Bitopic muscarinic agonists and antagonists and methods of synthesis and use thereof
09629833 ยท 2017-04-25
Assignee
Inventors
Cpc classification
A61K45/06
HUMAN NECESSITIES
C07D417/06
CHEMISTRY; METALLURGY
C07D295/14
CHEMISTRY; METALLURGY
C07D405/06
CHEMISTRY; METALLURGY
A61K31/443
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
International classification
A61K31/4439
HUMAN NECESSITIES
A61K31/443
HUMAN NECESSITIES
Abstract
The present invention is directed to a composition comprising the following compound: ##STR00001## The compound is associated with activity of a muscarinic receptor (e.g. one or more M.sub.1, M.sub.2, M.sub.3, M.sub.4 and M.sub.5.
Claims
1. A composition comprising a pharmaceutically acceptable carrier and a bitopic muscarinic antagonist having the formula: ##STR00020## in a therapeutically effective amount for activating or inhibiting activation of at least one muscarinic receptor selected from the group consisting of: M.sub.1, M.sub.2, M.sub.3, M.sub.4 and M.sub.5, in a subject.
2. The composition of claim 1, wherein the composition is formulated for oral or intravenous administration.
3. A kit comprising: (a) the composition of claim 1 in a therapeutically effective amount for activating or inhibiting activity of at least one muscarinic receptor in a subject; (b) instructions for use; and (c) packaging.
4. The kit of claim 3, wherein the at least one muscarinic receptor is selected from the group consisting of: M.sub.1, M.sub.2, M.sub.3, M.sub.4, and M.sub.5.
5. The kit of claim 3, wherein the subject is a human.
6. The kit of claim 3, wherein the composition is formulated for oral or intravenous administration.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
DETAILED DESCRIPTION
(5) Described herein are compositions, methods and kits for modulating muscarinic receptor activity and treating diseases and disorders associated with muscarinic receptor activity. A bitopic muscarinic antagonist of muscarinic M.sub.1, M.sub.2, M.sub.3, M.sub.4 and M.sub.5 receptors was discovered and is referred to herein as JB-D4. Structural analogs of JB-D4, as well as bitopic muscarinic agonists and analogs thereof, are also described herein.
JB-D4, a Bitopic Muscarinic Receptor Antagonist and Analogs Thereof
(6) A careful examination of the structural features of JB-D4 (also referred to herein as Compound A), reveals the classical NCCOC backbone of acetylcholine, the ammonium head-group, and a region of negative electrostatic potential. Not wishing to be bound by theory, the ammonium head group is believed to interact with the orthosteric site of muscarinic receptors while the p-butoxybenzoyl linker is believed to interact with the allosteric site in a bitopic manner. JB-D4 can be generated or synthesized using any suitable methods. An example of a method of synthesizing JB-D4 is described below in Example 2.
(7) ##STR00007##
(8) JB-D4 has been shown to slow down dissociation of both the antagonist N-methyl scopolamine (NMS) and the agonist acetylcholine in kinetics experiments on cell lines stably transfected with M.sub.1-M.sub.5 muscarinic receptors. To probe the ability of JB-D4 to allosterically modulate orthosteric ligand, its effect on the rate of orthosteric radioligand dissociation has been investigated. In these experiments, a single concentration (0.1 mM and 1 mM) of JB-D4 has been tested for effects on the control [.sup.3H] NMS and [.sup.3H] acetylcholine dissociation rate at all M.sub.1-M.sub.5 mAChR, in full time course assays. The presence of JB-D4 has significantly slowed the dissociation rate of both [.sup.3H] NMS and [.sup.3H] acetylcholine from all M.sub.1-M.sub.5 receptors.
