/petroleum ether 3+7) to yield 351 mg of a white solid (41% yield). .sup.1H NMR (200 MHz, DMSO) δ 7.67 (d, J=1.9 Hz, 1H), 7.40 (s, 2H), 7.35-7.04 (m, 8H), 6.96-6.80 (m, 2H), 6.21 (t, J=6.0 Hz, 1H), 4.35 (d, J=6.0 Hz, 2H), 3.81 (s, 3H). .sup.13C NMR (50 MHz, DMSO) δ 165.8, 158.0 (d, J=237.2 Hz), 153.1 (d, J=2.0 Hz), 142.8, 140.5, 139.5, 138.3, 128.8, 127.3, 127.3, 127.1, 117.4 (d, J=8.3 Hz), 116.1, 115.7, 52.9, 46.2. MS m/z 430
Step 4: 3-(Benzylamino)-2-(4-fluorophenoxy)-5-(hydroxymethyl)benzenesulfonamide (TEPS 87)
[0149] In a three necked flask 2 mmol of TEPS86 (860 mg) were dissolved in 8 mL anhydrous THF under argon atmosphere. Then 4 mL of a 1 M DIBAL-H solution in toluene were added. After one, two, three and four hours, respectively, another 2 mL of the 1M DIBAL-H solution in toluene were added each time and the reaction was stirred overnight. After TLC showed no remaining TEPS86 the mixture was cooled to 0° C. and quenched with 5% aqueous NH.sub.4Cl solution causing a gel-like substance to precipitate. The precipitate was then dissolved in 2 N HCl and extracted three times with ethyl acetate. The combined organic layers were washed three times with water, once with brine and dried over Na.sub.2SO.sub.4. The fluids were evaporated under reduced pressure and purified by recrystallization from ethanol to yield 665 mg of beige powder (83% yield). .sup.1H NMR (200 MHz, DMSO) δ 7.34-7.00 (m, 10H), 6.92-6.74 (m, 3H), 5.86-5.69 (m, 1H), 4.38 (s, 2H), 4.30 (d, J=5.2 Hz, 2H). .sup.13C NMR (50 MHz, DMSO) δ 157.7 (d, J=236.5 Hz), 153.7, 140.6, 140.0, 137.4, 135.5, 128.7, 128.1, 127.2, 117.2 (d, J=8.1 Hz), 115.7 (d, J=23.3 Hz), 113.5, 112.6, 63.0, 46.3. MS m/z 402
Step 5: 3-(Benzylamino)-2-(4-fluorophenoxy)-5-[(2,2,2-trifluoroethylamino)methyl]benzene-sulfonamide (TEPS 88)
[0150] 1.5 mmol (604 mg) of TEPS87 were dissolved in 5 mL thionyl chloride and heated to 80C.° for three hours. The thionyl chloride was evaporated under reduced pressure. The product was purified by column chromatography (ethyl acetate/petroleum ether 7+3) to yield 470 mg of brown solid (74% yield). 1 mmol (420 mg) of this intermediate benzyl chloride was dissolved in 5 mL of DMF, to this solution 2mmol (157 μl) of 2,2,2-trifluoroethylamine were added and the mixture was stirred at room temperature overnight in a sealed vial. After the reaction was completed, which was verified by thin layer chromatography, the fluid was evaporated under reduced pressure. This crude product was purified by column chromatography (ethyl acetate/petroleum ether 3+7) and recrystallization from ethanol, yielding 86 mg of white crystals (18% yield). .sup.1H NMR (200 MHz, MeOD) δ 7.32-7.11 (m, 6H), 7.09-6.71 (m, 5H), 4.34 (d, J=3.8 Hz, 2H), 3.84 (d, J=26.0 Hz, 2H), 3.05 (q, J=9.8 Hz, 2H). .sup.13C NMR (50 MHz, MeOD) δ 142.0, 139.0, 137.3, 128.1, 126.7, 116.5, 116.4, 115.5, 115.4, 115.0, 114.2, 52.1, 46.4. MS m/z 483
3-(Butylamino)-4-phenoxy-5-sulfamoyl-N-(3,3,3-trifluoropropyl)benzamide (TEPS 101)
[0151] ##STR00012##
[0152] 1 mmol (364 mg) of bumetanide was dissolved in in 5 mL dry tetrahydrofuran. 1.2 mmol (194 mg) 1,1-carbonyldiimidazole were added and the mixture was stirred for three hours. After the thin-layer chromatography showed that all bumetanide reacted, 2 mmol (300 mg) trifluoropropan-1-Amine were added and the mixture was stirred at room temperature overnight. After the reaction was completed 20 ml of 5% NaHCO.sub.3 were added and it was extracted three times with ethyl acetate. The collected organic phase was washed with brine and dried over Na.sub.2SO.sub.4. The solvent was then removed under reduced pressure. The crude product was purified via recrystallization from EtOH. Yield: 220 mg (47%).
