# Patent

## Title
Neuronal pain pathway modulators

## Abstract

The present invention relates to compounds that may be used to inhibit activation of protein kinase G (“PKG”). It is based, at least in part, on the discovery of the tertiary structure of PKG and the identification of molecules that either bind to the active site of PKG and/or are analogs of balanol.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation application of Ser. No. 11/674,965, filed Feb. 14, 2007 and claims priority to U.S. Provisional Application Ser. No. 60/773,691, filed Feb. 14, 2006, and U.S. Provisional Application Ser. No. 60/815,980, filed Jun. 23, 2006. The contents of these three referenced applications are incorporated herein by reference in their entireties.


GRANT INFORMATION
The subject matter of this application was developed at least in part under National Institutes of Health Grants NS12250 and NS35979, so that the United States Government has certain rights herein.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named 070050_5389_SL.txt and is 51,104 bytes in size.
1. INTRODUCTION
The present invention relates to compounds that inhibit the activated form of protein kinase G (“PKG”) and their use in the alleviation of pain, particularly in the context of chronic pain syndromes.
2. BACKGROUND OF THE INVENTION
Pain is perceived as a result of communication between the two main divisions—central and peripheral—of the nervous system. While the two divisions work together to produce our subjective experience, the central and peripheral nervous systems are anatomically and functionally different.
A painful stimulus impinging on a specialized pain receptor is propagated along a peripheral branch of a primary nociceptive sensory neuron whose cell body resides within a dorsal root ganglion (part of the peripheral nervous system) and then along a central branch of the neuron that enters the spinal cord (central nervous system). The signal is subsequently relayed to a second order neuron in the spinal cord that, in turn, transmits the signal to the opposite (“contralateral”) side of the spinal cord. The signal is then communicated to higher centers in the brain where it is perceived as painful.
Peripheral pain receptors, which respond to mechanical, thermal or chemical stimuli are located on nerve endings of the primary nociceptive neurons. Activation of these receptors results in pain that can be acute or chronic. Acute pain tends to be sharp and well-localized and is typically transmitted along the thinly myelinated axons of A delta sensory neurons. Chronic pain is usually dull and diffuse, and is conveyed along non-myelinated axons of C-type nociceptive neurons. Chemical mediators of inflammation such as bradykinin and prostaglandins stimulate pain receptors, and are important agents in chronic pain syndromes, such as the persistent pain associated with arthritis, ileitis or cystitis, to name but a few.
The perception of pain can be altered at various stages of the pain pathway. For example, administering a local anesthetic to the peripheral receptor can eliminate the painful stimulus. Drugs like opioids were classically known to intervene at the central nervous system stage of the pain pathway, and non-steroidal anti-inflammatory drugs at the peripheral stage (although it is now realized that there is some cross-reactivity of both). Likewise, what is perceived as chronic pain (not due to primary spinal cord injury) is typically associated with sensitization of peripheral pain receptors as well as changes in the excitability of the second order neurons, and therefore has both peripheral and central nervous system components. The peripheral and central components regulate “primary” and “secondary” hyperalgesia, respectively (Urban and Gebhart, 1999, citing Woolf, 1983 and La Motte et al., 1991). In secondary hyperalgesia, the second order neuron in the central nervous system undergoes changes in gene expression that contribute to the phenomenon of “central sensitization” or “spinal hyperalgesia”. Spinal N-methyl-D-aspartate (“NMDA”) receptors are believed to play an important role in this process (Urban and Gebhart, 1999, citing Urban and Gebhart, 1998; Palacek et al., 2003; Lee et al., 1993). Spinal cord injury (presumably) without activation of the peripheral nervous system can also produce spinal hyperalgesia resulting in a central pain syndrome (Zhang et al., 2005). Central neuropathic pain has been associated with phosphorylation of the transcription factor, cyclic AMP response element binding protein (“CREB”) (Cron et al., 2005).
Chronic pain is initiated in the periphery by either a nerve injury (“neuropathic pain”) or an inflammation and both sources result in pain that is a major clinical problem that has mostly resisted effective treatment. In humans (Gracely et al., 1992) and mammalian model systems (Millan, 1999), persistent pain after nerve injury is associated with long-term hyperexcitability (LTH) of those primary sensory neurons whose axons are in the affected nerve. LTH is manifested as increased sensitivity to electrical stimuli in the nociceptive sensory neuron cell body and axon at the injury site (Wall and Devor, 1983; Study and Kral, 1996; Zhang et al., 1997; Chen and Devor, 1998; Kim et al., 1998; Abdulla and Smith, 2001). These changes result in the discharge of action potentials from sensory neurons at rest or during innocuous stimulation, leading to continuing excitation of higher order neurons in the central nervous system, spinal hyperalgesia and persistent pain. Because the appearance of LTH involves alterations in gene expression (Waxman et al., 1994; Wang et al., 2002; Park et al., 2003), a central question is, how are such changes in the neuron nucleus induced by an injury that occurs far from the cell body? Answering this question has been extremely difficult using the complex mammalian nervous system.
An experimentally favorable alternative is the homogeneous cluster of nociceptive sensory neurons that reside in the bilateral pleural ganglia of the mollusk Aplysia californica (Walters et al., 2004). Noxious mechanical stimulation of the body wall (Walters et al., 1983a) or crushing sensory neuron axons in vivo or in vitro elicits an LTH with electrophysiological properties similar to those seen after axotomy of mammalian nociceptive neurons (Walters et al., 1991; Walters, 1994; Ambron et al., 1996; Bedi et al., 1998; Ungless et al., 2002; Sung and Ambron, 2004). The LTH appears after a delay, suggesting that its induction after nerve crush is attributable to a positive molecular injury signal (Walters et al., 1991; Ambron and Walters, 1996; Lin et al., 2003). Two studies support this idea. First, blocking axonal transport after nerve injury in excised nervous systems prevented the appearance of LTH (Gunstream et al., 1995). Second, LTH was induced in noninjured sensory neurons by injecting axoplasm from injured axons (Ambron et al., 1995). LTH was also elicited in the neurons after intrasomatic injection of an ERK (extracellular signal-regulated kinase) member of the MAPK (mitogen-activated protein kinase) family (Sung et al., 2001). Other experiments have suggested that cyclic GMP (cGMP) and PKG (cGMP-dependent protein kinase; protein kinase G) are probably involved (Lewin and Walters, 1999). However, despite these observations, it was only recently that the signal from the axon was identified.
U.S. Pat. No. 6,476,007 by Tao and Johns (“the '007 patent”) relates to a proposed signalling pathway in the central nervous system in which stimulation of an N-methyl-D-aspartate (“NMDA”) receptor leads to activation of nitric oxide synthase (“NOS”) and production of nitric oxide (“NO”), which then stimulates guanylate cylase (“GC”) and the production of cyclic guanoside monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase I (“PKG”). It was observed that administration of the PKG inhibitor Rp-8-[4-chlorophenyl)thio]-cGMPS triethylamine into the central nervous system by intrathecal administration, after the induction of an inflammatory response, produced significant attenuation of acute pain in rats 10 and 60 minutes later. Further, the inventors of the '007 patent noted an upregulation of PKG expression in the lumbar spinal cord 96 hours after noxious stimulation was blocked by administration of a neuronal NOS inhibitor, a soluble GC inhibitor, and a NMDA receptor antagonist.
However, the '007 patent is directed toward the mechanism of inflammatory hyperalgesia in the central nervous system; the role of the peripheral nervous system is not considered. Targeting the pain pathway in the central nervous system suffers from several important disadvantages. First the neuronal circuits in the spinal cord are highly complex and not well understood. Thus, drugs that might be predicted to relieve pain can have the opposite effect. Second, the neurons in the central nervous system are sequestered from the rest of the body by the blood-brain-barrier, which is a formidable obstacle that often prevents many therapeutic drugs from ever reaching their targets. The limited permeability means that treatment of spinal hyperalgesia according to the '007 patent would be problematic. Third, drugs that do penetrate the blood brain barrier have access to the entire central nervous system so that side effects can be severe. In contrast, there is no such barrier in the peripheral nervous system. Moreover, the anatomical disposition of the DRG means that it is possible to target specific populations of primary sensory neurons for treatment. Fourth, pain as a sensation is perceived only when signals from the periphery are communicated to higher centers in the brain. Consequently, since the DRG neurons are the portal for these signals, the present invention offers the advantage of intervening in subjective pain as it first arises. Finally, the 007 patent describes methods to prevent the activation of PKG; it does not address the inhibition of the already activated PKG.
Active PKG has a critical role in the initiation of pain. (See International Patent Application No. PCT/US2006/010107, Publication No. WO2006/102267). Following injury to a peripheral nerve there is an increase in nitric oxide synthase (“NOS”) activity that results in increased nitric oxide (“NO”) production. The NO activates soluble guanylyl cyclase (“sGC”), thereby increasing levels of cyclic guanosine monophosphate (“cGMP”) which results in the activation of protein kinase G (“PKG”) in the axons of the C-type and A-delta type nociceptive neurons. The activated PKG is then retrogradely transported from the site of injury along the axon to the neuron cell body, where it phosphorylates mitogen-activated protein kinase-erk (“MAPKerk”) (Sung et al., Aug. 25, 2004). The activated MAPKerk then translocates into the cell nucleus, where it modulates expression of the pain-related genes that mediate the appearance of LTH. Since inhibiting PKG attenuates pain and reduces the level of mRNAs for proteins that are involved in nociception, the focus of the present invention relates to modulators of the activated PKG.
Balanol is a known protein kinase C (PKC) inhibitor. Various balanol analogs which inhibit PKC have been previously identified by a retro-synthesis of balanol isolated from Verticillium balanoides (Lai et al. 1997). The retro-synthesis of the compound divided the compound into the following three main constituents: a tetrasubstituted benzophenone diacid, a trans-3,4-aminohydroxyperhydroazepine, and a 4-hydroxybenzoic acid. The balanol analogs were then synthesized with replacement of the perhydroazepine moiety. Specifically, Lai compared the activity of the analogs to balanol, the parent compound, and found that the analogs were more isozyme selective, demonstrating more selectivity between PKC and PKA than the parent compound (Lai et al. 1997). Lai concluded that the activity and the selectivity of the compounds was largely related to the conformation of the nonaromatic structural elements of the molecule. Ring size of the pyrrolidine nitrogen was found to greatly affect potency, with five molecules considered to have optimal potency.
While Lai was directed to analog development, the focus on the pyrrolidine ring, while valuable in its findings, is limited. The value of different or additional varying substituents at other ring sites within the compound, and the advantage of PKG selective inhibitory activity, were not considered prior to the present invention.
The prior art has demonstrated some additional compounds that exhibit PKC inhibitory action. For example, U.S. Pat. No. 5,432,198 by Jadgdmann et al. (“the '198 patent) discloses additional balanol analogues with different substituents, wherein the compounds have PKC inhibitory activity. The '198 patent discloses a balanol analogue without a pyrrolidine nitrogen, but instead has a carbon ring up to 7 members. Among other substitutions, the '198 compound also requires an alkyl substituted aromatic ring on the amine end of the compound.
U.S. Pat. No. 5,583,221 by Hu et al. (“the '221 patent”) similarly discloses compounds that exhibit PKC inhibitory activity. However, the '221 patent is limited in that it does not cover balanol derivatives or pyrrolidine-containing compounds. U.S. Pat. Nos. 6,376,467 and 6,686,334 by Messing et al. (“the '467 patent” and “the '334 patent”, respectively) disclose methods to lessen pain with compounds that are specifically directed to an inhibitor of the e isozyme of PKC. The '334 patent further discloses that the amount of inhibitor contemplated would not significantly inhibit other isozymes of PKC.
Thus, there remains a need in the art for unique compounds capable of selectively inhibiting active PKG in a peripheral nervous system. Inhibition of the active kinase would both prevent its transport from the periphery as well as block its activity in the cell body.
3. SUMMARY OF THE INVENTION
The present invention relates to compounds that may be used to inhibit the activated form of protein kinase G (“PKG”). It is based, at least in part, on the prediction of the tertiary structure of PKG and the identification of molecules that either are predicted to bind to the active site of PKG and/or are analogs of balanol.
In one set of embodiments, the present invention provides for pharmaceutical compositions comprising an effective (inhibitory) amount of these PKG modulator compounds.
In another set of embodiments, the present invention provides for methods of inhibiting PKG activity in a neuron by exposing the neuron to an effective inhibitory concentration of one of the PKG modulator compounds. Preferably, but not by way of limitation, the PKG inhibitor is administered to the peripheral nervous system and the neuron in which PKG activity is inhibited is a peripheral neuron.
In related embodiments, the present invention provides for a method of relieving chronic pain in a subject, comprising administering, to the subject, an effective inhibitory amount of one of the PKG modulator compounds of the invention.



4. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A-C. FIG. 1A-C provides a schematic diagram of Aplysia PKG (“apPKG”) showing the position of the conserved tandem cGMP binding domains, the ATP binding and catalytic sites, and the position of an autoinhibitory sequence. Bottom, Clustal W sequence alignment of the predicted apPKG amino acid sequence with Drosophila DG1 (GenBank accession number AAB03405) and DG2T3a (AAA28459), human Ia (BAA08297) and II (CAA64318), mouse 1β (AAD16044) and II (AAA02572), and rat II (CAA85284) PKGs. Conserved amino acids are shaded in black; similar amino acids are shaded in light gray.
FIGS. 2A-C. FIGS. 2A-C provide the structure for various balanol compounds. FIG. 2A is balanol-7R. FIG. 2B is 10′′ deoxybalanol. FIG. 2C is 14′′ decarboxy balanol.
FIGS. 3A-B. FIGS. 3A and 3B provide an overview of surface dermatomes. FIG. 3A provides the front view, and FIG. 3B depicts the back view.
FIGS. 4A-B. FIGS. 4A and 4B show schematic drawings of (A) PKA co-crystallized with balanol and (B) balanol docked to a homology model of PKG.
FIGS. 5A-B. FIGS. 5A and 5B provide a schematic showing differences in PKA and PKG active sites. In FIG. 5A, balanol is docked into the PKA active site. In FIG. 5B, balanol is docked into the PKG active site.
FIG. 6. FIG. 6 provides the structures for cyclopentane analogs of balanol.
FIGS. 7A-B. FIGS. 7A and 7B provide schematic diagrams of docked poses of (A) compound 8H (NOP47935) and (B) balanol in the active site of the PKG homology model, illustrating the sequence differences between PKG type 1 alpha (a) and PKA/PKB/PKC.
FIGS. 8A-L. FIGS. 8A-8L provide structures of compounds 8A-8L, either identified based on similarity to balanol (8A and 8B) or identified by docking to homology models of PKG (8C-8L).
FIGS. 9A-K. FIGS. 9A-9K provide schematic depictions of PKG bound to various compounds. FIG. 9A shows PKG bound to balanol. FIG. 9B shows PKG bound to compound 8C. FIG. 9C shows PKG bound to compound 8D. FIG. 9D shows PKG bound to compound 8E. FIG. 9E shows PKG bound to compound 8F. FIG. 9F shows PKG bound to compound 8G. FIG. 9G shows PKG bound to compound 8H. FIG. 9H shows PKG bound to compound 81. FIG. 9I shows PKG bound to compound 8J. FIG. 9J shows PKG bound to compound 8K. FIG. 9K shows PKG bound to compound 8L.
FIGS. 10A-B. FIGS. 10A and 10B show inhibition of PKG activity by (A) 1 micromolar of either compound 8J (NOP952668) or compound 8H (NOP479435); or (B) 10 micromolar of either compound 8J (NOP952668) or compound 8H (NOP479435).
FIG. 11. FIG. 11 shows the inhibition of PKG activity by increasing concentrations of compound 8H (NOP479435).
FIG. 12. FIG. 12 shows the inhibition of PKG activity toward a peptide substrate in presence of 1 or 10 micromolar of each of the indicated compounds.
FIGS. 13A & B. FIGS. 13A and 13B show the selective inhibition of PKG activity by increasing concentrations of compound 21 and compound 6.
FIG. 14A-E. FIG. 14A-E summarizes the rankings of structural linkages with respect to PKG and PKA.
FIGS. 15A-15L. FIGS. 15A-15L show Compound 6 with varying linkage structures.
FIG. 16. FIG. 16 provides a schematic of the synthesis of balanol analogues.
FIGS. 17A-E. FIGS. 17A-17E provides schematics of the synthesis of various elements in the synthesis of balanol analogues. FIG. 17A shows the synthesis of the benzophenone subunit of balanol. FIG. 17B shows the synthesis of the simplified benzophenone subunit. FIG. 17C depicts synthesis of amino alcohols, and FIG. 17D shows the synthesis of diamines. FIG. 17E shows the synthesis of indazole acids.
FIG. 18. FIG. 18 shows the structures of certain balanol analogues.
FIG. 19. FIG. 19 shows a schematic of the synthesis of simplified benzophenone acid (4-(2-Fluoro-3-methoxy-6-methoxymethoxy-benzoyl)-benzoic acid).
FIGS. 20A-20B. FIG. 20 provides the structures and inhibitory activity of various balanol type compounds. FIG. 20A represents the Series A compounds and FIG. 20B represents the Series B compounds.
FIG. 21. FIG. 21 shows the structures for compounds 6 and 46.



5. DETAILED DESCRIPTION OF THE INVENTION
For clarity of description, and not by way of limitation, this section is divided into the following subsections:

    
    
        (i) modulators of PKG;
        (ii) synthesis of modulators;
        (iii) methods of use of modulators of PKG;
        (iv) a PKG model system; and
        (v) linkers to modify the lead compounds.
    
    


The following are terms relevant to the present invention:
Long-term hyperexcitability (“LTH”), as defined herein, is increased, persistent, sensitivity of a primary sensory neuron cell body or axon to stimuli. During electrophysiological testing, LTH is manifested as a decrease in spike threshold, an increase in repetitive firing, broader spikes, and/or an increase in spike amplitude. In animals that perceive pain, LTH is associated with persistent (chronic) pain (see Sung and Ambron, Mar. 22, 2004).
Electrophysiological testing may be performed using methods known in the art. One specific, non-limiting example of electrophysiological testing using Aplysia californica (hereafter referred to as either “Aplysia californica” or simply as “Aplysia”) sensory neurons (SN) may be performed as follows (see Liao et al., 1999). Intracellular recordings from SN somata may be made with glass microelectrodes filled with 3 M potassium acetate (electrode resistance 8-20 M). Recordings may be made at 19-21° C. while the preparation is bathed in buffered artificial sea water (“ASW”), L15 medium, or a 1:1 mixture of ASW and L15, pH 7.6. Soma spike threshold may be measured with a standard series of 20 msec depolarizing pulses. Repetitive firing (spike accommodation) may be quantified by counting the number of spikes evoked by a 1 second intracellular depolarizing pulse using 2.5 times the threshold current determined with the 20 msec pulse. Repetitive firing may, for example, be examined by counting the number of spikes evoked by a series of 1 sec depolarizing pulses at 1.25, 2.5, and 5 times the threshold current, or by 1, 2, 3, and 5 nA. Input resistance (Rin) may be determined from the voltage change produced during injection of a 1 sec hyperpolarizing pulse (0.5 nA). Axon excitability may be tested by passing current between two compartments through a narrow, Vaseline-sealed opening containing nerves p7, p8, and p9. Threshold may be determined with a rapid series of 2 msec pulses.
Persistent pain (also referred to as chronic pain) includes pain that endures longer than the period of acute injury, and includes chronic pain syndromes such as, but not limited to, neuropathic pain (see Bennett et al., 2005). In specific, non-limiting embodiments, the duration of persistent pain is at least 1 day, at least one week, at least one month, or at least one year.
The terms aryl and heteroaryl include fused and unfused ring(s); and the term alkyl includes both branched and unbranched alkyls.
5.1 MODULATORS OF PKG
In various embodiments of the invention, the present invention provides for compounds that bind to the active site of PKG and preferably inhibit PKG activity. Non-limiting examples of compounds that may be used as PKG modulators were identified by docking compounds to two homology models of the ATP binding domain of PKG, as described in Example Section 6 and FIG. 8C-8L. A number of classes, subclasses and specific examples of PKG inhibitors are set forth herein.
One class of identified PKG modulators may be represented by general Formula I:

A-D-E  Formula I

wherein the following substituents are named with respect to Formula I:

A may be a substituted or unsubstituted ring structure which may comprise fused rings; for example, and not by way of limitation,
A may be substituted or unsubstituted chromanyl or isochromanyl, where the substituent may (without limitation) be one or more ketone, one or more hydroxyl, or a ketone, halogen, carbamoyl, amido, and hydroxyl group;
A may be a substituted or unsubstituted pyridyl, where the substituent may (without limitation) be (C1-C4) alkyl, halo, hydroxyl, carbamoyl, amido, amino, and carbonyl;
A may be a substituted or unsubstituted indole, isoindole, or indazole, where the substituent may (without limitation) be (C1-C4) alkyl, halogen, hydroxyl, carbamoyl, amido, amino, and carbonyl; or
A may be a substituted or unsubstituted phenyl, where the substituent may, without limitation, be hydroxyl, (C1-C4)alkoxy, (C1-C4) alkyl, preferably (p)hydroxyphenyl, and wherein more than one such substituent may be present;
D may be a 5-11 atom chain, preferably comprised of carbon and at least one heteroatom such as (without limitation) N, O, or S, optionally comprising one or more amide bond; and/or one or more (C4-C7) ring, said ring optionally comprising at least one unsaturated bond and optionally fused to A; and/or SO2; and
E may comprise (i) (C1-C4)alkyl; (ii)(C5-C13)cyclic or heterocyclic (including fused cyclic or heterocyclic); or (iii) (C1-C4)alkyl(C5-C13)heterocyclic; E may optionally comprise unsubstituted or substituted phenyl (e.g. fluorophenyl, chlorophenyl, hydroxyphenyl), and may comprise one or more of N, O, S, Br, Cl, F or I.
More particularly, in various non-limiting embodiments, the present invention provides a pharmaceutical composition for treating chronic pain in a subject using PKG inhibitor compounds, in an amount effective at inhibiting long-term hyperexcitability of sensory neurons in a subject to which it is administered, represented by Formula II:




wherein the following substituents are named with respect to Formula II:
wherein R1 may be H, (C1-C4)alkyl, (C2-C4)alkenyl, (C1-C4)alkoxy, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl or hydroxyl;
wherein R2 may be H, (C1-C4)alkyl, (C2-C4)alkenyl, (C1-C4)alkoxy, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl or hydroxyl or hydroxyl;
wherein R3 may be H, (C1-C4)alkyl, keto-, (C2-C4)alkenyl, (C1-C4)alkoxy, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl or hydroxyl or hydroxyl;
wherein R4 may be H, (C1-C4)alkyl, keto-, (C2-C4)alkenyl, (C1-C4)alkoxy, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl or hydroxyl or hydroxyl, and wherein if R4 is keto, x is a single bond, and wherein if R4 is not keto, x is a double bond;
wherein m may be 0-4;
wherein R5 may be amido, (C1-C4)alkylamido, amido(C1-C4)alkyl; carbonyl(C1-C4)alkylamido; (C1-C4)alkylcarboxy(C1-C4)alkylamido, or amido(C1-C4)alkylamido;
wherein n may be 0-4;
wherein R6 may be (C1-C4)alkylhydroxyphenyl or a (C5-C13)cyclic or heterocyclic ring preferably comprising N and one or more additional heteroatom selected from N, O, or S; and
wherein each of the aforesaid groups being capable to have one or more substituents may optionally be substituted with one or more substituents independently selected from halo, (C1-C4)alkyl, hydroxyl, amino, (C1-C4)alkoxy, or CF3.
Compound 8C, shown in FIG. 8C, is a non-limiting example of a compound of Formula II.
In other non-limiting embodiments, the present invention provides a pharmaceutical composition for treating chronic pain in a subject using PKG inhibitor compounds, in an amount effective at inhibiting long-term hyperexcitability of sensory neurons in a subject to which it is administered, represented by Formula III:




