Compositions of novel cannabinoid receptor modulators and methods of use thereof

Abstract

The present disclosure provides a compound of Formula (1), or a pharmaceutically acceptable salt thereof wherein R1 is hydrogen, alkyl, or substituted alkyl. Also provided are pharmaceutical compositions comprising the compound and methods of treating pain in a subject by administering an effective amount of the compound or pharmaceutical composition. The compound can have R1 as C1-C10 alkyl or substituted alkyl with substituents such as -NH-C(=N)-NH2, -NH-C(=O)-NH2, substituted cycloalkyl, substituted cycloalkenyl, aryl, amine, cyano, hydroxyl, or carboxyl. Methods of treating neuropathic, inflammatory, or chronic pain are disclosed.

Description

021053 / WO WSTL021053.WO COMPOSITIONS OF NOVEL CANNABINOID RECEPTOR MODULATORS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 672,963 filed on July 18, 2024, the disclosures of each are incorporated herein by reference in their entirety. STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under NS126036 awarded bythe National Institutes of Health. The government has certain rights in the invention. FIELD OF INVENTION

[0003] The present disclosure relates to cannabinoid receptor type 1 (CB1) agonists, andmore particularly to peripherally-restricted CB1 agonists with reduced β-arresting recruitment for the treatment of pain. BACKGROUND

[0004] Cannabinoid receptor type 1 (CB1) agonists have shown promise as potentialtreatments for chronic pain and other conditions. However, existing CB1 agonists face significant challenges that limit their therapeutic utility. Many current CB1 agonists readily cross the blood-brain barrier, leading to psychoactive side effects when they activate CB1 receptors in the central nervous system. These central effects can include catalepsy, hypothermia, and cognitive impairment. Additionally, repeated administration of CB1 agonists often results in the development of tolerance, reducing their analgesic efficacy over time.

[0005] Efforts to develop peripherally-restricted CB1 agonists have had limited success.While some compounds show reduced brain penetration compared to earlier CB1 agonists, they still exhibit signs of central CB1 activation at therapeutically relevant doses. This indicates that further improvements in peripheral selectivity are needed to fully separate the desired analgesic effects from unwanted central side effects.

[0006] Another challenge with existing CB1 agonists is their propensity to stronglyactivate β-arrestin signaling pathways in addition to G protein-mediated signaling. Excessive 1 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO β-arrestin recruitment is thought to contribute to the development of tolerance to CB1 agonists. However, most synthetic cannabinoid receptor agonists developed to date robustly stimulate both G protein and β-arrestin pathways.

[0007] There remains a need for CB1 receptor agonists with improved pharmacologicalprofiles that can maintain analgesic efficacy while minimizing both central side effects and the development of tolerance. Achieving this goal requires overcoming several obstacles, including enhancing peripheral selectivity, reducing β-arrestin recruitment, and maintaining potent activation of therapeutically relevant signaling pathways.

[0008] The design of such optimized CB1 agonists is complicated by the limitedstructural information available on the CB1 receptor binding pocket. While some crystal structures of CB1 have been solved, they can not capture all relevant receptor conformations or reveal cryptic binding sites that could be exploited for drug design. This highlights the need for new approaches to elucidate CB1 structure and dynamics to guide the development of next-generation cannabinoid therapeutics. SUMMARY

[0009] This summary is provided to introduce a selection of concepts in a simplifiedform that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0010] One aspect of the disclosure is a compound of Formula 1, or a pharmaceuticallyacceptable salt thereof: OMe 1) whereilkyl.

[0011] Another aspect of the disclosure is a compound of Formula 2, or apharmaceutically acceptable salt thereof: 2 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO 2) wherei ituted C1-C10 alkyl.

[0012] A further aspect of the disclosure is a compound of Formula 3, or apharmaceutically acceptable salt thereof: 3) whereiH-C(=O)-NH2, amino-dioxy cyclobutenyl, phenyl, amine, cyano, hydroxyl, or carboxyl; and n is an integer of 0 to 8.

[0013] Yet another aspect of the disclosure is a compound of Formula 4, or apharmaceutically acceptable salt thereof: 4) wherei, H2, -NH-C(=O)-NH2, amino-dioxy cyclobutenyl, phenyl, amine, cyano, hydroxyl, or carboxyl; and n is an integer of 0 to 8. 3 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0014] A further aspect of the disclosure is a pharmaceutical composition comprising acompound having the structure of any one of Formulae 1 to 4 and a pharmaceutically acceptable carrier.

[0015] Also an object of the disclosure are methods of treating pain in a subject in needthereof, comprising administering to the subject an effective amount of a compound having the structure of any one of Formulae 1 to 4 or a pharmaceutical composition comprising a compound having the structure of any one of Formulae 1 to 4.

[0016] The foregoing general description of the illustrative embodiments and thefollowing detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive. BRIEF DESCRIPTION OF FIGURES

[0017] Non-limiting and non-exhaustive examples are described with reference to thefollowing figures.

[0018] Figure 1. Computational structure-guided design of safe, effective CB1-targeted analgesics. a. Previous CB1 agonists, like MDMB-Fubinaca (FUB) (yellow), have promising analgesic effects, but their utility is limited by dangerous side effects such as catalepsy and because tolerance can develop to these drugs, limiting long-term effectiveness. To overcome these problems, we aimed to design a ligand with a charged group that binds at CB1’s D2.50site (a sodium-binding site at many GPCRs)(See Nature.2023 Jan;613(7945):767-774; ACS Cent Sci.2024 Jul 17;10(8):1490-1503; Nat Commun.2025 Mar 13;16(1):2518; Trends Pharmacol Sci.2025 Feb;46(2):98-101).

[0019] .We hypothesized that such a ligand would have both lower brain penetrance,reducing side effects, and lower arrestin signaling, reducing tolerance. b. In the cryo-EM structure of CB1R with FUB bound (left image, PDB:6N4B), the D2.50site is not accessible from the orthosteric binding pocket due to blockage by the toggle switch residues (F3.36and W6.48). However, molecular dynamics (MD) simulations reveal the transient opening of a previously unknown cryptic pocket (right image) that extends from the orthosteric binding pocket to the D2.50site. The distance between F3.36and W6.48during the simulation is plotted below and indicated by the purple dotted line in the structures. A dashed horizontal line on the plot indicates the distance in the cryo-EM structure, from which the simulations were initiated. c. Using the simulation frame identified in (b), we designed a ligand with a positively charged functional group predicted to engage the D2.50binding site. We attached a 4 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO flexible linker ending in a highly charged head group to FUB (drawing at left). Using docking and molecular modeling tools, we tested several design variables: the linker length, the attachment point on the scaffold, and the choice of charged head group. The final optimized molecule post synthesis and structure-based design (VIP36) is illustrated in the model at right.