[0153] .sup.1H NMR (200 MHz, MeOD) δ 7.74-7.61 (m, 2H), 7.40 (d, J=2.0 Hz, 1H), 7.29 (t, J=7.9 Hz, 2H), 7.10-7.03 (m, 2H), 6.96-6.85 (m, 2H), 3.64 (t, J=7.0 Hz, 2H), 3.12 (t, J=6.8 Hz, 2H), 2.70-2.37 (m, 2H), 1.42 (p, J=6.8 Hz, 2H), 1.24-1.05 (m, 2H), 0.81 (t, 3H). .sup.13C NMR (50 MHz, MeOD) δ 169.1, 157.8, 144.0, 140.6, 138.4, 132.9, 130.7, 127.96 (d, J=276.2 Hz), 124.0, 116.6, 114.9, 114.5, 43.7, 34.60 (q, J=4.0 Hz), 34.03 (q, J=27.8 Hz), 32.0, 20.8, 14.0. MS m/z 459
3-(Butylamino)-2-phenoxy-5-[(3,3,3-trifiuoropropylamino)methyl]benzenesulfonamide (TEPS 102)
[0154] ##STR00013##
[0155] 1.56 mmol (363 mg) of TEPS101 was dissolved in 20 mL of THF and 5.8mmol (0.556 ml) borane dimethylsulfid complex was added. The reaction mixture was then stirred at 86° overnight. Once TLC showed that no starting material was present, the mixture was cooled to room temperature and then quenched with 20 ml of half-saturated aqueous NaHCO.sub.3. It was extracted three times with 25 mL of ethyl acetate, washed brine and dried over Na.sub.2SO.sub.4. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (ethyl acetate/petroleum ether and TEA, 1:1+20 mL of TEA) and recrystallized from 70% EtOH to yield 178mg.(Yield 26%)..sup.1H NMR (200 MHz, CDCl.sub.3) δ 7.38-7.16 (m, 3H), 7.06 (t, J=7.3 Hz, 1H), 6.91 (d, J=7.3 Hz, 3H), 4.90 (s, 1H), 3.79 (s, 2H), 3.06 (q, J=6.7 Hz, 2H), 2.91 (t, J=7.1 Hz, 2H), 2.34 (qt, J=10.9, 7.1 Hz, 2H), 1.41 (p, J=6.8 Hz, 2H), 1.17 (dq, J=13.7, 6.9 Hz, 2H), 0.82 (t, J=7.2 Hz, 3H). .sup.13C NMR (50 MHz, CDCI.sub.3) δ 156.4, 142.4, 138.2, 135.7, 135.6, 133.0, 130.1, 126.79 (d, J=276.8 Hz), 123.5, 120.5, 115.5, 115.3, 114.3, 53.4, 43.2, 42.23 (q, J=3.3 Hz), 34.41 (q, J=27.7 Hz), 31.2, 19.9, 13.8. MS m/z 445
Example 2: NKCC1 Inhibitory Activity of the Compounds According to the Invention
[0156] The compounds of formula (I) according to the present invention are inhibitors of Na.sup.+—K.sup.+-2Cl.sup.−-cotransporters (NKCCs), particularly of NKCC1. The NKCC1 inhibitory activity of the compounds of the invention can be determined, for example, using the following NKCC1A activity assay.