(9) JB-D4 has been shown to slow down dissociation of NMS to the extent comparable with classical allosteric modulators like gallamine and alcuronium (Table 1,
(10) TABLE-US-00001 TABLE 1 Dissociation of [.sup.3H] NMS control +0.1 mM JB-D4 +1 mM JB-D4 k.sub.off [h.sup.1] k.sub.off1 [h.sup.1] f.sub.2 [%] k.sub.off2 [h.sup.1] k.sub.off1 [h.sup.1] f.sub.2 [%] k.sub.off2 [h.sup.1] M.sub.1 1.10 0.06 0.91 0.05 12 1 1.15 0.06 0.61 0.06 M.sub.2 3.7 0.2 2.8 0.3 23 2 3.2 0.2 1.19 0.03 12 2 3.0 0.4 M.sub.3 0.57 0.03 0.41 0.03 0.085 0.008 M.sub.4 0.54 0.02 0.38 0.03 0.076 0.006 M.sub.5 0.26 0.01 0.18 0.01 0.83 0.005
Eq. 1 was fitted to the data from kinetic experiments. Data are meansSD from 3 independent experiments performed in quadruplicates. Equations:
y=(100f.sub.2)*e.sup.(Koff1*X)+f.sub.2*e.sup.(Koff2*X)(Eq. 1)
(11) where y is the percentage of radioligand binding at time x of radioligand binding at time 0, k.sub.off1 and k.sub.off2 are rate dissociation constants and f.sub.2 is the percentage of sites with k.sub.off2 rate of dissociation.
y=f*e.sup.(Koff*X)(Eq. 2)
(12) where y is the percentage of radioligand binding at time x of radioligand binding at time 0, k.sub.off is rate dissociation constant and f is percentage of sites with k.sub.off rate of dissociation.
(13) JB-D4 has been shown to slow down dissociation of acetylcholine dissociation stronger than those of gallamine and alcuronium (Table 2,
(14) TABLE-US-00002 TABLE 2 Dissociation of [3H] Acetylcholine control +0.1 mM JB-D4 +1 mM JB-D4 f [%] k.sub.off [min.sup.1] f [%] k.sub.off [min.sup.1] f [%] k.sub.off [min.sup.1] M.sub.1 89 3 0.30 0.02 90 3 0.19 0.02 88 3 0.11 0.02 M.sub.2 63 3 0.97 0.06 64 4 0.56 0.04 67 3 0.039 0.005 M.sub.3 85 3 0.40 0.03 84 3 0.19 0.02 82 3 0.084 0.007 M.sub.4 83 3 0.38 0.02 81 4 0.18 0.02 86 3 0.049 0.005 M.sub.5 82 3 0.11 0.01 81 3 0.038 0.05 81 3 0.033 0.005
Eq. 2 was fitted to the data from kinetic experiments. Data are meansSD from 3 independent experiments performed in quadruplicates.
(15) JB-D4 has been shown to fully inhibit both QNB and NMS binding, in competition experiments (
(16) TABLE-US-00003 TABLE 3 Inhibition of NMS and QNB Binding Competition with [.sup.3H]NMS IC.sub.501 IC.sub.50 2 IC.sub.503 avg IC.sub.50 K.sub.D NMS K.sub.I rec [M] [M] [M] [M] SD [nM] [M] M1 14.3 13.8 15.1 14.4 0.65574385 0.25 2.88 M2 3.31 3.37 3.45 3.37666667 0.07023769 0.37 0.91194647 M3 16.6 17.2 16.9 16.9 0.3 0.23 3.1601626 M4 43.4 42 40 41.8 1.70880075 0.22 7.53770492 M5 12.5 11.2 13.7 12.4666667 1.25033329 0.3 2.87692308 Competition with [.sup.3H]QNB IC.sub.501 IC.sub.502 IC.sub.503 avg IC.sub.50 K.sub.D QNB K.sub.I rec [M] [M] [M] [M] SD [nM] [M] M1 8.72 9.42 8.19 8.77666667 0.61695489 0.134 1.0371017 M2 9.33 8.6 9.63 9.18666667 0.52974837 0.195 1.4990795 M3 39.5 20.6 28.16 29.42 9.51279139 0.173 4.33901108 M4 20.6 32.6 28.1 27.1 6.06217783 0.128 3.0751773 M5 5.73 6.76 7.34 6.61 0.81541401 0.143 0.82697288
(17) JB-D4 may be used as a neuromuscular blocker with potencies comparable or greater to gallamine and alcuronium due to its bitopic nature. The much weaker allosteric effects of gallamine and alcuronium on acetylcholine kinetics may be associated to the smaller molecular size of acetylcholine compared to the much larger antagonists NMS or QNB. Both gallamine and alcuronium may leave a slit at the binding site opening, small enough for small agonists like acetylcholine to slip through. This, however, was not observed for JB-D4. Thus, JB-D4 and analogs thereof may find use in anesthesiology applications, e.g., in compositions for anesthetizing a subject.