[0020] Fig. 2. cryoEM structure of VIP36-bound CB1 and ligand structure-activityrelationship. a,b. VIP36 is a Gi-biased agonist. (a) VIP36 (green curves) displays a nearlyequivalent extent of activation as FUB (orange curves) at activating the Gi pathway as evidenced by cAMP inhibition potency, but (b) shows ~3.5-fold lower activation of β-arrestin-2 pathway. c. We next determined a high-resolution cryoEM structure of VIP36bound to Gi-coupled CB1. d. A conserved set of interactions with the FUB portion of the agonist are present in the two structures, including direct packing of the second extracellular loop (ECL2) via F268ECL2and a π-π interaction with the key toggle switch residue F2003.36. The guanidino group of VIP36 forms cation-π interactions with both residues in the toggle switch, particularly W3566.48(4.0 Å), along with hydrogen bonding interactions with N3897.45(3.0 Å). These strong interactions preclude ionic interactions of the charged guanidino group and side chain of the conserved D1632.50. e. The toggle switch residue W3566.48in the “conventional active” conformation as observed in the FUB structure (blue) is incompatible with the guanidinium linker in VIP36, resulting in stabilization of the “active-open” conformation observed from simulation, along with subtle outward movement of TM3 (V2043.40). f. To better understand the mechanism of arrestin recruitment, additional analogs of VIP36 were designed to probe the role of engaging the D2.50residue. Plot shows BRET assay normalized to CP55,940. This data indicates that the positive charge of tail group dictates reduced arrestin efficacy, supporting the VIP36 interactions with D2.50as the mechanism for G-protein bias. Data is presented as dot plots and median, maximum and minimum for each drug is CP55940: 100%, 102%, 97%; VIP36: 46%, 69%, 42%, 47%; 47(- NH2): 48%, 55%, 45%, 43%; 43 (-CH3): 92%, 93%, 69%; 45(-CH2CH2-Ph): 119%, 160%, 101%, 52(-Squaramide): 171%, 186%, 147%, 2.50(-CO2H): 184%, 188%, 179%. (n=3 independent experiments done as quadruplets except for VIP50, n=2). n values and primary statistics for all panels are provided in Supplementary Table S4. g. cryoEM density comparing structures of VIP36(green)-bound CB1 (pink) (left hand side) with that observed for VIP2.33(yellow)-bound CB1 (brown) (right hand side). While the introduced linker and guanidino group in VIP36 displays strong density, consistent with a (largely) single 5 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO conformation, the tail and urea group of VIP2.33 is disordered, along with the W356 side chain, indicating conformational heterogeneity for these groups. Data for G-protein activity using cAMP and BRET-based assay for β-arrestin2 recruitment in CB1 transfected cell lines are mean^±^s.e.m. (n^=^3 experiments each done in quadruplets).

[0021] Figure 3 . Molecular mechanism of ligand signaling bias at CB1R. a. VIP36displays G-protein bias, whereas close analog VIP2.33 is arrestin-biased. Bias analysis for signaling was performed as described in Methods. b. Snapshots from simulations show that VIP36 and VIP2.33 interact differently with D2.50. Simulations were initiated with CB1R in the conformation observed in the cryo-EM structure with VIP36 bound (see Methods). Five independent simulations, each 2^µs in length, were performed for each ligand. c. Distributions of minimum distance in simulations between heavy atoms of ligand head-group and sidechain of D2.50. VIP36 displays a peak at short interaction distances, indicative of ionic and hydrogen bonding interactions, whereas VIP2.33 does not. d. Snapshots from simulations, viewed from the extracellular side, show that VIP36 and VIP2.33 favor different conformations of transmembrane helix 7 (TM7). The alternative active conformation is characterized by a counterclockwise twist at TM7, moving P7.50 toward D2.50. e. To analyze the conformation of TM7, we measured the distance between the Cɑ atoms of D2.50and P7.50. The distribution of distances shows that VIP36 generally favors the canonical active conformation, whereas VIP2.33 favors the alternative conformation. See SI table S3 for statistical analysis for panels in this figure.

[0022] Figure 4: Lead compound VIP36 produces analgesia with reduced adverseeffects and tolerance in animal models. a. VIP36 is peripherally restricted as brain and plasma ratio show marked preference for plasma over brain ratio over FUB at 3 mg / kg, IP dose at 30 min. Both bound as well as unbound drug levels for FUB and VIP36 in plasma and brain are shown. b. VIP36 ED50of 0.22 mg / kg dose and FUB ED50of 0.01 mg / kg based on reversal of mechanical hyperalgesia following SNI in the affected hindpaw. c. VIP36 effectively reverses NTG induced hyperalgesia in a model of chronic migraine. Mice repeatedly dosed with NTG over 9 days displayed significant cephalic hyperalgesia that was reversed 1 hour post administration of VIP361 mg / kg, Three-way ANOVA p<0.05 effect of time x NTG treatment x VIP treatment. ****p<0.0001 NTG-Veh compared to Veh-Veh in post treatment time point, #p<0.05 NTG-Veh compared to NTG-VIP-36 in post treatment time point. d. VIP36 reversal of hyperalgesia is dependent on peripheral CB1Rs, but not on CB2R. Mice with SNI exhibited hyperalgesia which was reversed by administration 6 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO of VIP36 and vehicle. Pre-administration of the peripherally-restricted CB1 antagonist AM6545 blocked VIP’s anti-hyperalgesic effect. Treatment with the CB2 receptor antagonist AM630 did not block VIP36’s anti-hyperalgesic effect. ****p<0.0001, VIP36-AM6545 compared to VIP36-Vehicle. e. VIP36 showed limited tolerance in the SNI model following twice-daily administrations over 9 days. Mechanical withdrawal thresholds were assessed every other day, 1–2 hours after the first intraperitoneal (IP) injection of Vehicle, VIP36 (1mg / kg) or FUB (0.1 mg / kg). Data are represented as mean ± SEM. Two-Way ANOVA with Tukey's multiple comparisons *p<0.05, **p<0.01, ***p<0.001 VIP vs VEH f. VIP36 produces catalepsy at 100x ED50. p<0.00011-way ANOVA, ****p<0.000130 mg / kg compared to 0 mg / kg dose, FUB produces catalepsy at 0.1 mg / kg. **p<0.00321-way ANOVA, ****p<0.00010.22 mg / kg compared to 0 mg / kg dose. Green dotted line in panel g / h / j shows the SNI ED50of VIP36 and orange dotted line in panel g / h / j shows SNI ED50dose of FUB. g. VIP36 produces hypothermia at 20 mg / kg dose and 30 mg / kg dose p<0.0001 1-way ANOVA ****p<0.000120 mg / kg and 30 mg / kg compared to 0 mg / kg dose, FUB produces hypothermia at 0.03 mg / kg dose 0.1 mg / kg and 0.3 mg / kg dose ***p<0.0002, p<0.00011-way ANOVA ****p<0.00010.03 mg / kg, 0.1 mg / kg and 0.3 mg / kg compared to 0 mg / kg dose. h. VIP36 does not produce changes in locomotion in an open field test at 3 mg / kg, IP. dose compared to vehicle 1. Unpaired student t-test p=0.8844. FUB at 0.01 mg / kg , 0.03 mg / kg , 0.1 mg / kg, I.P. produces significant reduced locomotion in an open field test. Vehicle 2 used for FUB at 0.01mg / kg and 0.03 mg / kg, IP. Vehicle 3 used for FUB at 0.1 mg / kg, IP. *p<0.0219, ****p<0.0001, ****p<0.0001 vehicle compared to FUB unpaired student t-test. i. VIP36 produces antinociception in tail flick at 100x ED50, p<0.00011-way ANOVA, ****p<0.000130 mg / kg compared to 0 mg / kg, and FUB produces analgesia in tail flick the same dose that reversed hyperalgesia. p<0.00011-way ANOVA, ***p<0.001 at 0.1 and 0.3 mg / kg, IP. compared to 0 mg / kg dose point. See SI table S4 for statistical analysis for panels in this figure.