wherein the following substituents are named with respect to Formula III:
wherein R7 may be H, (C1-C4)alkyl, (C2-C4)alkenyl, (C1-C4)alkoxy, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl or keto;
wherein R8 may be H, (C1-C4)alkyl, (C2-C4)alkenyl, (C1-C4)alkoxy, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl or keto;
wherein p may be 0-4;
wherein R9 may be amido, (C1-C4)alkylamido, amido(C1-C4)alkyl; carbonyl(C1-C4)alkylamido; (C1-C4)alkylcarboxy(C1-C4)alkylamido, or amido(C1-C4)alkylamido;
wherein q may be 0-4;
wherein R10 may be a substituted or unsubstituted carboline, having one or more substituent selected from (C1-C4)alkyl, hydroxy, (C2-C4)alkenyl, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl, and (C1-C4)alkoxy; and
wherein each of the aforesaid groups being capable to have one or more substituents may optionally be substituted with one or more substituents independently selected from halo, (C1-C4)alkyl, hydoxyl, amino, (C1-C4)alkoxy, or CF3.
Compound 8D, as shown in FIG. 8D, is a non-limiting example of a compound of Formula III.
In other non-limiting embodiments, the present invention provides a pharmaceutical composition for treating chronic pain in a subject using PKG inhibitor compounds, in an amount effective at inhibiting long-term hyperexcitability of sensory neurons in a subject to which it is administered, represented by Formula IV:




wherein the following substituents are named with respect to Formula IV:
wherein R11 may be a substituted or unsubstituted oxadiazole or triazole, wherein the substituent may be (C1-C4)alkyl, hydroxy, (C2-C4)alkenyl, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl, and (C1-C4)alkoxy;
wherein R12 may be a substituted or unsubstituted naphthyl, anthryl, phenanthryl, or quinolyl, wherein the substituent may be (C1-C4)alkyl, hydroxy, (C2-C4)alkenyl, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl, and (C1-C4)alkoxy;
wherein α is H, (C1-C4)alkyl, halo, (C1-C4)alkoxy or hydroxy; and
wherein each of the aforesaid groups being capable to have one or more substituents may optionally be substituted with one or more substituents independently selected from halo, (C1-C4)alkyl, hydoxyl, amino, (C1-C4)alkoxy, or CF3.
Compounds 8F and 8G, as shown in FIGS. 8F and 8G, are non-limiting examples of compounds of Formula IV.
In one set of non-limiting embodiments, the present invention provides a pharmaceutical composition for treating chronic pain in a subject using derivatives of balanol, in an amount effective at inhibiting long-term hyperexcitability of sensory neurons in a subject to which it is administered, where balanol is disclosed in International Patent Application No. PCT/US92/07124, Publication No. WO93/03730 and the following structural Formula V:




In particular nonlimiting embodiments of the invention, Formula V may be varied to provide “balanol variants” which inhibit PKG. Non-limiting examples of such balanol variants include cyclopentane analogs of balanol, as shown in FIG. 6.
In various embodiments, the present invention provides for PKG inhibitor compounds represented by Formula VI, and pharmaceutical compositions comprising said compounds for treating chronic pain in a subject. Formula VI is represented by the following formula:




wherein the following substituents are named with respect to Formula VI:
wherein R13 may be substituted or unsubstituted phenyl, indolinyl, or isoindolinyl, wherein the substituent may be (C1-C4)alkyl, hydroxy, (C2-C4)alkenyl, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl, and (C1-C4)alkoxy, and wherein more than one (e.g. 2 or 3) such substituents may be present;
wherein t may be 0-4;
wherein R14 may be (C1-C4)alkyl, hydroxy, (C2-C4)alkenyl, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl, and (C1-C4)alkoxy;
wherein R15 and/or R16 may be hydrogen, hydroxyl, (C1-C4)alkyl, (C1-C4)alkoxy, heteroaryl or heterocyclic aryl, or keto or substituted or unsubstituted phenyl, dimethoxyphenyl, or substituted or unsubstituted ethylenedioxyphenyl; preferably (but not by way of limitation) R15 or R16 is a ketone, and ring G optionally further contains at least one double bond; and
wherein each of the aforesaid groups being capable to have one or more substituents may optionally be substituted with one or more substituents independently selected from halo, (C1-C4)alkyl, hydoxyl, amino, (C1-C4)alkoxy, or CF3. Preferably, but not by way of limitation, rings G and J together form a substituted or unsubstituted chromenone, e.g. a chromen-4-one or a chromen-2-one, where the substituents, which may be singular or plural, of said chromenone may be as set forth above in this paragraph).
Compounds 8A and 8B, shown in FIGS. 8A and 8B, are non-limiting examples of compounds of Formula VI.
In various embodiments, the present invention provides for PKG inhibitor compounds represented by Formula VII, and pharmaceutical compositions comprising said compounds for treating chronic pain in a subject using PKG inhibitor compounds:




wherein the following substituents are named with respect to Formula VII:
wherein X and Y are at trans or cis-configuration; and
wherein Z represents one of the following groups, or groups represented by X or Y;

    
    
        Z═—O— —CH2O— —OCH2— —CH2CH2O— —OCH2CH2— —CH2OCH2— —N— —CH2N— —NC2— —CH2CH2N— —NCH2CH2— —CH2NCH2— —CH2— —CH2CH2— —CH2CH2CH2—
    
    


wherein X represents one of the following functional groups:




wherein Y represents one of the following functional groups:




wherein R17 may be a substituted or unsubstituted aryl, heteroaryl, wherein there may be more than one substituent and each substituent may be hydroxyl, —CN, —NO2, (C1-C4)alkoxy, halo, (C2-C4)alkenyl, (C2-C4)alkynyl, (C1-C4)alkyl, or —SO3H;
wherein R18 may be amide, sulfonamide, or urea group; examples of the R18 are listed below:




wherein R19 may be (C1-C5)alkyl, aryl or heteroaryl un-substituted or substituted by one or more lower-alkyl, lower-alkoxy, hydroxy, alkoxy, amino, alkylamino or halogen groups; and

wherein R20 represents aryl or heteroaryl groups un-substituted or substituted by one or more lower-alkyl, lower-alkoxy, hydroxy, (C2-C4)alkenyl, (C2-C4)alkynyl, carbamoyl, amido, carbonyl, amino or halo groups
wherein each of the aforesaid groups being capable to have one or more substituents may optionally be substituted with one or more substituents independently selected from halo, (C1-C4)alkyl, hydoxyl, amino, (C1-C4)alkoxy, or CF3.
Examples of the aryl or heteroaryl groups are listed below:
When Y is:




R20 is:




When Y is:




R20 is:




Examples of compounds having Formula VII include compounds 8A and 8B.
In various embodiments, the present invention provides for PKG inhibitor compounds represented by Formula VIII and pharmaceutical composition comprising said compounds:

Ari—X*—Ar2  Formula VIII

wherein the following substituents are named with respect to Formula VIII:
wherein X* represents, but not limited to, one of the following groups;




wherein Ar1=Aryl, or heteroaryl, substituted or unsubstituted, for example:




wherein one of δ, β, γ and λ—is the bond to X*, and the others are respectively H, (C1-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl, halo, carbonyl, amido, cyano, carbamoyl, or aryl;
wherein Ar2=aryl, or heteroaryl, substituted or unsubstituted, for example:




wherein the substitutent(s) of any of the foregoing groups, which are optionally present and may be singular or plural, include, but are not limited to, alkyl (e.g., (C1-C4)alkyl), aryl, alkoxy (e.g. (C1-C4)alkoxy), alkylcarbonyl (e.g., (C1-C4)alkylcarbonyl), phenyl, alkylphenyl, halo, alkenyl (e.g., (C2-C4)alkenyl), alkynyl (e.g., (C2-C4)alkynyl) or hydroxy;
wherein Rx is hydrogen, halo, (C1-C4)alkoxy or (C1-C4)alkyl and
wherein each of the aforesaid groups being capable to have one or more substituents may optionally be substituted with one or more substituents independently selected from halo, (C1-C4)alkyl, hydoxyl, amino, (C1-C4)alkoxy, or CF3.
One specific, non-limiting example of a compound having Formula VIII is compound 8H.
In various embodiments, the present invention provides for PKG inhibitor compounds represented by Formula IX and pharmaceutical compositions comprising said compounds:

Ar1*—X**—Ar2*  Formula IX

wherein the following substituents are named with respect to Formula IX:
wherein X** represents, but not limited to, one of the following groups:
x** is:




wherein n=1 to 4;
wherein Ar1*=substituted or unsubstituted group, which include, but are not limited to, alkyl (e.g., (C1-C4)alkyl), aryl, alkoxy (e.g. (C1-C4)alkoxy), alkylcarbonyl (e.g., (C1-C4)alkylcarbonyl), furan, pyrrole, pyridine, phenyl, alkylphenyl, alkenyl (e.g. (C2-C4)alkenyl), alkynyl (e.g. (C2-C4)alkynyl), halo, or hydroxy, wherein the substitutent(s) of Ar1* are optionally present and may be singular or plural, include one or more substituents independently selected from the following:




halo, (C1-C4)alkyl, hydoxyl, amino, (C1-C4)alkoxy, or CF3;

wherein Ar2*=aryl, or heteroaryl, substituted or unsubstituted, for example:




wherein G is H, (C1-C4)alkyl, hydroxy, (C2-C4)alkenyl, (C2-C4)alkynyl, halo, carbamoyl, amido, amino, cyano, (C1-C4)alkylcarbonyl, and (C1-C4)alkoxy; X is O, N, or S; R21 and R22 are respectively H, alkyl (e.g., (C1-C4)alkyl), aryl, alkoxy (e.g. (C1-C4)alkoxy), alkylcarbonyl (e.g., (C1-C4)alkylcarbonyl), phenyl, alkylphenyl, alkenyl (e.g., (C2-C4)alkenyl), alkynyl (e.g., (C2-C4)alkynyl), halo or hydroxy;
each of the aforesaid groups optionally having one or more substituents selected from the group consisting of halo, (C1-C4)alkyl, hydoxyl, amino, (C1-C4)alkoxy, and CF3.
One specific, non-limiting example of a compound having Formula IX is compound 8J.
In various embodiments, the present invention provides for PKG inhibitor compounds and pharmaceutical compositions thereof comprising said inhibitors in amounts effective at inhibiting long-term hyperexcitability of sensory neurons, represented by Formula X:

D ring-C ring-B ring-A ring  Formula X

wherein the following substituents are named with respect to Formula X:
wherein the D ring is a substituted or unsubstituted aromatic ring (for example, and not by way of limitation);
wherein the C ring is a substituted or unsubstituted aromatic ring (for example, and not by way of limitation);
wherein the B ring is a cycloalkyl, preferably a cyclopentyl, or pyrrolidine (for example, and not by way of limitation); and
wherein the A ring is a substituted or unsubstituted aromatic ring (for example, and not by way of limitation).
The rings of Formula X are connected by various linkage groups, including but not limited to amide, ester, alkoxy, or ketone groups (for example, and not by way of limitation). Additional linkages groups contemplated by the invention are discussed below.
In particular embodiments, the PKG inhibitor compounds of Formula X are represented by Formula XI.




wherein the following substituents are named with respect to Formula XI:
wherein R1 may be F, COOH, Cl, or I, hydrogen, lower alkyl (e.g., straight chain, branched or cyclic moiety having 1-6 carbons), aryl, alkylamino, arylamino, aryloxy or alkoxy, preferably lower alkyl (for example, and not by way of limitation),
wherein R2 may be CH3O, CH3CH2O, (C1-C4)alkoxy, or OH, hydrogen, lower alkyl (e.g., straight chain, branched or cyclic moiety having 1-6 carbons), aryl, alkylamino, arylamino, aryloxy or alkoxy, preferably lower alkyl (for example, and not by way of limitation),
wherein R3 may be a halogen, alkyl, aryl, cycloalkyl; alkoxy, cycloalkoxy (e.g. cyclo-pentane-O—, cyclo-pentane-(CH2)n-O—), allyl-O—, aryl-O—, amide (—NCO—R′) sulfonamide (—NSO2R′); alkylcarbonyl (—CO—R′) CH3OCH2O, CH3CO, CH3COO, CH2OCH, O(CH2)2CH3, hydrogen, lower alkyl (e.g., straight chain, branched or cyclic moiety having 1-6 carbons), aryl, alkylamino, arylamino, aryloxy or alkoxy, preferably lower alkyl, COOH, COOR′, CONR′1R′2, F, Cl, I,




wherein n=0-5, preferably 1,2; and A,B=O,S,N,CH2, C═O, preferably O,N;





wherein C,D=O, N, S preferably 0 or N; and E=O,S, preferably O;

F—R5′

wherein F═O, S,N, CH2, C═O, S═O, preferably O, N (for example, and not by way of limitation),

wherein R4 may be H, OH, CH3O, or (C1-C4)alkoxy, lower alkyl (e.g., straight chain, branched or cyclic moiety having 1-6 carbons), aryl, alkylamino, arylamino, aryloxy or alkoxy, preferably lower alkyl (for example, and not by way of limitation),
wherein R5 may be H, OH, CH3O, or (C1-C4)alkoxy, lower alkyl (e.g., straight chain, branched or cyclic moiety having 1-6 carbons), aryl, alkylamino, arylamino, aryloxy or alkoxy, preferably lower alkyl (for example, and not by way of limitation),
wherein X may be O or N (for example, and not by way of limitation), and
wherein Y may be N, NH or C (for example, and not by way of limitation), preferably Y is N.
In other non-limiting embodiments, the present invention provides for compounds having the structure of Formula XI, wherein the A ring may be




and wherein the A ring is connected to the B ring by an amide linkage as shown above.