[0157] To activate NKCC1A prior to the uptake experiment, hNKCC1A-expressing oocytes (Lykke, K., et al. 2016) or uninjected control oocytes are pre-incubated for 30 min at room temperature in a K.sup.+-free solution. To measure K.sup.+ influx, oocytes are exposed to an isosmotic test solution in which KCl is substituted for choline chloride and .sup.86Rb.sup.+ is added as a tracer for K. Bumetanide (positive control), a compound of formula (I) according to the invention (“drug”), or control vehicle (negative control) are added to the test solution. The uptake assay is then performed at room temperature with mild agitation for 5 min. The influx experiments are terminated and the radioactivity present is determined by liquid scintillation β-counting with Opti-Fluor scintillation using a Liquid Scintillation Analyzer. hNKCC1A-mediated K.sup.+ uptake is then assessed as ([flux.sub.NKCC1-expressing oocytes in presence of×μM drug]−[flux.sub.uninjected oocytes in presence of×μM drug]), in order to correct for endogenous NKCC activity. A reduction in hNKCC1A-mediated K.sup.+ uptake observed with a test compound is indicative of the compound inhibiting NKCC1. When the exemplary compounds of formula (I) described in Example 1 are subjected to this assay, it can be confirmed that they exhibit NKCC1 inhibitory activity.
Example 3: Effects of the Compounds According to the Invention on Reducing Brain Damage and Neurological Deficits After Ischemic Stroke in Mice
[0158] Introduction
[0159] Stimulation of the WNK-SPAK/OSR1 kinases and their substrate Na.sup.+—K.sup.+2Cl.sup.− cotransporter 1 (NKCC1) play critical roles in cerebral edema and neurological functional deficits after ischemic stroke. Either NKCC1 inhibitor bumetanide (BMT) or knockout of WNK3 or SPAK shows profound protective effects in a mouse model of ischemic stroke. In this study, the efficacy of two pharmacological inhibitors of NKCC1, i.e. the novel NKCC1 inhibitor STS66 which is an exemplary compound of formula (I) according to the present invention, as well as the lipophilic BMT prodrug STS5 (reference compound), on reducing ischemic stroke-induced brain damage in mouse were investigated.
[0160] Material and Methods
[0161] Materials
[0162] Bumetanide (BMT) and AngII were from Sigma (Sigma-Aldrich, St Louis, Mo., USA). STS5 is described in Erker et al., 2016 (where it is referred to as BUMS); and STS66 is described in Example 1 above.
##STR00014##
[0163] Animals
[0164] All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The manuscript adheres to the ARRIVE guidelines for reporting animal experiments. Wild type C57BL/6j genetic background mice at 8-14 weeks old (26-30 g body weight, male and female) were used in the study. Animals were housed in a temperature- and humidity-controlled animal facility with a 12-hour light-dark cycle. Food and water were available ad libitum.
[0165] Transient Focal Cerebral Ischemia Model (tMCAO)
[0166] Transient focal cerebral ischemia was induced in mice by intraluminal occlusion of the left middle cerebral artery (MCA) for 50 min as described previously (Chen H et al., 2005). Mice were anesthetized with 3% isoflurane in 70%: 30% N.sub.2O/O.sub.2 until they were unresponsive to the tail pinch test. Animals were then fitted with a nose cone blowing 1.5% isoflurane for anesthesia maintenance. The left common carotid artery was exposed and the occipital artery branches of the external carotid artery were isolated and coagulated. The internal carotid artery was isolated and the extracranial branch was dissected and ligated. A rubber silicon-coated monofilament suture (6-0) was introduced into the internal carotid artery lumen and gently advanced approximately 8-9 mm to block the MCA blood flow for 50 min. The rectal temperature was maintained at 37.0±0.5° C. during surgery through a temperature-controlled heating pad. Achievement of ischemia was confirmed by monitoring regional cerebral blood flow (rCBF) with laser speckle contrast imager (Pericam). For reperfusion, the suture was withdrawn after the MCAO. The incision was closed and the mouse recovered under a heating lamp to maintain the core temperature (36.0-37.0° C.) during the 30-60 min recovery period. After recovery, animals were returned to their cages with free access to food and water.