[0023] Figure 5 is a graph depicting signaling bias that was calculated for 30, 33**p=0.0036, VIP36(8) ***p=0.0002, 35, and 38. Bias analysis for signaling was performed as described in methods.

[0024] Figure 6: a. VIP36 reversed CFA induced hyperalgesia in a dose dependentresponse at the 30 minute, 1 hr, and 2 hr post VIP36 administration, repeated Two-way ANOVA measurement p<0.0001 effect of time x dosage. ++p<0.013 mg / kg dose compared to Vehicle at same time point, ***p<0.0011 mg / kg dose compared to Vehicle, *p<0.051 7 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO mg / kg dose vs Vehicle at same time point Tukey testing. b. VIP36 ED50was not determined and while FUB ED50 was 0.10 mg / kg based on reversal of hyperalgesia following CFA administration in hindpaw. c. VIP36 at 1 mg / kg reversed SNI induced hindpaw hyperalgesia that was Three-way ANOVA p<0.001 effect of Time, SNI vs Sham, VIP36 vs Vehicle, *p<0.05 SNI-VIP36 compared to SNI-Veh at same time point. d. VIP36 reversal of hyperalgesia is dependent on peripheral CB1Rs, but not on CB2R. Mice treated with CFA in the right hindpaw had significant hyperalgesia that was reversed by administration of VIP36 and vehicle. Mice co-administered VIP36 and the CB1 peripheral antagonist AM6545 had sustained hyperalgesia, while mice treated with VIP36 and the global CB2 receptor antagonist AM630 did not. *p<0.05, VIP36-AM6545 compared to VIP36-Vehicle. e. VIP36 and FUB did not alter mechanical thresholds in sham-surgery animals over a 9-day dosing period. Mechanical withdrawal thresholds were assessed every other day, 1–2 hours after the first intraperitoneal (I.P.) injection of Vehicle, VIP36 (1mg / kg) or FUB (0.1 mg / kg). Data are represented as mean ± SEM. f. Distance moved in the center in the open field test (cm) performed following VIP36, FUB, or Vehicle injection. Data normalized to the Vehicle group.

[0025] Figure 7: a. VIP36 was screened for 45 CNS receptors in binding assays by theNational Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP). Only the mu opioid receptor was found to be a target. Weak affinity at MOR was observedfor VIP36. b. No measurable potency and low efficacy were observed in BRET Gi1 assayson MOR (n=2 independent experiments done as quadruplets).

[0026] Figure 8: a. VIP36 was screened for broad range of receptors in PRESTOTANGO functional assays by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) SSTR4 was identified as another potential target. b and c. VIP36 is at least 94-fold selective for CB1over SSTR4 (n^=^3 experiments each done in quadruplets). d.VIP36 has minimal binding at hERG (n^=^3 experiments each done in quadruplets). e. No measurable efficacy and potency observed for VIP36 in 5-HT2breceptor (n^=^2 experiments each done in quadruplets). DETAILED DESCRIPTION

[0027] The following description sets forth exemplary aspects of the present disclosure.It should be recognized, however, that such description is not intended as a limitation on the 8 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein.

[0028] The term "cannabinoid receptor type 1 (CB1)" is used herein to mean a G protein-coupled receptor that is a target for cannabinoid ligands. CB1 receptors are expressed throughout the pain neuraxis and are involved in mediating analgesic effects. Activation of CB1 receptors in the central nervous system can result in undesired side effects including psychoactivity in humans, as well as catalepsy and hypothermia in rodents.

[0029] The present disclosure relates to novel cannabinoid receptor type 1 (CB1)agonists that can provide improved pain management with reduced side effects. These compounds can offer advantages over existing treatments by potentially addressing limitations such as central nervous system side effects and development of tolerance. The new CB1 agonists described herein can exhibit enhanced peripheral selectivity and biased signaling properties, which can contribute to their improved therapeutic profile.

[0030] In some cases, the compounds disclosed herein can demonstrate analgesicefficacy across multiple pain models, including inflammatory, neuropathic, and migraine pain. The compounds can show a wide therapeutic window between pain relief and centrally- mediated side effects. Additionally, these novel CB1 agonists can maintain analgesic effects with repeated administration, potentially offering sustained relief for chronic pain conditions. The compounds and compositions described in this disclosure can represent a promising approach for addressing various types of pain, including those associated with conditions such as diabetic neuropathy, postherpetic neuralgia, and chemotherapy-induced peripheral neuropathy.

[0031] The novel CB1 agonist compounds disclosed herein can comprise several keystructural elements that contribute to their unique properties and potential therapeutic benefits. These compounds can be represented by general Formula 1: 9 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO OMe O 1) ubstituent that plays a crucial role in determining thecompound's properties and activity. In some cases, R1can be hydrogen, alkyl, or substituted alkyl. The alkyl group can be a C1-C10alkyl, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl. In some cases, R1 can be methyl, n-hexyl, or n-octyl.