In another non-limiting embodiment, the present invention provides for compounds having the structure of Formula XI, wherein the A ring may be




wherein Ra may be an alkyl, alkoxy, substituted aromatic, CH3, CH3O, aromatic ring, CF3, a halogen, or NHCO—Rb, and

wherein the nitrogen of Ra is linked to an alkyl sulfonamide, formyl, or acetyl group.

In other embodiments, the A ring of Formula XI may have up to 7 members.
In yet other embodiments, the ketones (O†) of Formula XI are separated by 6 carbons, where there are 6 atoms carbonyl to carbonyl, with 4 atoms between the carbonyls.
In other non-limiting embodiments, the present invention provides for compounds having the structure of Formula XI, wherein the C and D rings may be:




In a preferred embodiment of a compound of Formula XI, R1 is F and R2 is OCH3, wherein this particular embodiment has been shown to have high potency. The specificity of these embodiments is achieved by the diether group, which decreases activity.
In an alternative embodiment of a compound of Formula XI, where R1 is not F and R2 is not OCH3, R4 and R5 are preferably OH groups.
In yet another embodiment of a compound of Formula XI, where R4 and R5 are OH groups R1 is COOH and R3 is OH. In another embodiment of a compound of Formula XI, where R4 and R5 are OH groups R1 is OH and R3 is COOH.
The compound of Formula XII (Compound-6) is a specific non-limiting example of a compound of Formula XI:




The compound of Formula XIII (Compound-21) is another non-limiting example of a compound of Formula XI:




The activity of the compound of Formula XII has shown to have high selectivity for PKG versus PKA. Specifically the alkoxy substituent (at the 10 position) in the D ring of Compound 21 has been shown to account for improved selectivity. See FIGS. 13A and 13B.
In other non-limiting embodiments, the present invention provides for compounds having the structure of Formula XIII, wherein the substituent at the 10 position in the D ring may be OCH2CH═CH2, O-Cyclopropane, OCH2—Cyclopropane, OSO2NR′, NHSO2NR′, NR′′′ wherein R′′′ are alkyl, cycloalkyl, or an aromatic substituent.
The compound of Formula XIV is another non-limiting example of a compound of Formula XI:




