Heteroaromatic compounds

Details Heteroaromatic compounds Heteroaromatic compounds

2000-02-01

2001-09-04

Fan, Jane (Department: 1626)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C546S269100, C546S269400

Reexamination Certificate

active

06284779

ABSTRACT:

The invention relates to heteroaromatic compounds, the process for their production and their use in pharmaceutical agents.
It has been reported that compounds with an affinity for the FK506 binding protein (FKBP) that inhibit that protein's rotamase activity also possess nerve growth stimulatory activity. [Lyons et al.,
PNAS,
91, pp. 3191-3195 (1994)]. Many of these such compounds also have immunosuppressive activity.
FK506 (Tacrolimus), an immunosuppressive drug, has been demonstrated to act synergistically with NGF in stimulating neurite outgrowth in PC
12
cells as well as sensory ganglia [Lyons et al. (1994)]. This compound has also been shown to be neuroprotective in focal cerebral ischemia [J. Sharkey and S. P. Butcher,
Nature,
371, pp. 336-339 (1994)] and to increase the rate of axonal regeneration in injured sciatic nerve [B. Gold et al.,
J. Neurosci.,
15, pp. 7509-16 (1995)].
The use of immunosuppressive compounds, however, has obvious drawbacks. In addition to compromising immune function, prolonged treatment with these compounds can cause nephrotoxicity [Kopp et al.,
J. Am. Soc. Nephrol.,
1, p. 162 (1991)], neurological deficits [P. C. DeGroen et al.,
N. Eng. J. Med.,
317, p. 861 (1987)] and vascular hypertension [Kahan et al.,
N. Eng. J. Med.,
321, p. 1725 (1989)].
More recently, sub-classes of FKBP binding compounds which inhibit rotamase activity, but which purportedly lack immunosuppressive activity have been disclosed for use in stimulating nerve growth [see U.S. Pat. Nos. 5,614,547; 5,696,135; WO 96/40633; WO 96/40140; WO 97/16190; J. P. Steiner et al.,
Proc. Natl. Acad. Sci. USA,
94, pp. 2019-23 (1997); and G. S. Hamilton et al.,
Bioorg. Med. Chem. Lett.,
7, pp. 1785-90 (1997)]. While these compounds supposedly avoid certain unwanted side effects of immunosuppressive FKBP binding compounds, they still bind to FKBP and inhibit its rotamase activity. This latter property may still lead to undesirable side effects due to other roles FKBP may play in mammals.
Surprisingly, it is now known that binding to FKBP is not necessary for neuronal activity. Co-pending U.S. patent application Ser. Nos. 08/748,447, 08/748,448 and 08/749,114, each of which is incorporated by reference in its entirety, each describe the use of non-FKBP binding, non-immunosuppressive compounds for stimulating nerve growth and preventing neurodegeneration. Due to their lack of affinity for FKBP, these compounds advantageously avoid any potential interference with FKBP-associated biochemical pathways. These compounds do, however, inhibit multi-drug resistance (“MDR”) through inhibition of the p-glycoprotein and MRP. While it appears that the dosages of those compounds necessary to stimulate nerve growth and prevent neurodegeneration are lower than those that effect MDR, it would still be desirable to obtain compounds which are specific for neuronal activity, without other significant mechanisms of action.
It is known that piperidine and pyrrolidine derivatives have immunosuppressive and non-immunosuppressive properties. For example, WO 96/40633 describes that N-glyoxyl-prolylester compounds, which have an affinity to FKBP receptors, have a neurotrophic action and stimulate neuronal regeneration as inhibitors of the FKBP-rotamase.
The stimulation of neurite growth in nerve cells with piperidine derivatives is described in WO 96/41609, which is incorporated by reference herein in its entirety. The clinical use of the previously known piperidine and pyrrolidine derivatives for stimulation of neurite growth does not increase the chances of success, since the compounds are unstable in plasma and do not pass through the blood-brain barriers in sufficient amounts. WO 99/10340 also discloses compounds having neuronal activity. Its entire disclosure is also incorporated by reference herein.
Applicants have identified several subclasses of compounds that have potent neuronal activity.