[0033] When R1 is a substituted alkyl, the substituent can be selected from variousfunctional groups that can modulate the compound's activity and properties. These substituents can include -NH-C(=N)-NH2, -NH-C(=O)-NH2, substituted cycloalkyl, substituted cycloalkenyl, aryl, amine, cyano, hydroxyl, or carboxyl. In some cases, the substituent can be -NH-C(=N)-NH2or -NH-C(=O)-NH2.

[0034] The substituted alkyl can have different chain lengths depending on the specificsubstituent. For example, when the substituent is aryl, such as phenyl, the substituted alkyl can be a C1-C3alkyl. When the substituent is carboxyl, the substituted alkyl can be a C4-C6alkyl. In some cases, the substituted alkyl can be a C3-C8 alkyl when the substituent is -NH2 or hydroxyl.

[0035] Another important structural feature of these compounds can be represented byFormula 2: 2) CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0036] In Formula 2, R2 can be hydrogen, C1-C10 alkyl, or substituted C1-C10 alkyl. Thesubstitutions and variations described forR2 in Formula 1 can also apply toR2 in Formula 2.

[0037] The compounds can also be represented by Formula 3:3)

[0038] In Formula 3, -NH-C(=O)-NH2, amino-dioxy cyclobutenyl, phenyl, amine, cyano, hydroxyl, or carboxyl. The variable n can be an integer of 0 to 8, providing flexibility in the length of the alkyl chain connecting the core structure to R3. In some cases, n can be an integer of 1 to 8, 2 to 6, 3 to 6, 3 to 5, or specifically 4.

[0039] A fourth structural representation of the compounds can be given by Formula 4:4)

[0040] In Formula 4, R4tituents as R3 in Formula 3, andn can have the same range of values.

[0041] In addition, the compounds of Formula 1 can have the following structure:11 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO12 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO F ,13 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO .abovecan be referred to as VIP36. This compound can have a 4-carbon linker between the indazole ring and the guanidine group, with the guanidine group attached at the 6-position of the indazole ring. VIP36 can show peripheral selectivity with a brain-to-plasma partition coefficient (Kp,uu,brain) of 0.0012.

[0043] The compounds described herein can be formulated into pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier. The carrier can be selected from liquid carriers, solid carriers, and semi-solid carriers. Liquid carriers can include water, saline, and pharmaceutically acceptable organic solvents.

[0044] These pharmaceutical compositions can be formulated for various routes ofadministration, including oral, parenteral, topical, or transdermal administration. In some cases, the pharmaceutical composition can further comprise an additional active agent. This additional agent can be selected from opioid analgesics, non-steroidal anti-inflammatory drugs, anticonvulsants, antidepressants, and local anesthetics. 14 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0045] The structural features and formulation options described above can contribute tothe unique properties of these novel CB1 agonist compounds, potentially offering improved therapeutic profiles for pain management with reduced side effects.

[0046] The novel CB1 agonist compounds disclosed herein can exhibit unique structuraland functional relationships that contribute to their potential therapeutic benefits. These relationships can involve interactions between key structural elements and their effects on receptor binding and signaling pathways.

[0047] In some cases, the compounds can demonstrate G-protein bias and reduced β-arrestin-2 recruitment compared to other CB1 agonists. For example, the compound VIP36 can exhibit enhanced G-protein activation while showing diminished β-arrestin-2 recruitment relative to the parent compound FUB. This biased signaling profile can contribute to the improved therapeutic properties of VIP36 and related compounds.

[0048] The structural basis for these functional properties can be elucidated throughvarious techniques, including cryoelectron microscopy (cryoEM) and molecular dynamics simulations. CryoEM studies of VIP36-bound CB1 can reveal important details about the compound's binding mode and its effects on receptor conformation.

[0049] In some cases, the guanidinium group of VIP36 can form specific interactionswith key residues in the CB1 receptor. These interactions can include cation-π interactions with toggle switch residues and hydrogen bonding with N3897.45. Such interactions can play a role in stabilizing certain receptor conformations and influencing signaling outcomes.

[0050] Molecular dynamics simulations can provide insights into the dynamic behaviorof the CB1 receptor and its interactions with ligands. These simulations can reveal the presence of a cryptic pocket in CB1 that extends from the orthosteric binding pocket to the D2.50site. The identification of this cryptic pocket can have implications for ligand design understanding receptor activation mechanisms.

[0051] The binding of VIP36 to CB1 can induce specific conformational changes in thereceptor. In some cases, VIP36 can stabilize an 'active-open' conformation of the toggle switch residue W3566.48. This conformational change can be distinct from that observed with other CB1 agonists and can contribute to the unique signaling properties of VIP36.

[0052] The relationships between these structural features and functional outcomes canbe complex and interconnected. For example, the ability of VIP36 to engage the cryptic pocket and stabilize specific receptor conformations can be linked to its G-protein biased 15 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO signaling profile. The reduced β-arrestin-2 recruitment observed with VIP36 can be a consequence of these structural interactions and conformational preferences.

[0053] Understanding these structure-function relationships can provide insights into themechanisms underlying the potential therapeutic benefits of these novel CB1 agonists. The combination of reduced β-arrestin-2 recruitment and engagement of specific receptor conformations can contribute to the compounds' ability to produce analgesia with potentially reduced side effects and tolerance development.

[0054] In some cases, the structural features that enable VIP36 and related compounds tointeract with the cryptic pocket and stabilize certain receptor conformations can also influence their pharmacokinetic properties. For instance, these structural elements can affect the compounds' ability to cross the blood-brain barrier, potentially contributing to their peripheral selectivity.

[0055] The relationships between the structural elements of these compounds and theirfunctional properties can extend beyond receptor binding and signaling. In some cases, the specific substituents and their positions on the core scaffold can influence the compounds' metabolic stability, solubility, and other physicochemical properties that are relevant to their potential therapeutic applications.

[0056] By elucidating these complex relationships between structural features andfunctional outcomes, it can be possible to further refine and optimize CB1 agonists for specific therapeutic applications. This structure-guided approach can lead to the development of compounds with tailored signaling profiles and improved safety and efficacy profiles for pain management and other potential indications. Formulation

[0057] The agents and compositions described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. 16 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0058] The term "formulation" refers to preparing a drug in a form suitable foradministration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0059] The term "pharmaceutically acceptable" as used herein can describe substances orcomponents that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc., can also be used.