Additional non-limiting examples of PKG inhibitor compounds contemplated by the present invention are identified in the Examples below.
In specific non-limiting embodiments of the invention, a modulator according to the invention binds more tightly to PKG than to other kinases, such as PKA, PKB, and/or PKC. Such modulators may, for example, selectively interact with particular amino acid residues found in PKG but not such other kinases (see FIG. 7). For example, the following residues are different between PKG and PKA: Gly370Ser (PKG amino acid/human PKG Type I alpha residue number/PKA amino acid), Ile406Thr, Val501Thr, Cys441Val, Ala440Tyr, Ile491Leu.
The present invention further provides for molecules of Formulas I-IX which are conjugated to one or more carrier peptide, one or more transport peptide, or one or more carrier peptide and one or more transport peptide (also referred to as balanol variants, or balanol double variants).
The present invention provides for pharmaceutical compositions comprising effective amounts of one or more compound having Formula I-IV, VI-XIV or otherwise described herein. An “effective amount” of compound is an amount which may be administered to produce an effective concentration of compound at the site of action, for example, the sensory neuron affected, wherein effectiveness refers to ability to inhibit PKG and/or produce a significant pain-inhibiting effect. In specific non-limiting embodiments, the concentration of a PKG modulator according to the invention administered to the neuron, for example via its axon, may be between about 1 and 500 nM, or between about 2 and 100 nM, depending on the potency of the compound.
In non-limiting embodiments of the invention, the concentration of a PKG modulator and in particular a PKG inhibitor disclosed herein in the peripheral blood of a subject being treated may be between about 1 nanomolar and 500 microM, or between about 100 nanomolar and 100 microM, or between about 1 and 500 nM, or between about 2 and 100 nM. For compounds of Formulas VIII and IX and compounds 8H (NOP479435) and 8J (NOP952668), the effective concentration may be, without limitation, between about 0.01 and 10 micromolar, and preferably between about 0.1 and 5 micromolar. For compounds of Formula X through XIII, the effective concentration in the peripheral blood may be, without limitation, between about 1 to about 100 nanomolar, and preferably between about 40 and about 70 nanomolar. For the compound of Formula XII (Compound 6), the concentration in the peripheral blood, may be without limitation, about 100 nM to about 500 microM, and preferably between about 100 nM and about 100 microM.
The present invention further provides for pharmaceutical compositions as follows. Compositions of the invention may comprise an inhibitor agent as described above, where the inhibitor agent optionally comprises a carrier molecule that facilitates its translocation through a neuronal cell or nuclear membrane. Examples of carrier molecules which may be used include but are not limited to HIV-1 tat protein (YGRKKRRQRRRPP; SEQ ID NO: 1) and peptides that are about 9-30 or about 9-20 residues long comprising its cores sequence RKKRRQRRR (SEQ ID NO: 2), Drosophila Antennapedia homeo-domain (RQIKIWFQNRRMKWKK; SEQ ID NO: 3). Other carrier molecules that may be used according to the invention may be largely comprised (contain at least 60 percent, at least 70 percent, or at least 80 percent) of positively charged amino acids such as arginine (Wender et al., 2000) and/or lysine (Mai et al., 2002). Also encompassed by the invention are peptides and derivatized peptides which are at least about 90 or about 95 percent homologous to the above-recited peptides, as determined using standard homology assessing software such as BLAST or FASTA. The inhibitor agent may optionally alternatively or additionally comprise a transport peptide, as described below.
The present invention provides for such inhibitor agents, in either lyophilized form or dissolved in a suitable pharmaceutical carrier. Compositions that comprise more than one inhibitor agent are encompassed by the invention.
In non-limiting embodiments, the invention provides for a pharmaceutical composition comprising one or more inhibitor agent, as set forth above, together with at least one agent that promotes uptake of the inhibitor agent into a peripheral nerve. Examples of such agents include membrane permeability enhancing agents such as dimethyl sulfoxide and/or 2 hydroxypropyl-b-cyclodextrin.
In other non-limiting embodiments, the invention provides for a pharmaceutical composition comprising one or more inhibitor agent, as set forth above, together with at least one agent that treats an underlying cause of the pain, including, but not limited to, an anti-inflammatory agent (such as aspirin, a non-steroidal anti-inflammatory agent such as ibuprofen, or a corticosteroid).
In other non-limiting embodiments, the invention provides for a pharmaceutical composition comprising one or more inhibitor agent, as set forth above, together with at least one agent having a local anesthetic effect, such as lidocaine.
In a further non-limiting embodiment, the present invention provides for a transdermal device, such as a patch or apparatus comprising one or more inhibitor agent, as set forth above, and optionally one or more additional agent which promotes the uptake of agent in a peripheral nerve, treats an underlying cause of the pain, and/or has local anesthetic effect, where exemplary compounds in each of these categories is provided above. The device may in general utilize transdermal patch technology known in the art to facilitate sustained release of its therapeutic agents through the skin of a subject. In specific, non-limiting embodiments, the device creates an electrical potential which promotes uptake of the inhibitor agent(s) into local tissue (iontophoresis) or improves drug transfer using ultrasound or radiofrequency waves (see Bryan, 2004; U.S. Pat. No. 5,405,614, U.S. Pat. No. 4,708,716).
5.2 Synthesis of Balanol-Related Modulators
Balanol structurally consists of three different parts: the tetra substituted benzophenone, the p-hydroxybenzaminde moiety, and the perhydroazepine ring. This convenient analysis serves well as a guideline in planning the total synthesis of balanol and its analogs and also identified three major subjects of the SARs study of these interesting molecules. In the present invention, a systematic study of the SARs of balanol and its analogues was conducted with an aim toward uncovering factors that would allow the preparation of potent and selective PKG inhibitors.
As shown in a typical synthesis (see FIG. 16), preparation of balanol analogs followed a uniform scheme in which the azepine or its replacement was condensed with 4-hydroxybenzoyl residue or its replacements, and then coupled to a suitably protected benzophenone subunit followed by deprotection to give the final product. Specifically, benzoic acid 2 was converted to the corresponding acid chloride and coupled with an azepine replacement at the amino site. Occasionally this resulted in concomitant acylation of the vicinal hydroxyl group, and the crude products were treated with NaOH to provide the desire alcohols. For diamino compound 1 of FIG. 16, the formation of undesirable dimmer was unavoidable and the starting material could be recovered by harsh hydrolysis of diamides. Benzophenone acid 4 was usually converted to the corresponding acid chloride immediately before use and was coupled to amido alcohol or amido amine 3 of FIG. 16. With these common synthetic steps to complete the syntheses, the major task was reduced to construction of the desired three subunits. The synthesis of these required elements are shown in FIGS. 17A-E.
FIG. 17A shows the synthesis of the benzophenone subunit of balanol. Benzophenone acid 16 in FIG. 17A was prepared according to the literature method. As outlined in FIG. 17A, the differentially protected aryl bromide 8 was readily prepared from acid 7 in three steps. First, acid 1 was perbenzylated, and second, the benzyl ester was hydrolyzed and finally the acid was re-esterified. Benzyl alcohol 9 was metalated by reaction with n-butyllithium, and the resulting aryllithium was allowed to react with 1.2-dibromo-1,1,2,2-tetrafluroethane to give aryl bromide 10. Bromo alcohol 10 was oxidized to aldehyde 11 with TEMPO and protected as cyclic ketal. The required 1,2,3-trisubstituted aldehyde 12 could then be generated by bromine-lithium exchange with n-butyllithium followed by a quench with DMF. Coupling of aryl bromide with aldehyde proceeded to provide carbinol 13 in moderate yield. The ketone could conveniently be generated by oxidation of alcohol with manganese dioxide. The resulting benzophenone was then deprotected by p-TSA-catalyzed acetal hydrolysis to afford the corresponding aldehyde 14. Aldehyde 14 was oxidized with sodium chlorite to carboxylic acid. Benzylation and tert-butyl deprotection as usual provided the desired benzophenone acid 16.
FIG. 17B shows the synthesis of the simplified benzophenone subunit. Compound 18 in FIG. 17B was conveniently prepared from commercial available 3-fluoro-4-methoxyacetophenone through Baeyer-Villiger oxidation followed by deacetation and subsequent protection with MOM group. MOM-directed ortholithiation followed by aryllithium addition to commercially available aldehyde 19 provided barbinol 20 in moderate yield. Similarly, Oxidation of barbinol 20 with active manganese dioxide gave the crucial intermediate 21. Unmasking the carboxyl group of 21 with NaOH afforded acid 22. To introduce the desired side chain R, 21 was first treated with HCl to remove MOM group and introduction of R followed by the treatment of NaOH gave the desired benzophenone acid 24. Benzisoxazole acid 25 was prepared from benzophenone 23 by a three-step sequence in which oxime formation was accomplished with hydroxyl-amine in ethanol and dehydration with diethylazodicarboxylate and triphenyl phosphine followed by hydrolysis of the methyl ester furnished the final product 25. See FIG. 17B.
As shown in FIG. 17C, mCPBA epoxidation of olefin 26 followed by stereospecific epoxide opening with sodium azide and reduction afforded the trans-vicinal amino alcohol 29. Condensation of (+)-L-tartaric acid with benzylamine provided homochiral 3.4-dihydroxypyrrolidinedione, which was easily reduced to the respective enantiopure (3S,4S)-dihydroxylpyrrolidine 30 with LiAlH4. Debenzylation in the presence of Boc2O afforded the Boc-protected Diol 31. Diol 31 was transformed into its mesyl diester followed by azide substitution and subsequent catalytic reduction to give the enantiopure N-tBoc-(3R,4R)-3.4-diaminopyrrolidine 32. See FIG. 17D.
As shown in FIG. 17E, carbon substitutions in the 3-position of the indazoles was achieved by anion addition of Grignard reagents to 5-bromo-2-fluorobenzaldehyde 33. The resulting alcohol was oxidized with manganese dioxide to give the corresponding ketone 34. The indazole 35 was then formed by refluxing the ketone in hydrazine. Bromine-lithium exchange with n-butyllithium followed by a quench with dry CO2 yielded the indazole acid 35. The unsaturated N atom of indazole acid was often protected with Ac before its coupling with other amines. All compounds were prepared in racemic form, with the exception of diamide compounds.
5.3 Methods of Use of Modulators of PKG
A PKG inhibitor of the invention may be administered to a sensory neuron in need of such treatment in an amount effective in inhibiting LTH. Where the SN to which the inhibitor is to be administered is a SN in vivo in an animal subject, the inhibitor may be administered systemically (e.g. by intravenous injection, oral administration, inhalation, etc.), may be injected locally (in proximity to the damaged nerve), may be applied topically (for example, together with a skin permeability enhancing agent, such as a chemical compound or an electrical stimulus, optionally in the form of a sustained-release transdermal patch) or may be administered by any other means known in the art. In preferred non-limiting embodiments, the compound would not be administered directly into the central nervous system (for example, via intrathecal administration). However, in other embodiments, administration into the central nervous system (e.g. by intrathecal administration or by access to the central nervous system of drug administered by another route) may be appropriate, either alone or in conjunction with delivery to the peripheral nerve and/or systemic administration.
The amount of inhibitor to be administered may be determined using methods known in the art, for example, by doing dose response studies in one or more model system, such as the Aplysia system described above or a mammalian model of peripheral neuropathic pain, followed by approved clinical testing in humans. Where concentrations are set forth below, they refer to the concentration to which the sensory neuron or any component thereof, including axon, cell body or receptor, is exposed.
In related embodiments, an effective amount of an inhibitor may be administered to a subject in need of such treatment, where the subject suffers from chronic pain. The chronic pain preferably has a peripheral nervous system (primary) hyperalgesia component, where the method inhibits pain mediated by the peripheral nervous system, but in specific non-limiting embodiments the present invention also encompasses the treatment of spinal hyperalgesia as either a component of or the basis of (e.g., chronic central neuropathic pain resulting from spinal cord injury) chronic pain. Any of the foregoing modes of administration may be used, but if a spinal hyperalgesia component is to be treated, the inhibitor, which is directed to a neuron having its cell body in the central nervous system and not in the dorsal root ganglion, should be administered intrathecally.
An effective amount is an amount of inhibitor which decreases the level of pain subjectively perceived by the subject, preferably amount determined, in controlled experiments, which is greater than placebo effect. For example, and not by way of limitation, in certain embodiments of the invention, where perceived pain can be quantified on a scale from 0 to 10, where 0 is no pain, 1-5 is progressively more intense mild pain, 6-7 is progressively more intense moderate pain, 8-9 is progressively more intense severe pain, and 10 is the worst pain possible (to the subject), an effective amount of inhibitor may decrease the pain scale quantification of perceived pain by at least 2 points, or by at least 3 points.
In specific, non-limiting embodiments, the present invention provides for a method for treating chronic pain in a subject comprising administering, to the location from which the pain arises, an effective amount of an inhibitor as set forth herein (alternatively referred to as an “LTH inhibitor”), where administration can be by local injection or topical application (e.g., via a cream, ointment, or transdermal device, which may be a patch or may be an apparatus or an apparatus containing or otherwise associated with a patch), and the location can be, as non-limiting examples, a wound site, tissue overlying an inflamed joint, or an area within the dermatome associated with the perceived pain (e.g., L4, L5, S1, C3, C4, C5, C6 or C7, see below and FIG. 3).
In specific, non-limiting embodiments, the present invention provides for a method for treating post-operative pain in a subject comprising administering an effective amount of an inhibitor as set forth herein. Since the PKG is activated at a peripheral site, an incision on the skin should sever the surrounding sensory nerve endings resulting in the local activation of the NOS-sGC-PKG pathway. Subsequently, active PKG is transported along the axotomized sensory axons to the corresponding DRGs, initiating the development of hyper excitability and concomitantly pain. Therefore, the use of PKG inhibitors may be an effective means of treating post-operative pain.
The present invention provides for a method for modulating and specifically inhibiting pain pathways comprising a PKG inhibitor compound as set forth herein to an axon of a sensory nerve such that the compound is retrogradely transported along the axon to the nociceptive sensory neuron cell body in the dorsal root ganglion. In one non-limiting example, the transport peptide is PKKKRK (SEQ ID NO: 4), or a peptide or derivatized peptide which is at least about 80 percent homologous thereto as determined using standard homology assessing software such as BLAST or FASTA and which facilitate axonal transport. In another non-limiting example, the transport peptide is the related peptide CTPPKKKRKV (SEQ ID NO: 5) (see Ambron, 1992), or a peptide or derivatized peptide which is at least about 70, at least about 80, or at least about 90 percent homologous thereto as determined using standard homology assessing software such as BLAST or FASTA and which facilitate axonal transport. In specific, non-limiting embodiments of the invention, the transport peptide is between 5 and 20 amino acids long and comprises the peptide KKKRK (SEQ ID NO: 6), PKKKRK (SEQ ID NO: 4), PPKKKRK (SEQ ID NO: 7), TPPKKKRK (SEQ ID NO: 8), or PKKKKRKV (SEQ ID NO: 9).
For example, the PKG inhibitor compound comprising a transport peptide may be delivered to a peripheral pain receptor at the site of injury or in the same dermatome as the injury, as sensory axons arising throughout the dermatome converge on the same dorsal root ganglion. FIG. 3A-B presents the sensory dermatomes (from The Merck Manual of Diagnosis and Therapy, Section 14, Chapter 165, FIG. 165-2, which references Keegan J J and Garrett F D, “Anatomical Record 102:409-437, 1948, used with permission of the Wistar Institute, Philadelphia, Pa.). As examples, arthritis pain associated with the fingers is communicated via axons whose cell bodies reside in DRGs at levels C5-T1 and pain from the knees is communicated via axons whose cell bodies reside in DRGs at levels L3-S2.
Accordingly, the present invention provides for a method of treating pain in a subject, where the pain is determined to be associated with a dorsal root ganglion at a particular spinal cord level, comprising topically applying a PKG inhibitor comprising a transport peptide to skin lying within the dermatome corresponding to the spinal cord level associated with the pain.
A PKG inhibitor compound may be comprised in a cream, ointment, or transdermal device (see above), applied to the appropriate dermatome.
For example, a person suffering from lower back pain as a result of compression of the nerve exiting a bony foramen in the lower spine (lumbar, sacral, or lumbosacral radiculopathy) could be treated with a transdermal patch containing a PKG inhibitor compound (comprising a transport peptide) applied to the dermatome corresponding to the spinal cord level from which the compressed nerve originates, which may be identified by the person's symptoms and physical exam. As one specific example, because the radiculopathy often involves nerves that supply the L4, L5 and/or S1 dermatomes, a transdermal patch according to the invention may be applied to the appropriate region of the thigh or leg of the patient. As another specific non-limiting example, a person having arthritis involving the finger joints, dermatomes C6-C8, could wear a patch according to the invention on the upper arm or shoulder.
5.4 A PKG Model System
The present invention provides for assays that identify modulators (inhibitors or promoters/inducers) of PKG. Such assays may be used to evaluate a test agent in order to determine whether the test agent is an agent that modulates PKG and thereby modulates LTH. An inhibitor of PKG may be used to inhibit LTH and may be used to inhibit and/or treat (lessen, delay or prevent) persistent pain in a sensory neuron and/or a subject. A promoter/inducer of PKG may be used to develop a model system for persistent pain, preferably in an animal which, like Aplysia, is believed to not subjectively experience pain.
The assays of the invention utilize homology models of PKG built based on crystal structures of the ATP catalytic domain of PKA with balanol and a balanol analog (1BX6 and 1SVE, respectively). Putative modulators of PKG were then identified by docking 3-dimensional structures of commercially available drug-like small molecules to the foregoing PKG homology models.
Once a compound is identified as putatively binding to the PKG active site (“a putative modulator”), it may be tested for physiologic activity in a suitable model system. One non-limiting example of a suitable model system comprises a test sensory neuron (“TSN”) under physiological conditions that at least approximate the in vivo environment in which the sensory neuron exists in nature. The TSN comprises a cell body that contains the nucleus as well as an axonal segment, which constitutes at least a portion of the TSN's axon and more preferably constitutes the complete axon. In certain non-limiting embodiments, the TSN is an Aplysia SN. In other non-limiting embodiments, the TSN is a vertebrate SN, preferably a mammalian SN. The TSN may be maintained isolated in a culture, as part of a group of neurons that may or may not all be SNs, or as an explanted nerve or section thereof (e.g., an excised segment of rat sciatic nerve). In alternate embodiments, the TSN may be retained in an animal in vivo. In still further non-limiting embodiments, the axonal segment may contain at least one ligation.
To test the activity of the putative modulator in said model system, the TSN may be injured. For example, and not by way of limitation, the injury may be created by crushing, cutting and/or chemically injuring the TSN using methods known in the art. Other methods include inducing an inflammatory response, ischemia, a reduction of the blood supply to neurons, and hyperglycemia. The putative modulator may be administered to the TSN, either prior to, concurrent with, or following injury, either comprised in culture medium, systemically administered, locally injected, or directly injected or otherwise introduced into the TSN. In non-limiting embodiments, the putative modulator may be administered to a particular cellular location of the TSN, such as the cell body or the axon. Preferably, the effects of the putative modulator on the TSN are compared to comparable values in a control SN (“CSN”), such as an injured CSN. Preferably within 48 hours of injury, the assay of the present invention determines whether the putative modulator modulates protein kinase G (“PKG”) activity in an injured TSN, preferably relative to PKG activity in an injured CSN to which test agent has not been administered. An ability to inhibit PKG activity associated with SN injury indicates that the test agent is an LTH inhibitor. An ability to promote a further increase in PKG activity relative to control values indicates that the test agent is an LTH promoter. PKG activity may be measured, for example and not by way of limitation, by measuring the kinase activity in a SN extract. For example, the amount of PKG activity in a SN extract may be determined by measuring transfer of 32P from [32P]-ATP to BPDEtide (Calbiochem, La Jolla, Calif.). Further, electrophysiologic testing may be performed to determine whether the putative modulator modulates the development of LTH in the injured neuron, as compared to a control, injured neuron not exposed to putative modulator.
5.5 Linkers to Modify the Lead Compound
The most active known PKG ligands have a chemical structure involving a linker:

    
    
        (tail) Ar—C:O—Ar—C:O—X-linker-Y—Ar (head)

The linkers known to work well at PKG have had at their core a saturated alicyclic or aliphatic ring, such as azepane (in balanol), cyclopentane and pyrrolidine, connected in a trans-substituted arrangement via ester or amide to the tail and via an amide to the head (X and Y above). The main function of the linker is to provide the appropriate distance and angle between the head and tail, while remaining compatible with the size, shape, and electrostatic properties of the receptor in this region. If the linker is positively charged, it may make also make a salt bridge with anionic sidechains bordering the pocket. The goal in designing new linkers is to retain or improve the activity by retaining the right spatial and geometric characteristics, and to find new linkers that are convenient to use.

