Positive allosteric modulators and silent allosteric modulators of the Mu opioid receptor

Abstract

Disclosed are positive allosteric modulators (PAMs) and silent allosteric modulators (SAMs) for mu (μ)-opioid receptors that may be useful for the treatment of pain, either alone or in combination with orthosteric opioid receptor agonists. Methods for treating pain and modulating mu (μ)-opioid receptors are also disclosed.

Claims

1. A method of treating pain in a patient in need thereof comprising administering to the patient a compound which is a positive allosteric modulator for the mu-opioid receptor wherein the compound is selected from the group consisting of ##STR00002##

2. The method of claim 1, further comprising administering an orthosteric agonist for the mu-opioid receptor.

3. A method of modulating the mu-opioid receptor comprising contacting the receptor with a compound that is effective to provide an increase in the receptor function in the presence of orthosteric exogenous or endogenous agonist wherein the compound is selected from the group consisting of ##STR00003##

4. The method of claim 3 wherein the increase in receptor function is observed in maximal effect, potency, or both.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1

(2) Chemical structure and effect of mu-PAMs on β-arrestin recruitment. Chemical structures for two apparent mu-opioid receptor selective positive allosteric modulators (mu-PAMs), designated as BMS-986121 (A) and BMS-986122 (B), were identified from a high-throughput β-arrestin recruitment screen. BMS-986121 (A) and BMS-986122 (B) were assayed at varying concentrations in agonist detection mode (with compound alone) and in PAM detection mode (compound in the presence of a low concentration (.sup.˜EC.sub.10) of orthosteric agonist). For mu-opioid receptor expressing cells (U2OS-OPRM1) endomorphin-I (20 nM) was the orthosteric agonist used, and for delta-opioid receptor expressing cells (U2OS-OPRD1) leu-enkephalin (0.4 nM) was the orthosteric agonist used. Data are represented as mean ±s.e.m. of three experiments. In agonist detection mode, 0 and 100% activity represent basal activity and an E.sub.max of endomorphin-I (in U2OS-OPRM1, a 6-fold signal) or leu-enkephalin (in U2OS-OPRD1, a 4-fold signal), respectively. In PAM detection mode, 0% activity is normalized to the low concentration (.sup.˜EC.sub.10) of orthosteric agonist (endomorphin-I in U2OS-OPRM1 cells, and leu-enkephalin in U2OS-OPRD1 cells). 100% activity represents the response to an E.sub.max concentration of these respective agonists.

(3) FIG. 2

(4) Effect of mu-PAMs BMS-986121 and BMS-986122 on endomorphin-I stimulated β-arrestin recruitment in U2OS-OPRM1 cells. Both BMS-986121 (A) and BMS-986122 (B), produced concentration-dependent leftward shifts in the β-arrestin recruitment response to the agonist endomorphin-I. Calculated EC.sub.50 values (nM) for endomorphin-I at each concentration of compound are shown in each figure legend. The fold leftward shift in EC.sub.50 values for endomorphin-I in the presence of increasing concentrations of PAM compound is presented (C). Data are represented as mean+/−s.e.m. of 4 experiments.

(5) FIG. 3

(6) Effect of mu-PAMs on inhibition of forskolin-stimulated cAMP accumulation in CHO-mu cells. Both BMS-986121 (A) and BMS-986122 (B) increased the effect of a low (.sup.˜EC.sub.10; 30 pM) concentration of endomorphin-I (PAM detection mode) in a concentration-dependent manner. However, both compounds also showed some agonist activity above basal activity when added alone (agonist detection mode). For agonist detection mode, 0% activity represents vehicle (basal) activity. For PAM detection mode, 0% is normalized to the response to a .sup.˜EC.sub.10 (30 pM) concentration of endomorphin-I. The 100% response represents the response to an Emax. concentration of endomorphin-I (10 nM) in both agonist and PAM detection modes. Data are represented as mean±s.e.m. of three experiments.