[0060] The term “pharmaceutically acceptable excipient,” as used herein, can includeany and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0061] A "stable" formulation or composition can refer to a composition havingsufficient stability to allow storage at a convenient temperature, such as between about 0 ºC and about 60 ºC, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0062] The formulation should suit the mode of administration. The agents of use withthe current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents can also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents can be in fluid or mechanical communication with the 17 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0063] Controlled-release (or sustained-release) preparations can be formulated to extendthe activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently, affect the occurrence of side effects. Controlled-release preparations can be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent can be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0064] Agents or compositions described herein can also be used in combination withother therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one can also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition. Therapeutic Methods

[0065] Also provided is a process of treating, preventing, or reversing chronic pain in asubject in need thereof via administration of a therapeutically effective amount a peripherally restricted CB1 agonist.

[0066] Methods described herein are generally performed on a subject in need thereof. Asubject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing chronic pain. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0067] Generally, a safe and effective amount a peripherally restricted CB1 agonist is,for example, an amount that would cause the desired therapeutic effect in a subject while 18 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO minimizing undesired side effects. In various cases, an effective amount a peripherally restricted CB1 agonist described herein can substantially inhibit, slow the progress of, or limit the development of chronic pain.

[0068] According to the methods described herein, administration can be parenteral,pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0069] When used in the treatments described herein, a therapeutically effective amounta peripherally restricted CB1 agonist can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to treat, prevent, or reverse chronic pain.

[0070] The amount of a composition described herein that can be combined with apharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0071] Toxicity and therapeutic efficacy of compositions described herein can bedetermined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50(the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0072] The specific therapeutically effective dose level for any particular subject willdepend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda- 19 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0073] Again, each of the states, diseases, disorders, and conditions, described herein, aswell as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes reversing or delaying the appearance of clinical symptoms in a mammal that can be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or a physician.

[0074] Administration a peripherally restricted CB1 agonist can occur as a single eventor over a time course of treatment. For example, a peripherally restricted CB1 agonist can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0075] Treatment in accord with the methods described herein can be performed prior toor before, concurrent with, or after conventional treatment modalities for chronic pain. 20 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0076] A peripherally restricted CB1 agonist can be administered simultaneously orsequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a peripherally restricted CB1 agonist can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a peripherally restricted CB1 agonist, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a peripherally restricted CB1 agonist, an antibiotic, an anti-inflammatory, or another agent. A peripherally restricted CB1 agonist can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a peripherally restricted CB1 agonist can be administered before or after administration of an antibiotic, an anti- inflammatory, or another agent.

[0077] Active compounds are administered at a therapeutically effective dosagesufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that can be predictive of efficacy in treating the disease in a human or another animal, such as the model systems shown in the examples and drawings.

[0078] An effective dose range of a therapeutic can be extrapolated from effective dosesdetermined in animal studies for a variety of different animals. In general, a human equivalent dose (HED) in mg / kg can be calculated in accordance with the following formula (see e.g., Reagan-Shaw et al., FASEB J., 22(3):659-661, 2008, which is incorporated herein by reference): HED (mg / kg) = Animal dose (mg / kg) × (Animal Km / Human Km)

[0079] Use of the Km factors in conversion results in more accurate HED values, whichare based on body surface area (BSA) rather than only on body mass. Kmvalues for humans and various animals are well known. For example, the Km for an average 60 kg human (with a BSA of 1.6 m2) is 37, whereas a 20 kg child (BSA 0.8 m2) would have a Kmof 25. Kmfor some relevant animal models are also well known, including: mice Km of 3 (given a weight of 0.02 kg and BSA of 0.007); hamster Kmof 5 (given a weight of 0.08 kg and BSA of 0.02); rat Kmof 6 (given a weight of 0.15 kg and BSA of 0.025) and monkey Kmof 12 (given a weight of 3 kg and BSA of 0.24).

[0080] Precise amounts of the therapeutic composition depend on the judgment of thepractitioner and are peculiar to each individual. Nonetheless, a calculated HED dose provides 21 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment, and the potency, stability, and toxicity of the particular therapeutic formulation.

[0081] The actual dosage amount of a compound of the present disclosure orcomposition comprising a compound of the present disclosure administered to a subject can be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. These factors can be determined by a skilled artisan. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. The dosage can be adjusted by the individual physician in the event of any complication.

[0082] In some cases, the peripherally restricted CB1 agonist can be administered in anamount from about 1 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 25 mg / kg, or about 1 mg / kg to about 15 mg / kg, or about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg, or about 3 mg / kg. In some cases, a peripherally restricted CB1 agonist such as one or more of the compounds / ligands described herein can be administered in a range of about 1 mg / kg to about 200 mg / kg, or about 50 mg / kg to about 200 mg / kg, or about 50 mg / kg to about 100 mg / kg, or about 75 mg / kg to about 100 mg / kg, or about 100 mg / kg.

[0083] The effective amount can be less than 1 mg / kg / day, less than 500 mg / kg / day, lessthan 250 mg / kg / day, less than 100 mg / kg / day, less than 50 mg / kg / day, less than 25 mg / kg / day or less than 10 mg / kg / day. It can alternatively be in the range of 1 mg / kg / day to 200 mg / kg / day.

[0084] In other non-limiting examples, a dose can also comprise from about 1 micro-gram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples 22 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. Administration

[0085] Agents and compositions described herein can be administered according tomethods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0086] As discussed above, administration can be parenteral, pulmonary, oral, topical,intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0087] Agents and compositions described herein can be administered in a variety ofmethods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 µm), nanospheres (e.g., less than 1 µm), microspheres (e.g., 1-100 µm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0088] Delivery systems can include, for example, an infusion pump which can be usedto administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In 23 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0089] Agents can be encapsulated and administered in a variety of carrier deliverysystems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product. Embodiments

[0090] Embodiment 1. A compound of Formula 1, or a pharmaceutically acceptable saltthereof: OMe 1) wherein R1is hydrogen, a, .

[0091] Embodiment 2. The compound of embodiment 1, wherein R1 is C1-C10 alkyl.

[0092] Embodiment 3. The compound of embodiment 1, wherein R1 is methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, or octyl.

[0093] Embodiment 4. The compound of embodiment 1, wherein R1 is methyl, n-hexyl,or n-octyl. 24 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0094] Embodiment 5. The compound of embodiment 1, wherein R1 is substituted alkyland the substituent is -NH-C(=N)-NH2, -NH-C(=O)-NH2, substituted cycloalkyl, substituted cycloalkenyl, aryl, amine, cyano, hydroxyl, or carboxyl.