[0167] Permanent Focal Cerebral Ischemia Model (pdMCAO)
[0168] Permanent focal cerebral ischemia was induced by permanent occlusion of the distal middle cerebral artery (pdMCAO) in mice (Suenaga J, et al., 2015). Under anesthesia as described above, a skin incision at the midline of the neck was made. After being separated from the vagal nerve, the left CCA was exposed and occluded by ligation and the skin was sutured. Another skin incision (1 cm) was made between the left eye and the ear using fine operation scissors. The temporal muscle was identified and detached from the skull in its apical and dorsal part without totally removing the muscle by using the forceps. The MCA below the transparent skull in the rostral part of the temporal area, dorsal to the retro-orbital sinus was identified. If the MCA bifurcation is not visible (due to an anatomical normal variation), the vessel most rostral was identified. The skull above the MCA branch was thinned out with the drill until it has a thin and translucent texture. The artery, proximal and distal to the MCA bifurcation, was coagulated with the electrocoagulation forceps (in a bipolar mode at 7 W). The temporal muscle was relocated to its position and the burr hole was covered with wax and the skin wound was sutured and infiltrated with analgesia bupivacaine (100 μl 0.25%) topically. The animal was placed in a cage and monitored for recovery from anesthesia.
[0169] Drug Treatment
[0170] Vehicle: DMSO (2 ml/kg body weight/day), the NKCC1 inhibitor bumetanide (BMT, 10 mg/kg body weight/day in DMSO), bumetanide's prodrug STSS (13 mg/kg body weight/day in DMSO), or the novel NKCC1 inhibitor STS66 according to the invention (12 mg/kg body weight/day in DMSO) was administered via intraperitoneal injection (i.p). The initial half dose of the drugs (BMT, STSS, STS66) was given at 3 h after the onset of reperfusion and the second half dose at 8 h reperfusion in the tMCAO model.
[0171] Angiotensin II (AngII) Infusion-Induced Hypertension
[0172] Mice received 14 days of infusion of either saline or AngII via osmotic minipumps (model 1002; Alzet, Cupertino, Calif., USA) implanted subcutaneously in the intrascapular region under isofluorane anesthesia. Sterile procedures were used to prevent postoperative infection at the site of implantation. The pumps were loaded either with saline for the vehicle (Veh control) group, or the Ang II peptide at a rate of 1000 ng/kg/min as described previously (Nagai M, et al., 2011; Lu H, et al., 2015). The pumps were removed after 14 days of infusion.
[0173] Neurological Function Tests
[0174] Neurological functional deficits in mice were assessed in a blinded manner with the following tests: neurological score, adhesive tape removal test, corner test, cylinder test, and grid walking foot-fault test. These tests are established for identifying and quantifying sensorimotor deficits and postural asymmetries (Bederson J B, et al., 1986; Zhang L, et al., 2002; Schaar K L, et al., 2010).
[0175] Neurological score: Neurological deficit grading system was used to evaluate neurological deficit at 1, 2, 3, 5, 7, 10 and 14 days after tMCAO as described previously (Bederson J B et al, 1986).
[0176] Corner test: Neurological functional deficits in mice were determined by the corner test. The apparatus consists of two cardboards (each size is 30 cm×20 cm) placed together at a 30° angle to form a narrow alley. The mouse was placed between the two angled boards facing the corner. When exiting the corner, uninjured mice will turn left or right randomly. After tMCAO, animals with unilateral brain damage will exhibit unidirectional turning. The numbers of left and right turns of each mouse during 10 trials were recorded, and turning movements that were not part of a rearing movement were not scored (Zhang L, et al., 2002).
[0177] Adhesive tape removal test: An adhesive tape removal test was used to measure somatosensory deficits. Two pieces of adhesive tape (4 mm×3 mm) were attached to the forepaws in an alternating sequence and with equal pressure by the experimenter before each trial. The removal time is defined as the time at which the animal removes the tape. The trial ended after the adhesive patch was removed or after 2 min had elapsed. Pre-operative training was carried twice per day for three days and were tested on day 1, 2, 3, 5, 7, 10 and 14 days after tMCAO (Bouet V, et al., 2009).