    
    


With numerous possibilities to investigate, one could specify rings with some similarities in terms of charge and size to the known rings, using medicinal chemistry and from looking at the binding mode of the known ligands at PKG. For consistency, and to limit the size of the search space, one tail end can be the focus, however, the combinatorics is easily expanded to different tails. Certain rings that can be tested include, but are not limited to, aminocyclopropane, aminopyridine, piperazine, diazepane, proline. Additional larger rings or cages with nitrogens (triazacyclononane, diazaadamantane), by way of example, can also be tested to determine if they fit. Each of these rings has several substitution points, which means there are many possible combinations, including stereochemistry. Rings could also be joined directly via a C—C bond, or via an amide —N—C:O— or —C:O—N— (See FIGS. 15A-L and Example 10 below).
In designing linkers, if the ring has a nitrogen, it may be synthetically convenient to link directly to that nitrogen via an amide, removing a stereocentre and making the system generally easier to handle. Simultaneously varying the head group from phenol to indazole is also of interest, since prior screens have turned up an active indazole, which is novel as a PKG inhibitor chemotype, and in many cases the tests showed superior scores when docked to the binding site.
Thus, having defined the space to investigate, the possibilities are enumerated into a 156x2 virtual combinatorial library (See FIG. 14). These are then converted into low energy three dimensional models using standard techniques (Monte Carlo conformational searching, MMFFs and OPLS2005 forcefields).
Previously, structures were prepared of the catalytic domain of PKA from the PDB: structures 1bx6, 1sve and 1rek, all of which have balanol or balanol analogues bound. Likewise, using comparative homology modelling in Schrodinger's Prime software, corresponding PKG(1bx6), PKG(1sve) and PKG(1rek) models were prepared. Scores from docking ligands to such models using Schrodinger's Glide XP docking-and-scoring software provides a positive correlation between score and experimental affinity. While the correlation is not perfect, this allows a large set of potential ligands to be prioritized for synthesis and testing. In particular, this is a good method for increasing the efficiency of resource usage by ruling out what not to make and test (e.g., prolines are not necessarily promising).
Each candidate in the virtual combinatorial library is docked with Schrodinger's Glide XP software four times (in four input conformations) to each of the above six receptors or receptor models. This provides thorough sampling of both the ligand and protein conformational flexibility, and a best score to be obtained. The raw scores are modified by a term that takes account of the ligand strain energy according to OPLS2005 forcefield. The purpose of adding the strain energy is to penalize linkers that need to fold into a high energy conformation or transfer that energy to the protein in order to bind, and thus would take a hit in terms of their affinity. The final result is prioritization of all of the structures in the library, in terms of their scores at PKG, their preference for PKG over PKA, and whether the phenol or indazole head performs better with that particular linker. This same method was carried with a smaller library to list priorities for swapping out the acid-labile methoxymethyl group on the tail of the selective lead compound Compound-6.
6. EXAMPLE
Modeling of PKG and Identification of Putative Modulator Compounds
6.1 Method A—Homology Model Generation and Ligand-Receptor Docking:
Two homology models of PKG were generated at Schrödinger Inc. (Portland, Oreg.; New York City, N.Y.) from structures of PKA co-crystallized with balanol and a balanol analog, 1bx6 and 1sve, using the protein structure prediction package Prime (version 1.5102) (http://www.schrodinger.com/; Jacobsen et al., 2004). Approximately 100,000 commercially available drug-like compounds were virtually screened against these homology models using increasingly accurate modes of the docking algorithm Glide (version 4.0108) (Friesner et al., 2004; Halgren et al., 2004). Compounds were selected for purchase and biological screening based on their resultant extra precision (XP) GlideScores and a brief visual inspection of the structures for chemical reasonableness.
6.2 Method B—Similarity Analysis and Docking:
Balanol was used as a probe to search a database of approximately 1.3 million commercially available compounds to identify similar compounds. Similarity was calculated using the atom-pair similarity measure described by Carhart et al. (1985) where atom pairs are defined in terms of the atomic environments of, and shortest path between, all pairs of atoms in the topological representation of the chemical structure. A similarity cutoff of 0.55 identified 4 compounds which were docked against the aforementioned PKG homology models using the XP mode of Glide (Friesner et al., 2004; Halgren et al., 2004). Two compounds were selected for purchase and biological screening based on their XP GlideScores and brief visual inspection.
6.3 Results
Using Method A above, two homology models of PKG were generated. Docking balanol to these structures produced docked poses very similar to that seen in the crystal structure of balanol bound to PKA [see FIG. 4A (1BX6) and FIG. 4B (balanol docked into PKG homology model based on 1BX6)]. FIGS. 5A-B and FIG. 7 highlight the residues that are different in PKG and PKA/PKB/PKC, and thus can be exploited in the identification and design of PKG selective modulators. For example, the residues that are different between PKG and PKA are: Gly370Ser (PKG amino acid/human PKG Type I alpha residue number/PKA amino acid), Ile406Thr, Val501Thr, Cys441Val, Ala440Tyr, Ile491Leu. A number of compounds were identified by docking as being predicted to be modulators of PKG, the structures of which are depicted in FIGS. 8C-L. These molecules are referred to herein as compounds 8C-8L, respectively. FIGS. 9A-K depicts the docked poses of various compounds and PKG, including balanol (FIG. 9A) and compounds 8C-8L (FIGS. 9B-K).
Further, using Method B, compounds 8A-8B were identified by atom-pair similarity followed by docking to the homology model of PKG based on 1BX6 (FIGS. 8A-B). FIG. 6 shows a series of cyclopentane analogs of balanol.
7. EXAMPLE
Selection of Compound 6 as a PKG Inhibitor Drug Candidate
7.1 Methods.
Two building blocks were required to make Compound 6: Benzofenone acid and the linkage with para-hydroxy benzene ring. Each block required 5 steps to make them respectively as discussed below. In total, there was a convergence of 13 steps to make Compound 6. See FIG. 19.
Acetic acid 3-fluoro-4-methoxy phenyl ester
A mixture of 25 g (0.15 mole) of 3-fluoro-4-methoxyacetophenone(1) and 40 g (0.2 mole) of 85% 3-chloropheroxybenzoic acid in 350 ml of methylene chloride was refluxed for 48 h, cooled and washed with 5% potassium carbonate solution (200 ml) three times. The organic phase was dried with MgSO4 and the solvent was evaporated. The product (24 g, 87.0%) was used next step without purification.
3-Fluoro-4-methoxyphenol
20 g of 2 (0.11 mole) obtained above was dissolved in 200 ml of ethanol and 100 ml of 20% of NaOH was added slowly. After addition, the reaction was stirred at room temperature for 3 h. The aqueous solution was washed with ether and acidified with 6N HCl. The oil which separated was extracted into ether and the extracts dried with MgSO4. Removal of the solvent left a solid residue which was recrystallized from hexane to give 13 g product (83.3%)
2-Fluoro-1-methoxy-4-methoxymethoxy-benzene
A mixture of 3.0 g (21 mmole) of 3 and 7 ml of N,N-diisopropylethylamine (73 mmole) was dissolved in 100 ml of methylene chloride. To this solution cooled with ice-water bath, 0.3 ml of Chloromethyl methyl ether (39.5 mmole) was added dropwise. After addition, the reaction was stirred at room temperature for 3 h. After removing solvent, the residue was purified by chromatography on silica gel (elute with methylene chloride) to give 3.9 g product as light yellow oil (100%).
4-[(2-Fluoro-3-methoxy-6-methoxymethoxy-phenyl)-hydroxy-methyl]-benzoic acid methyl ester
10 ml of 1.6M of n-BuLi in THF (16 mmole) was added to 2.5 g of 4 (13.4 mmole) in 50 ml of dry THF at −78° C. The solution was stirred at the same temperature for 40 minutes. To this solution, a solution of 2.2 g of 4-Formyl-benzoic acid methyl ester (13.4 mmole) in 50 ml of dry THF was added by canal slowly (internal temperature was kept under −65° C.). The reaction was stirred at −65° C. for 6 h and the reaction was allowed to warm up to room temperature in 15 h. The reaction was quenched with water and extracted with ethyl actate (100 ml) three times. Combined organics was dried with MgSO4 and after removing solvent, the residue was purified by chromatography on silica gel (elutes:ethyl actate:hexane=4:6) to give 3.1 g of product as yellow oil. (66.0%)
4-(2-Fluoro-6-hydroxymethoxy-3-methoxy-benzoyl)-benzoic acid methyl ester
2.0 g of 5 (5.7 mmole) was dissolved in 50 ml of methylene chloride and to this solution, 15 g of activated MnO2 was added in portions. The reaction was stirred at room temperature overnight. The solution was filtered through a celite pat and after removing the solvent, 1.7 g of product was obtained as yellow oil (86%).
4-(2-Fluoro-3-methoxy-6-methoxymethoxy-benzoyl)-benzoic acid
1.5 g of 6 (4.3 mmole) was dissolved in 15 ml of methanol and water was added dropwise until the solution became cloudy. 1.0 g of LiOH (2.4 mmole) was added slowly and the reaction was stirred at room temperature overnight. The reaction was quenched with 7.0 g of citric acid. After removing most of methanol, the aqueous solution was extracted with methylene chloride (30 ml) three times and combined organics was washed with brine twice, dried (MgSO4). 1.4 g of product was obtained as while solid after removal of the solvent (98.0%).
7.2 Results and Discussion
The following features and characteristics are predictions of Compound 6 based on data of similarly situated drug-like and nondrug-like molecules, based on QikProp software (by William L. Jorgensen, available at http://www.schrodinger.com/ProductDescription.php? mID=6&sID=10). The following predictions suggest that Compound 6 would be a good candidate for a drug compound.

    
    
        (1) The molecular weight of Compound 6 is 536. Additional variants at about this molecular weight value or lower would be desirable. The number of properties that fall outside the range of 95% of similar values for known drugs is zero.
        (2) The compound is predicted to have 2 metabolites, which would be favorable for plasma and gut stability.
        (3) The oral bioavailability is predicted to be about 76%. Log MDCK and log CACO-2 cell permeability values are predicted to be within acceptable limits.
        (4) The predicted log BB for crossing the blood brain barrier is −1.3. This value is on the lower end for drugs in general.
        (5) The predicted log aqueous solubility is estimated between −3 to −5.
        (6) The predicted log P is estimated at about 2.4.
        (7) The predicted log IC50 for HERG K+ channel blockage is estimated at about −6.5 micromolar, which would fall within the average value for known drugs.
        (8) As opposed to fluoro derivative compounds, Compound 6 polyphenols would be expected to be less drug-like.
    