[0095] Embodiment 6. The compound of embodiment 5, wherein R1 is substituted alkyland the substituent is -NH-C(=N)-NH2 or -NH-C(=O)-NH2.

[0096] Embodiment 7. The compound of embodiment 5, wherein R1 is substituted alkyland the substituent is substituted cyclobutenyl.

[0097] Embodiment 8. The compound of embodiment 7, wherein R1 is substituted alkyland the substituent is amino-dioxy cyclobutenyl.

[0098] Embodiment 9. The compound of embodiment 5, wherein R1 is substituted alkyland the substituent is aryl.

[0099] Embodiment 10. The compound of embodiment 9, wherein R1 is substituted alkyland the substituent is phenyl.

[0100] Embodiment 11. The compound of embodiment 5, wherein R1 is substituted alkyland the substituent is -NH2.

[0101] Embodiment 12. The compound of embodiment 5, wherein R1 is substituted alkyland the substituent is cyano.

[0102] Embodiment 13. The compound of embodiment 5, wherein R1 is substituted alkyland the substituent is hydroxyl.

[0103] Embodiment 14. The compound of embodiment 5, wherein R1 is substituted alkyland the substituent is carboxyl.

[0104] Embodiment 15. A compound of Formula 2, or a pharmaceutically acceptable saltthereof: 25 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO 2) wherein R2is hydrogen, C l.

[0105] Embodiment 16. The compound of embodiment 15, wherein R1 is methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, or octyl.

[0106] Embodiment 17. The compound of embodiment 15, wherein R1 is methyl, n-hexyl, or n-octyl.

[0107] Embodiment 18. The compound of embodiment 15, wherein R1 is substituted C1-C10 alkyl and the substituent is -NH-C(=N)-NH2, -NH-C(=O)-NH2, substituted cycloalkyl, substituted cycloalkenyl, aryl, amine, cyano, hydroxyl, or carboxyl.

[0108] Embodiment 19. The compound of embodiment 18, wherein R1 is substituted C3-C8 alkyl and the substituent is -NH-C(=N)-NH2 or -NH-C(=O)-NH2.

[0109] Embodiment 20. The compound of embodiment 18, wherein R1 is substituted C3-C5 alkyl and the substituent is substituted cyclobutenyl.

[0110] Embodiment 21. The compound of embodiment 20, wherein R1 is substituted C3-C5alkyl and the substituent is amino-dioxy cyclobutenyl.

[0111] Embodiment 22. The compound of embodiment 18, wherein R1 is substituted C1-C3 alkyl and the substituent is aryl.

[0112] Embodiment 23. The compound of embodiment 22, wherein R1 is substituted C1-C3alkyl and the substituent is phenyl.

[0113] Embodiment 24. The compound of embodiment 18, wherein R1 is substituted C3-C8alkyl and the substituent is -NH2. 26 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0114] Embodiment 25. The compound of embodiment 18, wherein R1 is substituted C4-C6alkyl and the substituent is cyano.

[0115] Embodiment 26. The compound of embodiment 18, wherein R1 is substituted C3-C8alkyl and the substituent is hydroxyl.

[0116] Embodiment 27. The compound of embodiment 18, wherein R1 is substituted C4-C6 alkyl and the substituent is carboxyl.

[0117] Embodiment 28. A compound of Formula 3, or a pharmaceutically acceptable saltthereof: 3) wherein R3is hydrogen, -ino-dioxy cyclobutenyl, phenyl, amine, cyano, hydroxyl, or carboxyl; and n is an integer of 0 to 8.

[0118] Embodiment 29. The compound of embodiment 28, wherein R3 is hydrogen andn is an integer of 1 to 8.

[0119] Embodiment 30. The compound of embodiment 28, wherein R3 is -NH-C(=N)-NH2or -NH-C(=O)-NH2.

[0120] Embodiment 31. The compound of embodiment 28, wherein R3 is amino-dioxycyclobutenyl or phenyl.

[0121] Embodiment 32. The compound of embodiment 28, wherein R3 is -NH2 or cyano.

[0122] Embodiment 33. The compound of embodiment 28, wherein R3 is hydroxyl orcarboxyl.

[0123] Embodiment 34. The compound of any one of embodiments 30 to 33, wherein nis an integer of 1 to 8. 27 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0124] Embodiment 35. The compound of any one of embodiments 30 to 33, wherein nis an integer of 2 to 6.

[0125] Embodiment 36. The compound of any one of embodiments 30 to 33, wherein nis an integer of 3 to 6.

[0126] Embodiment 37. The compound of any one of embodiments 30 to 33, wherein nis an integer of 3 to 5.

[0127] Embodiment 38. The compound of any one of embodiments 30 to 33, wherein nis an integer of 4.

[0128] Embodiment 39. A compound of Formula 4, or a pharmaceutically acceptable saltthereof: 4) wherein R4is hydrogen, -NHamino-dioxy cyclobutenyl, phenyl, amine, cyano, hydroxyl, or carboxyl; and n is an integer of 0 to 8.

[0129] Embodiment 40. The compound of embodiment 39, wherein R3 is hydrogen andn is an integer of 1 to 8.

[0130] Embodiment 41. The compound of embodiment 39, wherein R3 is -NH-C(=N)-NH2or -NH-C(=O)-NH2.

[0131] Embodiment 42. The compound of embodiment 39, wherein R3 is amino-dioxycyclobutenyl or phenyl.

[0132] Embodiment 43. The compound of embodiment 39, wherein R3 is -NH2 or cyano.

[0133] Embodiment 44. The compound of embodiment 39, wherein R3 is hydroxyl orcarboxyl.

[0134] Embodiment 45. The compound of any one of embodiments 41 to 44, wherein nis an integer of 1 to 8. 28 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0135] Embodiment 46. The compound of any one of embodiments 41 to 44, wherein nis an integer of 2 to 6.

[0136] Embodiment 47. The compound of any one of embodiments 41 to 44, wherein nis an integer of 3 to 6.

[0137] Embodiment 48. The compound of any one of embodiments 41 to 44, wherein nis an integer of 3 to 5.

[0138] Embodiment 49. The compound of any one of embodiments 41 to 44, wherein nis an integer of 4.

[0139] Embodiment 50. The compound of embodiment 1, selected from:021053 / WO WSTL021053.WO F ,30 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO ..position comprising a compound of anyone of embodiments 1 to 51 and a pharmaceutically acceptable carrier.