(7) FIG. 4

(8) Effect of mu-PAMs on mu-opioid agonist stimulated [.sup.35S]GTPγS binding in membranes from C6mu cells and mouse brain, and DAMGO binding affinity in C6mu cell membranes. [.sup.35S]GTPγS binding in C6mu membranes was determined as described in the Methods and Materials. The EC.sub.50 of DAMGO to stimulate [.sup.35S]GTPγS binding was shifted to the left 4-fold in the presence of 10 uM of the mu-PAM BMS-986121 (A). BMS-986122 increased the DAMGO potency 7-fold (B). The maximal stimulation by DAMGO was not affected by BMS-986122 or BMS-986121. The mu-PAMs did not significantly affect basal values (vehicle control basal =3.2 ±0.2 fmol bound/mg protein). The EC.sub.50 of morphine to stimulate [.sup.35S]GTPγS binding was shifted to the left 2.5-fold in the presence of 10 uM BMS-986121 (C). BMS-986122 (10 uM) increased the morphine potency 3-fold (D). The maximal effect of morphine compared to DAMGO was increased by BMS-986121 (C) and BMS-986122 (D). BMS-986122 (10 uM) produced a 6-fold leftward shift in DAMGO affinity in DAMGO competition binding studies with [.sup.3H]diprenorphine (E), but had no effect on [.sup.3H]diprenorphine binding affinity (FIG. 7, Table 6). The EC.sub.50 of DAMGO to stimulate [.sup.35S]GTPγS binding in membranes from mouse brain was shifted to the left 4.5-fold in the presence of 10 uM BMS-986122 (F). Basal [.sup.35S]GTPγS binding (4.8±0.4 fmol bound/mg protein) was not affected by 10 uM BMS-986122. Shown are the combined mean ±s.e.m. data from 3-7 separate assays, each performed in duplicate.

(9) FIG. 5

(10) Characterization of functional SAMs in the β-arrestin recruitment assay and in DAMGO-mediated [.sup.35S]GTPγS binding. (A) BMS-986123 and BMS-986124 inhibited PAM responses to a .sup.˜EC.sub.80 concentration of BMS-986122 (12.5 uM) plus a .sup.˜EC.sub.20 concentration of endomorphin-I (30 nM) (SAM detection mode) in the β-arrestin recruitment assay in U2OS-OPRM1 cells. 100% activity represents the activity of the combined BMS-986122 plus endomorphin-I, and 0% activity represents the activity of the .sup.˜EC.sub.20 concentration of endomorphin-I alone. Graphs show the mean ±s.e.m. of three experiments. BMS-986123 and BMS-986124 showed no activity in agonist or PAM detection modes in either U2OS-OPRM1 cells or U2OS-OPRD1 cells (FIG. 8). Similarly, these two compounds showed no NAM/antagonist activity (in the presence of a .sup.˜EC.sub.80 (300 nM) concentration of endomorphin-I) in U2OS-OPRM1 cells (FIG. 9). (B) DAMGO potency to stimulate [.sup.35S]GTPγS binding in C6mu membranes was increased 8-fold in the presence of the mu-PAM BMS-986122 (10 uM). Co-incubation of the SAM BMS-986124 (50 uM) with BMS-986122 (10 uM) resulted in only a 2-fold increase in potency for DAMGO suggesting that BMS-986124 can antagonize the BMS-986122 PAM effect. Shown are the combined mean ±s.e.m. data from 3-7 separate assays, each performed in duplicate. EC.sub.50 values were compared by Student's t-test using GraphPad Prism. (**) represents p<0.01. Concentration response curves for DAMGO-stimulated [.sup.35S]GTPγS binding under the various conditions are shown in FIG. 10.

(11) FIG. 6

(12) Effect of endomorphin-I on inhibition of 1 uM forskolin-stimulated cAMP accumulation in CHO-mu cells. Endomorphin-I induced a 17-fold reduction in forskolin-stimulated cAMP accumulation with an EC.sub.50 of 76 (60-96) pM. Data are represented as the mean±s.e.m. of three experiments.

(13) FIG. 7

(14) Effect of the mu-PAM, BMS-986122, and the SAM, BMS-986123, on [.sup.3H]diprenorphine saturation binding in membranes from C6mu cells. BMS-986122 (A) had no significant effect on [.sup.3H]diprenorphine binding affinity but induced a 6-fold increase in the affinity of DAMGO in competition binding studies (see FIG. 5, Compound C, Table S1). BMS-986123 (B) produced a small (.sup.˜2-fold) but significant decrease in [.sup.3H]diprenorphine affinity, but had no significant effect on DAMGO affinity (see Table S1). Data are represented as the mean ±s.e.m. of 3-7 experiments.