    


8 EXAMPLE
Compounds 8H and 8J Significantly Inhibited Pkg Activity In Vitro
Compounds identified by in silico screening, including compounds of FIG. 8, were tested to determine whether they inhibit active recombinant PKG in an assay that measured the transfer of gamma-labeled 32P from radiolabeled ATP to a peptide substrate (RKISASEFDRPLR, SEQ ID NO: 10) in the absence or presence of compound. Compounds 8H (NOP479435) and 8J (NOP952668) were found to exhibit significant inhibitory activity at these concentrations (see FIGS. 10A-B), whereas other compounds tested did not show, under the assay conditions, significant inhibition at these concentrations. The results shown in FIGS. 10A-B were calculated from the average values of two independent experiments, and show the percentage PKG activity in the presence of the putative inhibitors relative to the activity in the absence of inhibitor. Of the compounds tested, 10 μM compound 8H (NOP479435) inhibited almost 100 percent of PKG activity. This activity is comparable to the inhibition of PKG by Rp-8-pCPT-cGMPS, which is the most widely used commercially available non-peptide inhibitor. Compound 8J (NOP952668) showed weaker inhibition.
The ability of compound 8H (NOP479435) to decrease PKG 1α activity was assessed by assaying PKG activity towards BPDEtide in the absence or presence of various concentrations of the compound. FIG. 11 shows the average values of duplicate experiments, plotted as the percentage of PKG activity in the absence of inhibitor. The IC50 value calculated for compound 8H is 2 μM at 30 μM ATP. FIG. 7A shows compound 8H (NOP47935) docked to PKG; FIG. 7B shows balanol in the corresponding site, illustrating the sequence differences between PKGα and PKA/PKB/PKC.
9. EXAMPLE
Comparison of Selectivity of Compound Derivatives
Compounds including compound 6, were tested to determine whether they selectively inhibit PKG according to the methods described above.
Compounds 1 and 2, as known in the art (Lai et al. 1997), at high concentrations (5-10 times higher than comparative tests described below) demonstrate the ability to inhibit PKC and PKA. These compounds, along with compounds 6 and 7 (diagrammed below) were compared for their ability to inhibit PKG and PKA. See Table 1.









TABLE 1






PKG




activity



Compound
(IC50)
PKA activity (IC50)


















1   



7-8
nM
30 nM (compound 24, Lai et al. 1997)


 


2   



7
nM
4-33 nM (compound 22, Lai et al. 1997)


 


6   



40
nM
950 nM


 


7   



40
nM
38 nM









The data clearly show that Compound 6 was more selective than compound 7 and compounds 1 and 2 with respect to PKG. Compound 6 had an IC50 value that was about 20 times higher to inhibit PKA, whereas compound 7 had approximately the same IC50. Compounds 1 and 2 only exhibit a 3 fold difference.

Additional compounds (Series A and Series B compounds, including compounds 6 and 7 discussed above) were tested and their inhibitory activity measured. The structures of Series A and B compounds are summarized in FIGS. 20A and 20B. The inhibitory activity results of these compound is provided below in Table 2.







TABLE 2







SERIES A


Compounds (at 10 μM) screened for ability to inhibit PKG at 30 μM ATP








Identity Number
Activity





1
ca. 37% inhibition


2
no inhibition


3
no inhibition


4
ca. 10% inhibition


5
no inhibition


6
ca. 30% inhibition


7
no inhibition


8
no inhibition


9
no inhibition


10
no inhibition


11
no inhibition


12
no inhibition


13
no inhibition


14
no inhibition










SERIES B


Balanol Derivatives; Screened as decribed at 30 μM ATP








# - Former Number
Activity





 1 - (NOP0317106)
IC50 = 7-8 nM (from literature: 30 nM PKA)


 2 - (NOP0403206)
IC50 = 7 nM (from literature: 4-33 for PKC



isoforms; 70 for PKA)


 3 - (NOP-0403306)
2 isomers - pM-μM


 4 - (NOP-0413406)
IC50 = 8 μM


 5 - (NOP-0413506)
IC50 = >10 μM


 6 - (NOP-0414606)
IC50 = 40 nM vrs PKG, 950 nM vrs PKA


 7 - (NOP-0414706)
IC50 = 40 nM vrs PKG, 38 nM vrs PKA


 8 - (NOP-4/14806)
IC50 = >10 μM


 9 -
no inhibition


10 -
≧5 μM


11 -
no inhibition


12 -
no inhibition


13 -
no inhibition


14 -
≧10 μM


15 -


16 -


17 -









The results of the screening tests demonstrate how changing substituents affect the inhibitory action of the compounds. The conclusions are summarized below.
(1) The substitution of the 6-membered ring (B) in balanol for a 5-membered ring (compound 2) reduces inhibition of PKG 5-fold, but that to other kinases by much more.
(2) Changing the 5-membered ring in compound 1 to a ring containing N, in compound 2, further reduces inhibition of PKA relative PKG.
(3) In Table 1, Compound 7 as compared to compound 2, where the F in the D ring is substituted for HOOC at 14′′ and the addition of H3CO at 13′′ and removal of both OH groups from ring C reduces inhibition of PKG and reduces selectivity. However this change is not due to changes in ring D but likely due to removal of the OH moieties.

(4) In Table 1, Compound 6 demonstrates superior inhibition of PKG relative to PKA. This may be attributed to the N group in the ring (as compared with compound 4) and the presence of the —O—O— (as compared with compound 5).

These results showed dramatic modifications of linkage between rings C and D destroyed activity (as evidenced by series A). Altering ring A eliminated (e.g., derivative 13) or markedly reduced (e.g., derivative 14) inhibition. Modifying the linkage between rings A and B generally (derivatives 11 and 12), but not always (derivative 10) eliminated inhibition. Lastly, separate isomers of compound 3 were identified.
Derivative compound 6 selective inhibition showed promising results. Additional modifications would include:
a) adding OH groups at positions 4′′ and 6′′ in ring C to lower the IC50 toward PKG back to the 7-8 nM range;
b) altering substituents at the 10′′ position in ring D; and
c) creating an amide linkage between rings B and C for stability.
10. EXAMPLE
Determining Linkages in PKG Inhibitor Compound 6
The design of alternative linkers to the established azepane, cyclopentane, and pyrrolidine rings for connecting the diarylketone of balanol analogues to the hinge-binding motif was studied in Compound 6 (Table 1). Compound 6 was analyzed for alternative linkers by looking at the structures and their relative rankings (1 to >157) at PKG and PKA with both phenol and indazole as the hinge-binding group (See FIGS. 14 and 15A-15L). In FIG. 14, lower numbers indicate better predicted binding at PKG and PKA, or more preference for PKG over PKA (PKG vs. PKA rank). However, neither the range of relative scores nor the receptor structure suggests large differences in selectivity can be obtained by varying the linker.
The data demonstrate that indazole as a hinge-binding motif continues to score better on average than phenol by a couple of kcal/mol (50-fold) for equivalent structures, although there's a range because the position of the linker with respect to charged residues E127 and D184 is sensitive to the size of the aromatic. Lower numbers in the final column indicate preference for indazole and higher numbers a preference for phenol.
It is apparent that the pyrrolidine linker of Compound 6 is already highly optimized for the receptor, and only a minority of the linkers tested score as well or better than this structure. Though the main role of a linker is to put the rest of the pharmacophore elements at the right distance and in the right orientation, optionally making a salt bridge, doing so in an ideal manner has been difficult.
The focus of this study was on amide, rather than ester, containing linkers, due to the known metabolism issue. The amide analogue of Compound 6 (and its indazole analogue) bind and scored similarly to the esters.
2-substituted piperazine derivatives (Z1-26) were also analyzed in this study. Specifically, Z24, Z14 and Z6 with phenol and Z14, Z15 and Z5 with indazole were found to be competitive with Compound 6, which would make them promising candidates.
Some derivatives of aminocyclopropane (C1-C4) scored fairly strongly and are comparable to Compound 6. Prolines did not appear to be worth pursuing at this stage. The indazolyl L-proline Compound 13 is in the set, P10, and its score is mediocre. The other proline isomers reviewed were generally poor at PKG (though one D-proline P12 with indazole received a high score at PKA, as did urea P24 with phenol). The best scoring proline at PKG was the indazolyl P16, a positional isomer/analogue of Compound 11.
Some of the 1,4-diazepane derivatives (D1-62, 7-membered ring with two nitrogens) scored very well with indazoles (D34, D59, D24, D12, D39, D7) or phenols (D59, D22, D46, D27, D6) but they appear harder to make than the piperazines.
Additionally, some aminopyridinium cations (A1-32) scored reasonably well, though the pKis of A1-4 and A7 are likely to be too low for the ring to be protonated. A8 with indazole and A12, A7 and A21 with phenol performed best. A variety of rings containing two nitrogens where both were functionalized as amides (X1-4, D21, D62, Z9, Z26) are predicted to be inactive.
The method used was ensemble docking with XP4.0 using four input conformations, to three PKA structures and three PKG homology models from pdb codes 1bx6, 1sve & 1rek. The final rankings were based on the best strain-corrected glidescore, treating indazoles and phenols, PKG and PKA separately.
11. EXAMPLE
Understanding SARs of Balanol Analogues
The present Example provides a description of the initial understanding the SARs of balanol analogues aimed at determining whether the azepine portion of balanol can be replaced by simpler and more readily accessible cyclic arrays such as pyrrolidine and cyclopentane without significant loss of PKG inhibitory activity. The results of these efforts are shown in Table 2.
Table 3 shows the PKG and PKA inhibition by balanol and its analogs with simple azepine replacement (IC50 in μM).















TABLE 3







Compd
balanol
B-1
B-2
B-10
B-29
B-31









PKG
0.0016
0.007
0.007
>5
>10
>10



PKA
0.0039
0.03 
0.07 






































The conformational flexible seven-membered azepine ring appeared to be replaceable, as can be seen from Table 3 in which Compound B-2 with a five-membered pyrrolidine ring was found to be almost as potent as Balanol itself against PKG. In contrast to the azepine nitrogen atom in Balanol, which has been shown to be significant for its activities, the pyrrolidine nitrogen atom seemed dispensable, at least for PKG, since Compound B-1 with a cyclopentane ring that is otherwise identical to Compound B-2 was equally active. Consistent with the report that stereochemistry around azepine or its replacements is critical to bioactivities of Balanoids, all three analogues (Compounds B-10, B-29, B-31) with favorable ring sizes, but without a trans-vicinal amino alcohol substructure, lost activities. This substructure has been shown in computational modeling to guarantee a favorable spatial projection of the aromatic side chains R1 and R2.
In summary, the SARs studies of the perhydroazepine moiety of balanol indicate that the azepine ring can be replaced by five-membered rings as long as the replacement is able to raise the two aromatic side chains in a stereochemically correct manner. The two five-membered ring analogs 1 and 2 are attractive not only for their impressive potency but also for their ease of preparation. For example, by using epoxide-opening reactions (FIG. 17C), it took only three steps to reach the required pyrrolidine ring from commercially available materials. This compared very favorably to a seven-step synthesis of the corresponding azepine amino alcohol. As the five-membered ring system was much more easily accessible, they were generally used in preference to the azepane in later synthetic work.
Although recent work has provided direct evidence that balanol retained activities in cellular assays, it is expected that some attenuation of the polar nature of the benzophenone moiety will be highly desirable to obtain compounds with the overall physical properties suitable for continued pharmaceutical development. More importantly, Koide et al. demonstrated that removing certain benzophenone functionality could lead to a marked differentiation in protein kinase selectivity. Considering its tedious chemical synthesis, simplification of benzophenone portion is also of practical interest. Recently, Breitenlechner et al. reported potent balanol-like PKB inhibitors that bear a simplified benzophenone subunit with two hydroxyl groups removed from its internal benzene ring and the carboxylic acid functionality on the external benzene ring replaced by fluorine.
Table 4 shows PKG and PKA inhibition by balanol analogs with simplified benzophenone subunits (IC50 in μM).