[0142] Embodiment 53. The pharmaceutical composition of embodiment 52, furthercomprising an additional active agent. 31 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0143] Embodiment 54. The pharmaceutical composition of embodiment 53 wherein theadditional active agent is selected from the group consisting of an opioid analgesic, a non- steroidal anti-inflammatory drug, an anticonvulsant, an antidepressant, and a local anesthetic.

[0144] Embodiment 55. The pharmaceutical composition of any one of embodiments 52to 54, wherein the pharmaceutically acceptable carrier is selected from the group consisting of a liquid carrier, a solid carrier, and a semi-solid carrier.

[0145] Embodiment 56. The pharmaceutical composition of embodiment 55, wherein theliquid carrier is selected from the group consisting of water, saline, and a pharmaceutically acceptable organic solvent.

[0146] Embodiment 57. The pharmaceutical composition of any one of embodiments 52to 56, formulated for oral, parenteral, topical, or transdermal administration.

[0147] Embodiment 58. A method of treating pain in a subject in need thereof,comprising: administering to the subject an effective amount of a compound of any one of embodiments 1 to 51 or a pharmaceutical composition of any one of embodiments 52 to 57.

[0148] Embodiment 59. The method of embodiment 58, wherein the pain is selectedfrom the group consisting of neuropathic pain, inflammatory pain, and chronic pain.

[0149] Embodiment 60. The method of embodiment 59, wherein the neuropathic pain isassociated with a condition selected from the group consisting of diabetic neuropathy, postherpetic neuralgia, and chemotherapy-induced peripheral neuropathy.

[0150] Embodiment 61. The method of any one of embodiments 58 to 60, wherein thecompound or pharmaceutical composition is administered orally, parenterally, topically, or transdermally.

[0151] Embodiment 62. The method of any one of embodiments 58 to 61, furthercomprising administering an additional active agent selected from the group consisting of an opioid analgesic, a non-steroidal anti-inflammatory drug, an anticonvulsant, an antidepressant, and a local anesthetic.

[0152] Examples of peripherally restricted CB1 agonist agents are described herein.Agents can include one or more compounds and ligand described herein, or pharmaceutically acceptable salts thereof.

[0153] The formulas, analogs, and R groups can be optionally substituted orfunctionalized with one or more groups independently selected from the group consisting of hydroxyl; C1-10alkyl hydroxyl; amine; C1-10carboxylic acid; C1-10carboxyl; straight chain or branched C1-10alkyl, optionally containing unsaturation; a C2-10cycloalkyl optionally 32 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C1-10alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C1-10alkyl hydroxyl; amine; C1-10carboxyl; C1-10carboxylic acid; C1-10carboxyl; straight chain or branched C1-10alkyl, optionally containing unsaturation; straight chain or branched C1-10alkyl amine, optionally containing unsaturation; a C2-10cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C1-10alkyl amine; heterocyclyl; heterocyclic amine; aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms; and the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C1-10alkyl hydroxyl; amine; C1-10carboxylic acid; C1-10carboxyl; straight chain or branched C1-10alkyl, optionally containing unsaturation; straight chain or branched C1-10alkyl amine, optionally containing unsaturation; a C2-10cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; heterocyclyl; straight chain or branched C1-10alkyl amine; heterocyclic amine; and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms. Any of the above can be further optionally substituted.

[0154] The term “imine” or “imino”, as used herein, unless otherwise indicated, caninclude a functional group or chemical compound containing a carbon-nitrogen double bond. The expression “imino compound”, as used herein, unless otherwise indicated, refers to a compound that includes an “imine” or an “imino” group as defined herein. The “imine” or “imino” group can be optionally substituted.

[0155] The term “hydroxyl”, as used herein, unless otherwise indicated, can include -OH. The “hydroxyl” can be optionally substituted.

[0156] The terms “halogen” and “halo”, as used herein, unless otherwise indicated,include a chlorine, chloro, Cl; fluorine, fluoro, F; bromine, bromo, Br; or iodine, iodo, or I.

[0157] The term “acetamide”, as used herein, is an organic compound with the formulaCH₃CONH₂. The “acetamide” can be optionally substituted.

[0158] The term “aryl”, as used herein, unless otherwise indicated, include a carbocyclicaromatic group. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, or anthracenyl. The “aryl” can be optionally substituted. 33 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0159] The terms “amine” and “amino”, as used herein, unless otherwise indicated,include a functional group that contains a nitrogen atom with a lone pair of electrons and wherein one or more hydrogen atoms have been replaced by a substituent such as, but not limited to, an alkyl group or an aryl group. The “amine” or “amino” group can be optionally substituted.

[0160] The term “alkyl”, as used herein, unless otherwise indicated, can includesaturated monovalent hydrocarbon radicals having straight or branched moieties, such as but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl groups, etc. Representative straight-chain lower alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, - n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; while branched lower alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2- methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3- methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, unsaturated C1-10 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, - 3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, or -3-methyl-1 butynyl. An alkyl can be saturated, partially saturated, or unsaturated. The “alkyl” can be optionally substituted.

[0161] The term “carboxyl”, as used herein, unless otherwise indicated, can include afunctional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group (-COOH). The “carboxyl” can be optionally substituted.

[0162] The term “carbonyl”, as used herein, unless otherwise indicated, can include afunctional group consisting of a carbon atom double-bonded to an oxygen atom (C=O). The “carbonyl” can be optionally substituted.

[0163] The term “alkenyl”, as used herein, unless otherwise indicated, can include alkylmoieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety. An alkenyl can be partially saturated or unsaturated. The “alkenyl” can be optionally substituted.

[0164] The term “alkynyl”, as used herein, unless otherwise indicated, can include alkylmoieties having at least one carbon-carbon triple bond wherein alkyl is as defined above. An alkynyl can be partially saturated or unsaturated. The “alkynyl” can be optionally substituted. 34 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0165] The term “acyl”, as used herein, unless otherwise indicated, can include afunctional group derived from an aliphatic carboxylic acid, by removal of the hydroxyl (– OH) group. The “acyl” can be optionally substituted.