(18) JB-D4 has the potential to be an effective modulator for the treatment of OAB syndrome due to the fact that it significantly slows down both acetylcholine and NMS dissociation at M.sub.3 receptors (Tables 1 and 2). The efficacy of antimuscarinic ligands in the treatment of OAB syndrome is believed to arise through inhibition of bladder M.sub.3, and to a lesser extent M.sub.2, muscarinic receptors on detrusor smooth muscle cells, urothelium and bladder afferent nerves. Experimental research has shown that for treatment of OAB, slow dissociation of antagonists from the M.sub.3 receptor is more important than selectivity based on affinity. Therefore, M.sub.3 antagonists are desirable agents for the symptomatic treatment of OAB. JB-D4 (1 mM) substantially and significantly reduced the dissociation rate of both NMS and acetylcholine from M.sub.3 receptors, K.sub.off (NMS) 0.0850.008 (control 0.570.03) and K.sub.off (Acetylcholine) 0.0840.007 (0.400.03).
(19) JB-D4 has the potential to be an effective M.sub.5 antagonist for the treatment of drug addiction and withdrawal due to the fact that it significantly slows down both acetylcholine and NMS dissociation at M.sub.5 receptors (Tables 1, 2). JB-D4 (1 mM) substantially and significantly reduced the dissociation rate of both NMS and acetylcholine from M.sub.5 receptors, K.sub.off (NMS) 0.0830.005 (control 0.260.01) and K.sub.off (acetylcholine) 0.0330.005 (0.110.03). The areas of the brain associated with rewarding properties of opiate-based analgesic drugs contain M.sub.5 receptors expressed in dopamine containing neurons of the ventral tegmental area (VTA). Since the VTA plays a dominant role in the rewarding system of the brain, it has been hypothesized that M.sub.5 antagonists could reduce the pleasurable effects associated with such drugs. Therefore, the discovery of M.sub.5 antagonists could be of great therapeutic value in the treatment and prevention of substance abuse.
(20) Analogs and derivatives of JB-D4 are encompassed by the present invention. Examples of structural analogs of JB-D4 that are expected to display muscarinic bitopic properties similar to JB-D4 are shown as Compounds B-M in
(21) ##STR00008##
wherein: R.sub.1 is Me, Et, Pr, Bu, pentyl, or hexyl; R.sub.2 is H or Me; X is C, O, or S; Y is O, S or no group; W is C, O, N, or S Z is C, O, or S; n is 1-5 CH.sub.2-group; and m= is 0 or 1; and wherein the B-ring can be a 5 (m=0) or 6 (m=1)-membered saturated or unsaturated ring containing one or more double bonds between any 2 carbon atoms and with WC, O, N, or S at any position.
(22) ##STR00009##
(23) Some examples of analogs are described herein (e.g., Compounds B-M of
(24) A second series of compounds is synthesized and tested for muscarinic binding and functional activity. In this series of compounds, the ester functionality is replaced by an ether or thio-linkage (YS, O) to improve metabolic stability and functional selectivity, Compounds E-G. Esters can be hydrolyzed by choline acetylcholine esterase, the enzyme that breaks down acetylcholine at synaptic gaps after neurotransmission. In all compounds, R.sub.1 are one- to six-carbon alkoxy substituents and R.sub.2 can either be a hydrogen or methyl group. These structural analogs of the invention may display similar muscarinic bitopic biological properties.