The term “neuronal activity,” as used herein, includes stimulation of damaged neurons, promotion of neuronal regeneration, prevention of neurodegeneration and treatment of a neurological disorder. The compounds of this invention have activity in both peripheral nerves and the central nervous system.
Applicants have discovered diverse genera of compound with neuronal activity which do not bind to FKBP, and which do not have multi-drug resistance reversal activity. Without being bound by theory, applicants believe that the compounds disclosed in this application exert their neuronal activity by increasing cytoplasmic Ca
2+
concentrations. This is likely achieved by interaction, either direct or indirect, with a calcium release channel, such as the ryanodine receptor or the inositol 1,4,5-trisphosphate receptor, in the endoplasmic reticulum of the nerve cell.
Thus, according to one embodiment, the invention provides a method of stimulating nerve growth or preventing neurodegeneration by contacting nerve cells with a compound that:
a. increases cytoplasmic Ca
2+
concentration or binds to the ryanodine receptor;
b. does not bind to FKBP; and
c. does not possess MDR reversal activity.
According to a related embodiment, the present invention provides a compounds that:
a. has neuronal activity;
b. increases cytoplasmic Ca
2+
concentration or bind to the ryanodine receptor;
c. does not bind to FKBP; and
d. does not possess MDR reversal activity.
The term “increases cytoplasmic Ca
2+
concentration,” as used herein means a detectable increase in channel current recorded in the single channel recording assay described below in the presence of such a compound as compared to an appropriate control. Alternatively, the term “increases cytoplasmic Ca
2+
concentration,” as used herein means a detectable shift in the fluorescence spectrum in the cell assay described herein.
The term “binds to the ryanodine receptor,” as used herein, means that the compound specifically competes with ryanodine for binding to microsomes in the assay described below.
The term “does not bind FKBP,” as used herein means that the compound demonstrates a Ki of 10 &mgr;M or greater in at least one of the rotamase inhibitory assays described below.
The term “does not possess MDR reversal activity,” as used herein means that at a concentration of 2.5 &mgr;M, the compound has an MDR ratio of less than 7.0, and preferably less than 3.0 in at least one of the MDR assays described below.
Single-channel recording experiments are useful to determine if the compounds of this invention cause the requisite increase in cytoplasmic Ca
2+
concentration. These experiments are conducted as described in E. Kaftan et al.,
Circulation Research,
78, pp. 990-997 (1996), the disclosure of which is herein incorporated by reference. Single channel recordings are conducted under voltage clamp conditions with a pair of Ag/AgCl electrodes contacting the solutions via CsCl junctions. Vesicles are added to the cis chamber and fused with planar lipid bilayers composed of phosphatidylethanolamine/phosphatidylcholine (3:1, 30 mg/ml in decane, Avanti Polar Lipids). The trans chamber contains 250 mM HEPES and 53 mM B(OH)
2
, pH 7.35; the cis chamber contains 250 mM HEPES-Tris pH 7.35. Compounds dissolved in methanol are added to the cis chamber. Channel currents are amplified using a bilayer clamp amplifier (BC-525A, Warner Instruments) and recorded on VHS tape (Dagen Corp.). Data are filtered to an eight-pole Bessel filter (Frequency Devices) to 500 Hz, digitized at 2 kHz, transferred to a personal computer, and analyzed with pclamp version 6.0 (Axon Instruments). Single channel recordings are done at least 3 times for each compound condition.
Ryanodine binding may be measured by incubating microsomal protein with
3
H-ryanodine in buffer containing 36 mM Tris pH 7.2 and 50 mM KC
1
in the absence or presence of test compounds. Controls for maximum binding were done in the presence of 0.72 mM ATP and 36 &mgr;M CaCl
2
. Nonspecific binding was measured in the pre

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