[0178] Grid walking foot-fault test: The grid walking test is sensitive to deficits in descending motor control. Each mouse was placed on a stainless steel grid floor (20×40 cm with a mesh size of 4 cm.sup.2) elevated 1 m above the floor. Every animal was tested for three 1-min trials. The data were expressed as the number of foot fault errors made by the forelimbs contralateral to the injured hemisphere as a percentage of total steps (Jun Zhang, et al., 2017).
[0179] Brain Infarct Volume and Swelling Measurement
[0180] At 24 h post-reperfusion, mice were anesthetized with 5% isoflurane and then decapitated. Coronal brain slices (2 mm thickness) were stained with 1% 2,3,5-triphenyltetrazolium chloridemonohydrate (TTC) for 20-30 min and brain slices were scanned (Begum G et al., 2015). Ischemic lesions were traced in each slice in a blinded manner, and the total volume of infarction was calculated with correction for edema, as described by Swanson et al., 1990 using ImageJ software. Infarct areas were summed across all slices and multiplied by slice thickness to yield total infarct volume (mm.sup.3). Brain swelling was determined with the following formula. For tMCAO: swelling (% contralateral hemispheric volume)=[(ipsilateral hemispheric volume)−(contralateral hemispheric volume)]/(contralateral hemispheric volume)×100, as described before (Guo Q G et al., 2009; Sunghee Cho et al., 2005).
[0181] Statistics
[0182] The animal subjects were randomly assigned into different studies and surgical procedures, and data analyses were performed by investigators who were blinded to the experimental conditions. Values are expressed as means SD or SEM. Statistical analysis was performed using the multiple comparisons, ANOVA multiple comparisons test (Graphpad, Prism? software). A p-value less than 0.05 was considered statistically significant.
[0183] Results
[0184] Efficacy of BMT, STS5 and STS66 on Reducing Brain Infarction and Edema in Normotensive Mice After Ischemic Stroke
[0185] Ischemic stroke in C57B6/j mice via tMCAO and post-stroke administration of vehicle DMSO control, BMT, STS5 or STS66 were performed as illustrated in
[0186] Animal body weight and animal survival rates were also monitored among four groups during 1-14 days after tMCAO. As demonstrated in
[0187] Effects of BMT and STS66 on Improving Neurological Function in Mice After Ischemic Stroke
[0188] A series of neurological behavioral tests were conducted to assess changes of sensorimotor function deficits in mice treated with either BMT or STS66 following ischemic stroke. According to 1-14 days monitor and behavior test, BMT and STS66 treatments improved all sensorimotor deficits in normotensive mice after stroke. Both BMT- and STS66-treated mice exhibited progressive decrease in neurological deficit score. Importantly, a faster improvement of neurological deficit scores was detected in the STS66-treated groups, from day 1 to day 14 post-reperfusion (neurological score 4.0±0.0 to 1.2±0.4), as also shown in
[0189] Efficacy of STS66 on Preventing AngII-Induced Hypertensive Mice from Developing Worsened Infarct and Cerebral Edema After Ischemic Stroke
[0190]
[0191] These findings show that the compounds of formula (I), including in particular the compound STS66, are particularly well suited for the treatment of stroke, as reflected by the observed reduction of brain infarction and cerebral swelling after stroke, as well as a considerably improved therapeutic outcome, including improved survival and improved sensorimotor functional recovery after stroke. The observed neuroprotective effect further supports a prophylactic therapy (prevention) of stroke. The compounds of formula (I) can hence advantageously be used for the treatment or prevention of stroke as well as other neurological diseases/disorders involving NKCCs, such as traumatic brain injury, spinal cord injury, peripheral nerve injury, brain edema, or glioma.