(15) FIG. 8

(16) Activity of BMS-986122 and 18 analogs in a β-arrestin recruitment assay in U2OS-OPRD1 and U2OS-OPRM1 cells. Compounds were tested in U2OS-OPRD1 cells in agonist detection mode (in the absence of leu-enkephalin) and PAM detection mode (in the presence of an .sup.˜EC.sub.10 concentration of leu-enkephalin). Compounds were also tested in U2OS-OPRM1 cells in agonist detection mode (in the absence of endomorphin-I) and PAM detection mode (in the presence of an .sup.˜EC.sub.10 concentration of endomorphin-I). Finally, compounds that exhibited no agonist or PAM activity in U2OS-OPRM1 cells were tested in SAM detection mode (in the presence of an .sup.˜EC.sub.20 concentration of endomorphin-I plus an .sup.˜EC.sub.80 of the PAM BMS-986122). Graphical curve fit data are representative of three combined experiments. EC.sub.50 and E.sub.max values are represented in Table S2. For agonist detection mode, 0% and 100% activity represent basal activity and an Emax concentration of orthosteric agonist, respectively. For PAM detection mode, 0% activity represents the response to a low (.sup.˜EC.sub.10) concentration of agonist alone, and 100% activity represents the response to an E.sub.max concentration of agonist. For SAM detection mode, 0% inhibition represents the response to a low (.sup.˜EC.sub.20) concentration of endomorphin-I combined with an .sup.˜EC.sub.80 concentration of the mu-PAM BMS-986122. 100% inhibition represents the response to a low (.sup.˜EC.sub.20) concentration of endomorphin-I alone.

(17) FIG. 9

(18) Effect of BMS-986123 and BMS-986124 on β-arrestin response to an .sup.˜EC.sub.80 concentration of endomorphin-I (antagonist/NAM detection mode) in U2OS-OPRM1 cells. BMS-986123 and BMS-986124 had no significant effect on endomorphin-I (300 nM) mediated β-arrestin activity in U2OS-OPRM1 cells. 100% activity is normalized to the response to endomorphin-I (300 nM) alone. 0% activity represents basal activity. Data are represented as the mean+s.e.m. of three experiments.

(19) FIG. 10

(20) Effect of BMS-986124 on BMS-986122-mediated PAM activity to DAMGO-stimulated [.sup.35S]GTPγS binding in C6mu cell membranes. DAMGO-stimulated [.sup.35S]GTPγS binding potency in C6mu membranes was increased 8-fold in the presence of the mu-PAM BMS-986122 (10 uM) (A). DAMGO-stimulated [.sup.35S]GTPγS binding potency was not affected by incubation with 50 uM BMS-986124 (B). Co-incubation of BMS-986124 (50 uM) with BMS-986122 (10 uM) resulted in a rightward shift in DAMGO potency when compared to incubation with BMS-986122 alone (C). The potency of DAMGO, in the presence of both BMS-986122 and BMS-986124 was shifted leftward by only 2-fold compared with DAMGO potency in the presence of the vehicle control. These data suggest that BMS-986124 can antagonize the BMS-986122 PAM effect. Shown are the combined mean+s.e.m. data from 3-7 separate assays, each performed in duplicate. EC.sub.50 values are given in the Results & Discussion section and in FIG. 5B.

(21) FIG. 11

(22) Effect of the SAMs BMS-986123 and BMS-986124 on DAMGO-stimulated [.sup.35S]GTPγS binding above basal activity in membranes from C6mu cells. DAMGO potency (EC.sub.50 of 222 (179-274) nM) was not significantly affected by BMS-986123 (A) (EC.sub.50 of 321 239-432) nM) or BMS-986124 (B) (EC.sub.50 of 223 (150-331) nM. The maximal stimulation by DAMGO (control max=232 (223-242) %, was not affected by BMS-986124 (243 (224-262) %. Maximal stimulation was decreased slightly by BMS-986123 (206 (193-218) %. The modulators did not significantly affect the basal values (vehicle control basal=3.2±0.2 fmol bound/mg protein). Shown are the combined data from 3-7 separate assays, each performed in duplicate.