(25) A third series of compounds is synthesized in which the classical NCCOC backbone of acetylcholine is partly incorporated within a cyclic dioxolane moiety, Compounds H-J. Either oxygen atom of the 1,3-dioxolane moiety can serve as the region of negative electrostatic potential. These compounds are also expected to be more resistant to enzymatic hydrolysis. In all compounds, R.sub.1 are one- to six-carbon alkoxy substituents and R.sub.2 can either be a hydrogen or methyl group. These structural analogs of the invention may display similar muscarinic bitopic biological properties.
(26) A fourth series of compounds is synthesized in which the 3-alkoxy-1,2,5-thiadiazo moiety is coupled with morpholine, piperidine and tetrahydropyridine, Compounds K-M. In all compounds, R.sub.1 are one- to six-carbon alkoxy substituents and R.sub.2 can either be a hydrogen or methyl group. These structural analogs of the invention may display similar muscarinic bitopic biological properties.
Compositions for Modulating Muscarinic Receptor(s) Activity
(27) Compositions for modulating muscarinic receptors include a bitopic muscarinic receptor antagonist or agonist, or analog or derivative thereof, as described herein. Typically, the composition includes a pharmaceutically acceptable carrier and a bitopic muscarinic antagonist or agonist having the formula:
(28) ##STR00010##
wherein: R.sub.1 is Me, Et, Pr, Bu, pentyl, or hexyl; R.sub.2 is H or Me; X is C, O, or S; Y is O, S or no group; W is C, O, N, or S; Z is C, O, or S; n is 1-5 CH.sub.2-group; and m= is 0 or 1; and wherein
the B-ring can be a 5 (m=0) or 6 (m=1)-membered saturated or unsaturated ring containing one or more double bonds between any 2 carbon atoms and with WC, O, N, or S at any position, or an analog or derivative thereof. The bitopic muscarinic antagonist or agonist is in a therapeutically effective amount for activating or inhibiting activation of at least one muscarinic receptor (e.g., one or more of M.sub.1, M.sub.2, M.sub.3, M.sub.4 and M.sub.5) in a subject. In a particular embodiment, the composition includes a pharmaceutically acceptable carrier and a bitopic muscarinic antagonist having the formula:
(29) ##STR00011##
(30) Compositions for use in anesthetizing a subject (e.g., a human in need thereof) will typically include a pharmaceutically acceptable carrier and a bitopic muscarinic antagonist that acts as a neuromuscular blocking agent. The bitopic muscarinic antagonist is in an amount effective to specifically activate M.sub.3 and M.sub.4 muscarinic receptors on smooth muscle tissues in the subject. An example of such a bitopic muscarinic antagonist is Compound A (JB-D4). However, suitable analogs or derivatives of Compound A may also be used.
(31) The compositions described herein may be formulated for any suitable route of administration. Compositions may be administered orally, or parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation and may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
(32) Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that modulates activity of a muscarinic receptor(s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, and/or dispersing agents.
(33) As indicated above, compositions (e.g., pharmaceutical compositions) described herein may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
(34) Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof). The bitopic muscarinic receptor agonists and antagonists described herein may be formulated as transdermal formulations, which may be administered using a variety of devices which have been described in the art (e.g., those described in U.S. Pat. Nos. 3,598,122, 3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636, 3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084, 4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, and 4,292,303 each of which is hereby incorporated by reference in its entirety).
(35) Formulations for oral use include tablets containing the active ingredient(s) (e.g., a bitopic muscarinic receptor agonist or antagonist or a derivative thereof) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches such as potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.
(36) The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material, such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.
(37) Optionally, a composition as described herein may be administered in combination with any other appropriate therapy; such methods are known to the skilled artisan and described in Remington: The Science and Practice of Pharmacy, supra. Combinations are expected to be advantageously synergistic. Therapeutic combinations that specifically activate or specifically inhibit activation of one or more muscarinic receptors are identified as useful in the methods described herein.