Example 4: Effects of the Compounds According to the Invention on Glioma
[0192] Materials and Methods
[0193] Materials
[0194] Bumetanide (BMT, #B3023) and Temozolomide (TMZ, #T2577) were purchased from Sigma-Aldrich (St. Louis, Mo.). Dulbecco's Modified Eagle Medium (DMEM/HEPES, Cat #12430-054) and Penicillin/streptavidin (Cat #15240062) were from Gibco (Carlsbad, Calif.). Fetal bovine serum (FBS) was obtained from Invitrogen (Carlsbad, Calif.). Anti-phospho-NKCC1(Thr206) antibody was developed by Dr. Yang (Taiwan National University) (Moriguchi et al. 2005; Yang et al. 2010). Monoclonal antibody against total NKCC (T4) was from the Developmental Studies Hybridoma Bank (Iowa City, Iowa). Antibody against a-tubulin (Cat #2125), rabbit antibody against Ki67 (Cat #9129S) and antibody against cleaved caspase-3 (Cat #9661S) were from Cell Signaling (Beverly, Mass.). Goat antibody against NKCC1 (Cat #ab99558) was from Abcam Ltd (Cambridge, Mass.). BCA Protein Assay Kit (Cat #23227) was from Thermo Scientific (Rockford, Ill.).
[0195] Cell Cultures and Authentication
[0196] Immunogenic mouse glioma GL26 and non-immunogenic mouse SB28-GFP glioma cells were used as previously described (Kohanbash et al. 2017). GL26 and SB28-GFP glioma cells obtained from Prof. Gary Kohanbash, PhD, were derived as described previously (Kosaka et al. 2014) and maintained in DMEM/HEPES containing 10% heat-inactivated FBS, 2 mM L-glutamine, 1× Penicillin/streptavidin and 1 mM sodium pyruvate. Cultures were passaged approximately every 4 days with fresh medium at a density of 106 cells/75 cm2 in a culture flask. Passage 10-30 of glioma cells were used in the study. All cell lines were authenticated by short tandem repeat (STR) DNA fingerprinting (by IDEXX BioResearch, Columbia, Mo.) in the past 6 months. In addition, PCR analysis was performed to confirm the absence of mycoplasma infection in all cell cultures.
[0197] Rubidium Uptake Assay
[0198] GL26 and SB28-GFP cells were seeded in 24-well plates and the rubidium uptake assay was performed on cells that were 60% confluent. TMZ was added to cells for 48 hr incubation. The medium was then removed from the wells and washed with wash buffer (Rb.sup.+ free). After wash buffer was aspirated, isotonic and hypertonic solutions (contain Rb.sup.+) with BMT were added, and cells were incubated at 37° C. for 5 min. After this incubation period, cells were washed with isotonic or hypertonic solutions (RID.sup.+ free). After washing, cell lysis buffer (200 μl/well) was added to the plate to release intracellular RID.sup.+. The RID.sup.+ concentration was measured using an automated atomic absorption spectrophotometer (Ion Channel Reader, ICR-8000; Aurora Biomed, Vancouver, Canada).
[0199] BrdU Proliferation Assay
[0200] Cell proliferation of GL26 and SB28-GFP cells was measured by quantifying BrdU incorporation. GL26 cells (5×103 cells/well) or SB28-GFP cells (1×103 cells/well) were seeded in 96-well plates in 100 μL media. After 24 hours in culture, cells were incubated with fresh medium plus the following reagents: DMSO vehicle (Con-Veh), BMT (B, 10 μM), TMZ (100 μM), or TMZ+B for 48 h. BrdU was added in the last 4 h period of the whole 48 h incubation. The incorporation of BrdU into newly synthesized DNA of proliferating cells was detected by using a peroxidase-conjugated antibody which reacts with the thymidine analogue BrdU. Bound anti-BrdU-peroxidase conjugated antibody was measured by a substrate reaction, and then quantified calorimetrically by an ELISA plate reader (Spectra MAX 190, Molecular Devices, Sunnyvale, Calif.) at dual wavelength of 450/550 nm.