TABLE 4





Comp
PKG
PKA







B-4 
   8



B-5 
>10



B-6 
    0.04
     0.95


B-7 
    0.04
     0.04


B-11
>10



B-12
>10



B-21
   0.2
>10


B-24
>10



B-25
>10



B-28
>10



B-32
>10



B-33
>10



B-40
   0.8



B-43
>10



B-44
     0.0025
     0.0031


B-45
   0.5
    3.7








B-6, R′ = CH3OCH2—


B-7, R′ = H


B-21, R′ = CH3(CH2)2—


B-40, R′ = CH3O(CH2)2—





B-43, R′ = CH3(CH2)2—, X = Boc


B-44, R′ = H, X = H


B-45, R′ = CH3(CH2)2—, X = H





B-32, R′ = CH3OCH2—


B-33, R′ = H





B-4, R′ = CH3OCH2—


B-5, R′ = H





B-11, R′ = CH3OCH2—


B-12, R′ = H





B-25, R′ = CH3OCH2—


B-28, R′ = H


















Compared with balanol, this fluorinated benzophenone system is less polar and more synthetically straightforward, and therefore, is a good starting point for modification. Further encouraged by computational studies that it scores as well as balanol itself against PKG, a series of balanol analogues were made based on this benzophenone motif. As can be seen in the activity of Compound B-7, new benzophenone only led to a slight reduction of potency. Intriguingly, Compound B-6, the precursor of Compound B-7 with the MOM-protected hydroxyl group, retained PKG inhibitory activity but lost a substantial portion of its potency on PKA, and then, provided the first selective PKG inhibitor in this investigation.
According to the X-ray crystallography studies from Breitenlechner et al, the free hydroxyl group of the benzophenone formed a COO . . . HO hydrogen bond with the side chain of Glu91 in PKA. In addition, a variety of residues make van der Waals contacts with the benzophenone moiety. It is reasoned that the MOM side chain in Compound B-7 increases van der Waals contact that is enough to offset the loss of a hydrogen bond in its binding to PKG but not enough for PKA. Computational modeling on this particular site indicates that hydrophobic side chains will be favorable for selectivity. Therefore, the present invention sought to replace the MOM side chain with propane, which, as in Compound B-21, resulted in a dramatic increase of specificity for PKG over PKA coupled with an acceptable reduction of potency. As shown by Compound B-40, increasing the size of side chains will further decrease compounds' potency. These results reinforced the notion that despite the high homology in the catalytic domains of AGC-family kinases, there is enough difference to allow for the development of potent and selective inhibitors acting in this region
One compound with a planar benzophenone, Compound B-24, was prepared to see if a more rigid conformation could lead to active compounds. This modification was found to be essentially devoid of activity.
Interestingly, with the new benzophenone ring, the pyrrolidine nitrogen now becomes critical, as in Compound B-5 and Compound B-43, deleting N atom or masking with a protecting group resulted in a totally lost of activity. The published structure of a balanol-PKA complex revealed that the benzophenone fragment of balanol could correspond to the triphosphate region of ATP and interacted extensively with the kinase glycine-rich loop, also called the triphosphate subsite. In the complex, the azepane ring occupied the catalytic loop or the ribose subsite in which the azepane N formed hydrogen bonds with the carbonyl oxygen atom of residue Glu170. It is speculated that the polar and nonpolar interactions generated by the new benzophenone moiety couldn't make up for the loss of hydrogen bonds involving the pyrrolidine nitrogen atom. Since the previous SAR results are not completely applicable to the new benzophenone system, the modification of the azepine part was further investigated. All analogues with benzophenone attached to rings other than pyrrilidine showed poor activities and may well be another example of significance of positioning benzophenone properly regardless its modification.
The presence of an ester functionality has prompted concern over the metabolic stability of the compound. There is evidence suggesting that replacing labile esters with amide linkage could increase compounds' plasma stability. Unfortunately, this replacement often resulted in substantial loss of potency in the case of PKC studies. To investigate its effect on PKG inhibitory activity, diamide compounds, Compounds B-44 and B-45 were prepared. Two compounds are even slightly more potent than their ester counterparts. It should be noted in considering these data that diamide compounds were made in optically pure form, with the same (1R,2R) configuration as is found in naturally occurring balanol. The natural balanol has been shown to be more potent than its unnatural enantiomer. Nevertheless, we were able to achieve plasma stability without losing potent inhibition.
A key site on the benzophenone part was identified through which hydrophobic chains regulating compounds' affinity for PKG over other kinases can be introduced. However, substantial loss of activity was observed in most cases as a compensation for achieving sufficient selectivity. Since there was a need to improve potency through other part of balanol molecules, attention was given to the modification of the 4-hydroxybenzamido moiety.
Table 5 shows PKG and PKA inhibition by balanol analogs with the replacements of hydroxybenzamido moiety (IC50 in μM).









TABLE 5





Comp
PKG
PKA







B-13
>10



B-14
>10



B-22
>10



B-23
>10



B-26
>10



B-30
    0.085



B-35
>10



B-46
    0.007
5  


B-53
     0.0025
1.2


B-54
>10








B-22, R′ = CH3(CH2)2—


B-23, R′ = CH3OCH2—


B-26, R′ = H





B-30, X = O, R′ = CH3OCH2—, R′′ = H


B-35, X = NH, R′ = CH3OCH2—, R′′ = 4-F—Ph


B-46, X = NH, R′ = CH3(CH2)2—, R′′ = H


B-53, X = NH, R′ = CH3(CH2)2—, R′′ = Me





B-13, R′ = CH3OCH2—


B-14, R′ = OH





B-54, R′ = CH3(CH2)2—












As indicated by available crystal structures of ATP-PKA and balanol-PKA complexes, a planar moiety occupies the adenine subsite and PKA residues whose atoms participate in hydrogen bonds to balanol are the same as those that interact with ATP. Specifically, the carbonyl oxygen atom of Glu121 and the backbone nitrogen atom of Val123 form hydrogen bonds in both balanol-PKA and ATP-PKA complexes. In balanol-PKA complex, the single hydroxyl group of balanol's 4-hydroxybenzamido moiety serves as both H-bond donor and acceptor, donating and accepting electrons to form hydrogen bonds with Glu121 and Val123, respectively; in the ATP-PKA complex, the purine ring N1 atom donates electrons to the Val123 amide hydrogen atom while the purine ring N6 atom accepts electrons from the Glu121 backbone carboyl oxygen atom. The H-bond to Val123 or homologue is thought to be nearly universal among protein kinase inhibitor complexes and is apparently critical for tight binding inhibitors. Replacing 4-hydroxybenzamido moiety with pyridine has been shown to be successful for potent PKB inhibitors and as revealed by following X-ray studies, pyridine could form a N . . . OCNH hydrogen bond to Val123 through its N atom. As shown by Compounds B-22, B-23, and B-26, this replacement was totally unsuccessful for PKG.
Comparing with 4-hydroxybenzamido moiety of balanol or adenine moiety of ATP, pyridine lacks a H-bond donor, which may diminish its affinity to PKG. Aromatic heterocycles as adenine mimics, containing both H-bond donor and acceptor, frequently appear in kinase inhibitors and may be a good replacement for 4-hydroxybenzamido moiety. Suggested by computational modeling, 4-hydroxybenzamido moiety was replaced with indazole. As can be seen in the activity of Compound B-30, this modification did not lead to an improvement although retained comparable potency against PKG. Surprisingly, indazole replacement, as in Compound B-45, dramatically improved activities of diamide compounds. Stable diamide compounds are more desirable in terms of stability in vivo. Contradicting to computational prediction, adding hydrophobic groups on 3 position of indazole increased neither activity nor selectivity, as shown by balanol-50 and 55 in Table 4.
F is often used as a substitute for hydroxyl group in medicinal chemistry, however, replacing hydroxyl group with F, as in Compound B-54, resulted in loss of activity. In the screening of unrelated compounds including byproducts and intermediates generated during the synthesis, it was found that symmetrical diamide Compound B-48 showed moderate PKG inhibitory activity. This is surprising in light of previous SARs indicating that the benzophenone scaffold is crucial to bioactivities. Further modification yielded the most potent Compound B-50 in this series with an IC50 of 70 nm. This simple molecule showed no selectivity over PKA. Since high molecular weight is a concern, these simple motifs may provide a new ground for further medicinal chemistry efforts.
Table 6 shows PKG and PKA inhibition by balanol analogs with new scaffolds (IC50 in μm).














TABLE 6







Comp
B-48
B-49
B-50
B-51
B-52









PKG
~0.2
~1
0.07
>10
>10



PKA


0.07










B-48, R = R1



B-49, R = R2(X = H)



B-51, R = R2(X = 4-F—Ph)



B-52, R = R2(X = Me)



















J. M. Defauw et al. reported a class of acyclic balanol analogs that are highly potent and selective for PKC. As shown in FIG. 18, a series of compounds with a flexible ethylenediamine bridge was prepared in hope of generating new scaffolds for PKG. These compounds are generally inactive with an exception of Compounds B-41 and B-42, which showed moderate potency (data not shown).
12. EXAMPLE
Testing Compounds 6 and 46
Compounds 6 and 46 exhibited superior inhibitory activity toward PKG and selectivity toward PKA. The structures for compounds 6 and 46 are provided in FIG. 21. The IC50 of compound 6 with PKG and PKA is 40 nM and 950 nM, respectively; and the IC50 of compound 46 with PKG and PKA is 7.5 nM and 2 μM, respectively.
Both compounds were further tested for their efficacy in reducing complete Freund's adjuvant (CFA)-induced thermal hyperalgesia in the hindpaws of rats. The hindpaws of rats were first tested for their response to a thermal probe as a baseline reference. Then the right hindpaw was injected with CFA (100 μl). The injected site developed edema and redness, indicating an inflammatory reaction, within 12 hours. 24 hours later, the latency of both hindpaws withdraw to a thermal stimulus was determined, the injected paw of all of the rats showed a significantly more rapid withdrawal time in response to the thermal stimulus relative to the contralateral uninjected side. The rats were then given either compound 6 or 46 at different times after CFA injection via different routes as shown in the tables below. Each hindpaw was then tested for its response to a thermal stimulus on successive days. Tables 7 and 8 provide a summary of the data.











TABLE 7







Compound




Compound
Number
administration




6
of
after

Effec-


Conc.
animals(a)
CFA injection
Route
tiveness*







  25 μM**
1
24 hours
Intrathecal
>90% 6 days





pump
later


0.25 μM**
1
72 hours
Intrathecal
>50%





pump
recovery






10 days


82.5 μM**
2
24 hours
Intrathecal
No effect





single injection


 3.3 nmol
2
60 hours
Subcutaneous
>90%





pump
recovery






5 days later




















TABLE 8







Compound




Compound
Number
administration


46
of
after

Effec-


Conc.
animals(a)
CFA injection
Route
tiveness*







0.25 mM**
2
24 hours
Intrathecal
No effect





single injection


 0.5 nmol
2
24 hours
Intra-peritoneal
No effect





single injection


  4 nmol
2
24 hours
Subcutaneous
No effect





pump





(a)Equal number of control animals


*Effectiveness compares the differential latency (CFA injected paw-contralateral paw) between the control animals that received CFA injection and comparable vehicle that the compound was dissolved in.


**Final concentration is calculated based on the assumption that there is 400 μl of cerebral spinal fluid in the rat. (Neurotherapeutics: Emerging Strategies By Linda M. Pullan, Jitendra Patel).






The single dose injection of compound 6 failed to reduce thermal hyperalgesia; this may be due a potential instability in the structure. These preliminary results suggest that compound 6 exhibits analgesic effects comparable to that achieved with the most potent commercially available inhibitor of PKG (RP-G: Rp-8-pCPT-cGMPS). Animals exposed to compound 6 did not exhibit any adverse behavioral effects with regard to eating, sleeping, defecating, micturation, balance, exploring, or socialization. Modification of compound 6 to increase affinity and selectivity, and perhaps plasma half-life, as described above, will be important. In contrast, compound 46, at the doses tested, did not exhibit any analgesic effects and failed to alleviate CFA induced thermal hyperalgesia within the testing dosages and with different modes of delivery (intrathecally or subcutaneously, e.g., via single dose injection and by osmotic pump for a continuos delivery). Two rats that received intrathecal compound 46 exhibited signs of motor dysfunction after injection. For example, the animals had difficulty in changing from the supine to prone positions. They also exhibited ataxia. The effects lasted for at least 24 hours and disappeared after 3 days.
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Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.

## Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of a compound represented by the following structure:



where Z represents —NH;
X represents




Y represents




R17 is phenyl, unsubstituted or substituted with one or more of hydroxyl, (C1-C4)alkoxy, alkoxylalkoxy, or halo;
R18 together with R19 is





and
R20 is

2. A method of treating chronic pain in a subject, comprising administering, to the subject, a pharmaceutically effective amount of a compound represented by the following structure:



where Z represents —NH;
X represents




Y represents




R17 is phenyl, unsubstituted or substituted with one or more of hydroxyl, (C1-C4)alkoxy, alkoxylalkoxy, or halo;
R18 together with R19 is





and
R20 is