Methods of Treating a Disease or Disorder Associated with Activity of a Muscarinic Receptor
(38) Described herein are methods of treating a disease or disorder associated with activity of a muscarinic receptor(s) in a subject. Typically the method includes administering to the subject a composition including a pharmaceutically acceptable carrier and a bitopic muscarinic antagonist or agonist having the formula:
(39) ##STR00012##
wherein: R.sub.1 is Me, Et, Pr, Bu, pentyl, or hexyl; R.sub.2 is H or Me; X is C, O, or S;
(40) Y is O, S or no group; W is C, O, N, or S; Z is C, O, or S; n is 1-5 CH.sub.2-group; and m= is 0 or 1; and wherein
the B-ring can be a 5 (m=0) or 6 (m=1) -membered saturated or unsaturated ring containing one or more double bonds between any 2 carbon atoms and with WC, O, N, or S at any position, or an analog or derivative thereof. The bitopic muscarinic antagonist or agonist is in a therapeutically effective amount for activating or inhibiting activity of at least one muscarinic receptor in the subject and alleviating or eliminating the disease or disorder in the subject. In a typical embodiment, the subject is a human and the disease or disorder is, for example, a CNS disorder (e.g., Parkinson's disease, Schizophrenia, AD, drug addiction and/or withdrawal), OAB syndrome, COPD, or asthma. The composition can be administered by any suitable route, e.g., orally or intravenously.
(41) In one embodiment, in which the composition includes a bitopic muscarinic antagonist, administration of the composition results in inhibition of activation of at least one muscarinic receptor such as M.sub.1, M.sub.2, M.sub.3, M.sub.4, or M.sub.5. In this embodiment, the bitopic muscarinic antagonist can be Compound A, for example. In one example, when used to treat drug addiction and withdrawal, the composition can include a bitopic muscarinic antagonist that specifically inhibits activation of M.sub.5. In some embodiments, administration of the composition results in inhibition of activation of multiple muscarinic receptors (e.g., two or more of M.sub.1, M.sub.2, M.sub.3, M.sub.4, and M.sub.5). For example, in anesthesiology applications, the bitopic muscarinic antagonist blocks activation of M.sub.3 and M.sub.4 muscarinic receptors. In another example, when used to treat OAB, the composition can include a bitopic muscarinic antagonist that specifically inhibits activation of M.sub.3, and optionally, M.sub.2.
(42) In another embodiment, in which the composition includes a bitopic muscarinic agonist, administration of the composition results in activation of at least one muscarinic receptor such as M.sub.1, M.sub.2, M.sub.3, M.sub.4, or M.sub.5. In this embodiment, the bitopic muscarinic agonist can be used for a number of neurological and psychiatric diseases including AD, Schizophrenia.
(43) The therapeutic methods of the invention (which include prophylactic treatment) in general include administration of a therapeutically effective amount of a composition described herein to a subject in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects at risk can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, genetic marker, family history, and the like).
(44) The administration of a composition including a bitopic muscarinic antagonist or agonist, or a derivative thereof, for the treatment of a disease or disorder associated with activity of a muscarinic receptor(s) may be by any suitable means that results in a concentration of the therapeutic that, (e.g., in some embodiments, when combined with other components), is effective in inhibiting or promoting, respectively, activation of the muscarinic receptor(s). The bitopic muscarinic antagonist or agonist, or derivative thereof, may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. As described above, the composition may be administered locally or systemically (e.g., parenteral, orally, subcutaneously, intravenously, intramuscularly, or intraperitoneally).
(45) In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of change in one or more suitable parameters or markers depending upon the disease or disorder being treated, using, for example, one or more diagnostic markers or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with activity of one or more muscarinic receptors in which the subject has been administered a therapeutic amount of a composition as described herein. The level of marker determined in the method can be compared to known levels of marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of marker in the subject is determined prior to beginning treatment according to the methods described herein; this pre-treatment level of marker can then be compared to the level of marker in the subject after the treatment commences, to determine the efficacy of the treatment.