[0201] Immunoblotting
[0202] GL26 and SB28-GFP cells were washed with ice-cold PBS and incubated in RIPA buffer containing 1 pill of phosSTOP and 2 mM protease inhibitors as described before (Algharabli et al. 2012). Cells were lysed by sonication at 4° C. Protein content of the cellular lysate was determined with BCA Protein Assay Kit. Samples and sample buffer (Thermo Scientific, Rockford, Ill., USA) were boiled at 95° C. for 5 min. The samples were then electrophoretically separated on 10% SDS gels. After transferring to PVDF membranes, the blots were blocked in 10% nonfat dry milk in TBS-T (Tris-buffered saline, 0.05% Tween-20) for 1 hour at room temperature and then incubated with appropriate primary antibodies (pNKCC1, 1:300 and tNKCC1, 1:3000) at 4° C. overnight. After rinsing with TBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary IgG (1:2000) for 1 hr at RT. Bound antibody was visualized with an enhanced chemiluminescence assay. Protein band signal intensities were analyzed using ImageJ and normalized to α-tubulin expression.
[0203] Mouse Syngeneic Glioma Model
[0204] All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
[0205] Six to eight-week-old female albino C57BL/6 mice were anesthetized with 2% isoflurane. Once in the anesthetic plane, mice were mounted on a stereotactic frame and an 1 cm incision was made along the midline of the cranium to expose the skull. Using a precision power drill with a fine tip needle, a single hole was made on the skull in right hemisphere (coordinates from bregma: +0.5 mm AP, +2.1 mm ML, and −3.2 mm DV). 1×105 GL26 or 4×104 GL26-mCitrine or 0.5×105 SB28-GFP cells (in 2 μL of serum-free DMEM) were injected into the right striatum in 4 min (at a rate of 0.5 μL/min), using a micro-pump injector and a 5-μl Hamilton syringe equipped with a 33-gauge needle. Cells were allowed to settle for 5 min and the needle was withdrawn slowly. The incision was closed with surgical staples. Ketofen (2 mg/kg, i.p.) was administrated once prior to surgery and daily for two days after the surgery and then daily if animals exhibit signs of pain. Animals were then allowed to recover in their cages under a heat lamp and access to water and wet chow.
[0206] Drug Treatment Regimens
[0207] Starting 7 days after tumor cell implantation (d.p.i.), mice were randomly assigned to each treatment group and received the therapy for 5 consecutive days: vehicle control (1.25% DMSO in PBS, 10 ml/kg/day, i.p.), NKCC1 inhibitor BMT (B, 5 mg/kg, twice a day, i.p.), TMZ therapy (50 mg/kg/day, once a day, i.p.), or TMZ+NKCC1 inhibitor BMT (T of 50 mg/kg/day+B of 5 mg/kg, twice a day, i.p.) combination treatment.
[0208] Animal Survival Test
[0209] Overall survival was evaluated in all mice. Tumor bearing animals were monitored daily for signs of pain, discomfort or neurological impairment. Signs of chronic pain, such as hunched posture, weight loss, absence of grooming behavior, and of neurological impairment, like seizures, weakness, difficulty walking, an inability to right themselves, circling behavior, and unusual aggressiveness or timidity were used to infer tumor development. In tumor cell injected mice, a loss of 20% body weight, severe neurological impairment, or major loss in body scoring index (<2.0 on a 5-point scale) were used as the humane endpoint. All other surviving mice were sacrificed at 90 days after glioma cell injection.
[0210] Results
[0211] The results of these experiments are shown in
[0212] pNKCC1 and tNKCC1 expression did not show significant decrease when treated with STS66 alone compared to BMT. TMZ did not trigger the NKCC1 upregulation. However, the combination treatment T+S significantly decreased the expression of NKCC1 (see
[0213] In the BrdU proliferation assay, STS66 showed more inhibition of GL26 and SB28-GFP cell proliferation than BMT (see
[0214] When the combinatorial regimen of BMT, STS66 and TMZ was tested in glioma bearing mice, it was found that T+S did not improve the survival of the mice, as shown in
[0215] These results indicate that the compounds of formula (I), including in particular STS66, are suitable for the therapy of glioma and exhibit a more pronounced activity than bumetanide.
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