(23) FIG. 12

(24) Effect of the SAMs BMS-986123 and BMS-986124 on morphine-stimulated [.sup.35S]GTPγS binding in membranes from C6mu cells. The EC.sub.50 of morphine to stimulate [.sup.35S]GTPγS binding (110 (71-171) nM) was not significantly affected by BMS-986123 (A) (140 (67-293) nM; 67-293) nM), but was decreased by BMS-986124 (B) (245 (161-372) nM). The maximal effect of morphine compared to DAMGO (30 uM) (control max=42 (38-45) %) was increased to a small degree by BMS-986123 (62 (52-73) %) and BMS-986124 (58 (53-64) %). Shown are the combined data from 3-7 separate assays, each performed in duplicate.

(25) FIG. 13

(26) Effect of mu-PAM BMS-986121 on inhibition of forskolin-stimulated cAMP accumulation, mediated by different orthosteric agonists, in CHO-mu cells. BMS-986121 (100 uM) produced leftward shifts in agonist potency for each of the three orthosteric ligands used ((A) endomorphin-I=4-fold, (B) morphine=6.5-fold, and (C) leu-enkephalin=4.5-fold)). EC.sub.50 values for the agonists at each BMS-986121 concentration are shown in the legend. Data represent the mean±s.e.m. of 3 experiments.

(27) TABLE-US-00001 TABLE S1 Effect of the mu-PAM, BMS-986122, and the SAM, BMS-986123, on [.sup.3H]diprenorphine saturation binding and DAMGO competition binding in membranes from C6mu cells. [.sup.3H]diprenorphine DAMGO Compound added Kd (nM) Ki (nM) (10 uM) mean (95% CI) mean (95% CI) Vehicle control 0.27 (0.21-0.32) 340 (208-552) BMS-986122 0.35 (0.18-0.51)  56 (41-76) BMS-986123 0.71 (0.57-0.86) 270 (179-406)

(28) TABLE-US-00002 TABLE S2 Structure activity relationship of BMS-986122 and analogs tested in the β-arrestin recruitment assay in U20S-OPRM1 cells, in PAM detection mode. Compounds exhibiting PAM activity were described based on their efficacy as Full, Strong, Moderate, or Weak PAMs. 0% activity represents the response to a low (~EC.sub.10) concentration of endomorphin-I alone and 100% activity represents the response to an E.sub.max concentration of endomorphin-I. The 2 compounds that showed no PAM activity were additionally tested in SAM detection mode (inhibition of BMS-986122 (~EC.sub.80) response in the presence of a low concentration (~EC.sub.20) of endomorphin-I) where the compounds were shown to inhibit the BMS-986122 response (FIG. S3). Calculated K.sub.b values are provided from IC.sub.50 values. Concentration response curves for the SAM compounds, BMS-986123 and BMS-986124, are shown in FIG. 5A. Data are represented as the mean of 3 experiments. β-arrestin β-arrestin PAM PAM β-arrestin SAM Detection detection Detection Mode mode Mode Substance R1 R2 R3 R4 R5 (% E.sub.min) (EC.sub.max uM) Description (K.sub.b, uM) BMS-986122 H Cl OMe Br H 79 3 Full PAM (ref) Analog 1 H Nitro OMe Br H 53 2 Moderate PAM Analog 2 H Me OMe Br H 18 4 Weak PAM Analog 3 H OMe OMe Br H 16 6 Weak PAM Analog 4 Me H OMe Br H 20 9 Weak PAM Analog 5 H Cl OMe Nitro H 45 4 Moderate PAM Analog 6 H Br OMe Nitro H 45 2 Moderate PAM Analog 7 (BMS-986123) H Me OMe Nitro H — — SAM 1 Analog 8 H Br OMe OMe H 18 14 Weak PAM Analog 9 H Br OMe H H 44 7 Moderate PAM Analog 10 H H OMe H H 17 23 Weak PAM Analog 11 H Cl H Cl H 65 5 Strong PAM Analog 12 H Cl Br H H 26 6 Weak PAM Analog 13 (BMS-986124) H Cl Br H OMe — — SAM 2 Analog 14 H OMe Br H H 3 7 Weak PAM Analog 15 H OMe OMe H H 9 37 Weak PAM

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