Methods of Anesthetizing a Subject
(46) As described above, JB-D4 has the potential to be a neuromuscular blocker with potencies comparable or greater to gallamine and alcuronium due to its bitopic nature. JB-D4 has been shown to slow down dissociation of NMS to the extent comparable with gallamine and alcuronium (Table 1,
(47) ##STR00013##
(48) The bitopic muscarinic antagonist is in an amount (a concentration) effective for inhibiting activation of M.sub.3 and M.sub.4 muscarinic receptors on smooth muscle tissues in the subject (e.g. a human in need of anesthesia). Such a composition can further include an anesthesia agent. Examples of anesthesia agents include gallamine and alcuronium. In another embodiment, the composition can be administered with a second composition that includes an anesthesia agent. In this embodiment, the composition can be administered prior to, simultaneous to, or subsequent to administration of the second composition.
Effective Doses
(49) The compositions described herein are preferably administered to an animal (e.g., rodent, human, non-human primates, canine, bovine, ovine, equine, feline, etc.) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., inhibiting or promoting activation of a specific muscarinic receptor(s) in the subject, anesthetizing a subject, etc.). Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, body weight, age, the particular composition to be administered, time and route of administration, general health, the clinical symptoms of the disease or disorder and other drugs being administered concurrently (if any). A composition as described herein is typically administered at a dosage that sufficiently inhibits or promotes activation of a specific muscarinic receptor(s) for treating, alleviating or preventing the diseases and disorders described herein or for anesthetizing a subject. As an example, a typical dose of JB-D4 for modulating activity (e.g., activation) of at least one muscarinic receptor in a subject is in the range of about 1 mg/day to about 1000 mg/day for a mammal. Such a dose is typically administered daily. However, depending on the subject and the disease or disorder being treated (or the anesthesia regimen), a dose may be administered multiple times a day, hourly, weekly, as-needed, etc.
Kits for Treating a Disease or Disorder Associated with Activity of a Muscarinic Receptor(s) in a Subject and for Anesthetizing a Subject
(50) Described herein are kits for treating a disease or disorder associated with activity of a muscarinic receptor(s) in a subject and for anesthetizing a subject. A typical kit includes a composition including a therapeutically effective amount of a bitopic muscarinic antagonist or agonist, or a derivative thereof, for modulating activity (e.g., activation) of at least one muscarinic receptor in a subject, packaging, and instructions for use. In a kit, the composition may further include a pharmaceutically acceptable carrier in unit dosage form. If desired, a kit for anesthetizing a subject may also contain an effective amount of an additional anesthesia agent (e.g., gallamine, alcuronium, etc.). In some embodiments, the kit includes a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
EXAMPLES
(51) The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.
Example 1
Synthesis of JB-D4 and Structural Analogs
(52) Method 1p-alkoxy ester N-substituted piperidine and morpholine salts (Compounds B, D): Compound 1 is formed from the reaction of 2-bromoethanol with piperidine or morpholine. Compound 2 is obtained from the reaction of 1 with p-alkoxy benzoyl chloride in anhydrous ether. The corresponding hydrochloride and methyl iodide salts are obtained by reacting 2 with either HCl gas or methyl iodide, respectively.
(53) ##STR00014##
(54) Method 2p-alkoxy ester N-substituted tetrahydro pyridine salts (Compound C): Compound 4 is formed from the reaction of 2-bromoethanol with pyridine. Compound 5 is obtained from the reduction of 4 with sodium borohydride. Reaction of 5 with p-alkoxy benzoyl chlorides in anhydrous ether yields compounds 6. The corresponding hydrochloride and methyl iodide salts 7 are obtained by reacting 6 with either HCl gas or methyl iodide, respectively.
(55) ##STR00015##
(56) Method 3p-alkoxy ether and thioether N-substituted piperidine and morpholine salts (Compounds E, G): Reaction of morpholine or piperidine with 2-bromoethanol affords compounds 8. Reaction of compounds 8 with mesylchloride produces compounds 9. Reaction of compounds 9 with either p-alkoxyphenoxide or thio salts yields compounds 10. Reaction of compounds 10 with HCl or methyl iodide produces salts 11.
(57) ##STR00016##
(58) Method 4p-alkoxy ether and thio ether N-substituted tetrahydro pyridine salts (Compound F): Reaction of pyridine with 2-bromoethanol affords compound 12. Reaction of compound 12 with sodium borohydride followed by mesylchloride produces compound 13. Reaction of compound 13 with either p-alkoxyphenoxide or thio salts yields compounds 14. Reaction of compounds 14 with HCl or MeI produce salts 15.
(59) ##STR00017##
(60) Method 5p-alkoxy-1,3-dioxoxolane N-substituted piperidine and tetrahydro pyridine (Compounds H, I): Reaction of p-alkoxy benzaldehyde and 3-bromo-1,2-ethanediol can afford compound 16. Reaction of 16 with piperidine, followed by HCl gas or iodomethane can yield salts 17. Reaction of 16 with pyridine, followed by reduction with NaBH.sub.4 and then HCl or iodomethane can produce salts 18.
(61) ##STR00018##
(62) Method 63-alkoxy-1,2,5-thiadiazo N-substituted tetrahydropyridine salts (Compound L): Oxidation of alcohol 5, followed by condensation with ammonium ion produces compound 20. Reaction of 20 with S.sub.2Cl.sub.2 provides compound 21. Reaction of 21 with an alcohol in the presence of sodium metal affords compound 22. Reaction of 22 with iodomethane or hydrogen chloride produces compounds 23.
(63) ##STR00019##
Example 2
Synthesis of JB-D4
(64) Route 1:
(65) [2-Bromoethyl p-butoxybenzoate] To a 100-mL round bottom flask were added 2-bromoethanol (0.625 g, 0.005 mol), triethylamine (0.505 g, 0.005 mol) and 5 mL of dry anhydrous ether. The flask was cooled in an ice-bath and p-butoxybenzoylchloride (1.06 g, 0.005 mol) was added dropwise. The mixture was then refluxed for 30 minutes. The solution was filtered and 15 mL of 1M HCl was added to the filtrate. The mixture was extracted several times with water. The ether layer was dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum to yield compound 1 (0.41 g, 27%). .sup.1H NMR (300 MHz, CDCl.sub.3) 8.0 (2H), 7.0 (2H), 4.6 (2H), 4.2 (2H), 3.6 (2H), 1.8 (2H), 1.6 (2H), 1.0 (3H). MS: m/z 300 (M.sup.+), 121, 138 (base peak).
(66) [2-(N-piperidine ethyl) p-butoxybenzoate]. Compound 1 (0.41 g, 0.00113 mol), 20 mL of acetonitrile, 1.0 g sodium carbonate and piperidine (0.116 g, 0.00113 mol) were added to a 100-mL boiling flask and left to stir overnight. The solution was filtered and concentrated under vacuum to yield 0.39 g of crude compound 2. The crude material was chromatographed over silica with a 10:1 mixture of CH.sub.2Cl.sub.2/MeOH to give compound 2 (0.36 g, 88%). .sup.1H NMR (300 MHz, CD.sub.3COCD.sub.3) 8.0 (2H), 7.1 (2H), 4.5 (2H), 4.2 (2H), 2.8 (2H), 2.6 (4H), 1.8 (2H), 1.6 (4H), 1.5 (2H), 1.0 (5H). MS: m/z 305 (M.sup.+), 165, 98, 138 (base peak).
(67) [2-(N-piperidine ethyl) p-butoxy benzoylester N-methyl Iodide] (JB-D4) Compound 2 (0.36 g, 0.00118 mol) and 1 mL of iodomethane were added to a 25 mL boiling flask and stirred overnight. The mixture was concentrated to yield crude salt 3 (0.50 g, 63%). The salt was re-crystallized from n-butanol, m.p. 111-113 C. .sup.1H NMR (300 MHz, D.sub.2O) 7.9 (2H), 7.0 (2H), 4.6 (2H), 4.1 (2H), 3.8 (2H), 3.5-3.3 (4H), 3.1 (3H), 1.9-1.8 (4H), 1.7-1.5 (4H), 1.45-1.35 (2H), 0.9-0.8 (3H). Anal. Calcd. For C.sub.19H.sub.30NO.sub.3I: C, 51.03%; H, 6.71%; N, 3.13%; I, 28.38%. Found: C, 50.48%; H, 6.59%; N, 3.19%; I, 30.29%.
(68) Route 2:
(69) [N-(2-hydroxyethyl)piperidine]2-bromoethanol (1.40 g, 0.0112 mol), sodium carbonate (2.00 g, 0.019 mol) and 15 ml of acetonitrile were transferred to a 100 ml boiling flask. A mixture of piperidine (0.952 g, 0.0112 mol) in 5 mL of acetonitrile was added dropwise to the bromoethanol solution and stirred for two days at room temperature. The reaction mixture was filtered, concentrated and washed several times with anhydrous ether. The ether extracts were combined and concentrated to afford 0.53 gram of compound 1, a colorless liquid (36.8%). .sup.1H NMR (300 MHz, CDCl.sub.3) 1.4-1.5 (2H), 1.5-1.65 (4H), 2.35-2.45 (4H), 2.45-2.5 (t, 2H), 3.25-3.35 (bs, 1H), 3.55-3.6 (t, 2H).
(70) [2-(N-piperidine ethyl) p-butoxybenzoate hydrochloride]N-(2-hydroxyethyl)piperidine (1.05 g, 0.00815 mol), p-butoxybenzoyl chloride (1.7 g, 0.0080 mol) and 50 mL of anhydrous ether were added to a 100 mL boiling flask and stirred to form a white precipitate within minutes. The reaction mixture was concentrated and precipitate washed several times with anhydrous ether to afford 0.77 grams of compound 2 (28.2%). .sup.1H NMR (300 MHz, D.sub.2O) 7.9 (2H), 6.9 (2H), 4.5 (2H), 4.0 (2H), 3.8 (2H), 3.6-3.2 (4H), 1.9-1.8 (4H), 1.7-1.5 (4H), 1.45-1.35 (2H), 0.9-0.8 (3H).
(71) [2-(N-piperidine ethyl) p-butoxybenzoate] Compound 2 (0.77 g, 0.00226 mol) was neutralized with saturated aqueous sodium carbonate. The solution was extracted with ether and combined extracts was dried over anhydrous magnesium sulfate. The solution was filtered, filtrate was concentrated to afford 0.40 gram of compound 3 (58.8%). .sup.1H NMR (300 MHz, CD.sub.3COCD.sub.3) 8.0 (2H), 7.1 (2H), 4.5 (2H), 4.2 (2H), 2.8 (2H), 2.6 (4H), 1.8 (2H), 1.6 (4H), 1.5 (2H), 1.0 (5H). MS: m/z 305 (M.sup.+), 165, 98, 138 (base peak).
(72) [2-(N-piperidine ethyl) p-butoxybenzoate N-methyl iodide] (JB-D4) Compound 3 (0.40 g, 0.00131 mol) was dissolved in 6 ml of HPLC-grade acetone and excess methyl iodide was added and the mixture stirred for four hours at room temperature. The reaction mixture was concentrated to afford 0.55 gram of compound 4 (94%), m.p. 108-110 C. Compound 4 was recrystallized from t-butyl alcohol to yield 0.311 gram (56.5%) of a white powder, m.p. 124-125 C. .sup.1H NMR (300 MHz, D.sub.2O) 7.9 (2H), 7.0 (2H), 4.6 (2H), 4.1 (2H), 3.8 (2H), 3.5-3.3 (4H), 3.1 (3H), 1.9-1.8 (4H), 1.7-1.5 (4H), 1.45-1.35 (2H), 0.9-0.8 (3H). Anal. Calcd. For C.sub.19H.sub.30NO.sub.3I: C, 51.03%; H, 6.71%; N, 3.13%; I, 28.38%. Found: C, 50.48%; H, 6.59%; N, 3.19%; I, 30.29%.
Other Embodiments
(73) Any improvement may be made in part or all of the compositions, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.