[0166] The term “alkoxyl”, as used herein, unless otherwise indicated, can include O-alkyl groups wherein alkyl is as defined above and O represents oxygen. Representative alkoxyl groups include, but are not limited to, -O-methyl, -O-ethyl, -O-n-propyl, -O-n-butyl, - O-n-pentyl, -O-n-hexyl, -O-n-heptyl, -O-n-octyl, -O-isopropyl, -O-sec-butyl, -O-isobutyl, -O- tert-butyl, -O-isopentyl, -O-2-methylbutyl, -O-2-methylpentyl, -O-3-methylpentyl, -O-2,2- dimethylbutyl, -O-2,3-dimethylbutyl, -O-2,2-dimethylpentyl, -O-2,3-dimethylpentyl, -O-3,3- dimethylpentyl, -O-2,3,4-trimethylpentyl, -O-3-methylhexyl, -O-2,2-dimethylhexyl, -O-2,4- dimethylhexyl, -O-2,5-dimethylhexyl, -O-3,5-dimethylhexyl, -O-2,4dimethylpentyl, -O-2- methylheptyl, -O-3-methylheptyl, -O-vinyl, -O-allyl, -O-1-butenyl, -O-2-butenyl, -O- isobutylenyl, -O-1-pentenyl, -O-2-pentenyl, -O-3-methyl-1-butenyl, -O-2-methyl-2-butenyl, - O-2,3-dimethyl-2-butenyl, -O-1-hexyl, -O-2-hexyl, -O-3-hexyl, -O-acetylenyl, -O-propynyl, - O-1-butynyl, -O-2-butynyl, -O-1-pentynyl, -O-2-pentynyl and -O-3-methyl-1-butynyl, -O- cyclopropyl, -O-cyclobutyl, -O-cyclopentyl, -O-cyclohexyl, -O-cycloheptyl, -O-cyclooctyl, - O-cyclononyl and -O-cyclodecyl, -O-CH2-cyclopropyl, -O-CH2-cyclobutyl, -O-CH2- cyclopentyl, -O-CH2-cyclohexyl, -O-CH2-cycloheptyl, -O-CH2-cyclooctyl, -O- CH2- cyclononyl, -O-CH2-cyclodecyl, -O-(CH2)2-cyclopropyl, -O-(CH2)2-cyclobutyl, -O-(CH2)2- cyclopentyl, -O-(CH2)2-cyclohexyl, -O-(CH2)2-cycloheptyl, -O-(CH2)2-cyclooctyl, -O- (CH2)2-cyclononyl, or -O-(CH2)2-cyclodecyl. An alkoxyl can be saturated, partially saturated, or unsaturated. The “alkoxyl” can be optionally substituted.

[0167] The term “cycloalkyl”, as used herein, unless otherwise indicated, can include anaromatic, a non-aromatic, saturated, partially saturated, or unsaturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 1 to 10 carbon atoms (e.g., 1 or 2 carbon atoms if there are other heteroatoms in the ring), preferably 3 to 8 ring carbon atoms. Examples of cycloalkyls include, but are not limited to, C3-10 cycloalkyl groups include, but are not limited to, -cyclopropyl, -cyclobutyl, - cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4- cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. The term “cycloalkyl” also can include -lower alkyl-cycloalkyl, wherein lower alkyl and cycloalkyl are as defined herein. Examples of -lower alkyl- cycloalkyl groups include, but are not limited to, -CH2-cyclopropyl, -CH2-cyclobutyl, -CH2- 35 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO cyclopentyl, -CH2-cyclopentadienyl, -CH2-cyclohexyl, -CH2-cycloheptyl, or -CH2-cyclooctyl. The “cycloalkyl” can be optionally substituted. A “cycloheteroalkyl”, as used herein, unless otherwise indicated, can include any of the above with a carbon substituted with a heteroatom (e.g., O, S, N).

[0168] The term “heterocyclic” or “heteroaryl”, as used herein, unless otherwiseindicated, can include an aromatic or non-aromatic cycloalkyl in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S, and N. Representative examples of a heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1,4)- dioxane, (1,3)-dioxolane, 4,5-dihydro-1H-imidazolyl, or tetrazolyl. Heterocycles can be substituted or unsubstituted. Heterocycles can also be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heterocyclic ring). A heterocyclic can be saturated, partially saturated, or unsaturated. The “heterocyclic” can be optionally substituted.

[0169] The term “indole”, as used herein, is an aromatic heterocyclic organic compoundwith formula C₈H₇N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. The “indole” can be optionally substituted.

[0170] The term “cyano”, as used herein, unless otherwise indicated, can include a -CNgroup. The “cyano” can be optionally substituted.

[0171] The term “alcohol”, as used herein, unless otherwise indicated, can include acompound in which the hydroxyl functional group (-OH) is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms. The “alcohol” can be optionally substituted.

[0172] The term “solvate” is intended to mean a solvate form of a specified compoundthat retains the effectiveness of such compound. Examples of solvates include compounds of the disclosure in combination with, for example, water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.

[0173] The term “mmol”, as used herein, is intended to mean millimole. The term“equiv”, as used herein, is intended to mean equivalent. The term “mL”, as used herein, is intended to mean milliliter. The term “g”, as used herein, is intended to mean gram. The term “kg”, as used herein, is intended to mean kilogram. The term “µg”, as used herein, is intended 36 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO to mean micrograms. The term “h”, as used herein, is intended to mean hour. The term “min”, as used herein, is intended to mean minute. The term “M”, as used herein, is intended to mean molar. The term "µL", as used herein, is intended to mean microliter. The term “µM”, as used herein, is intended to mean micromolar. The term “nM”, as used herein, is intended to mean nanomolar. The term “N”, as used herein, is intended to mean normal. The term “amu”, as used herein, is intended to mean atomic mass unit. The term “°C”, as used herein, is intended to mean degree Celsius. The term “wt / wt”, as used herein, is intended to mean weight / weight. The term “v / v”, as used herein, is intended to mean volume / volume. The term “MS”, as used herein, is intended to mean mass spectroscopy. The term “HPLC”, as used herein, is intended to mean high performance liquid chromatograph. The term “RT”, as used herein, is intended to mean room temperature. The term "e.g.", as used herein, is intended to mean example. The term “N / A”, as used herein, is intended to mean not tested.

[0174] As used herein, the expression “pharmaceutically acceptable salt” refers topharmaceutically acceptable organic or inorganic salts of a compound of the disclosure. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt can involve the inclusion of another molecule such as an acetate ion, a succinate ion, or another counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt can have more than one charged atom in its structure. In instances where multiple charged atoms are part of the pharmaceutically acceptable salt, the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion. As used herein, the expression “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the disclosure. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. As used herein, the expression “pharmaceutically acceptable hydrate” refers to a compound of the disclosure, or a salt thereof, that further can include a 37 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. Examples

[0175] Chronic pain impacts over 10% of the U.S. population and leads to considerabledisabilities.4Current non-opioid treatments for chronic pain such as gabapentinoids, selective serotonin reuptake inhibitors (SSRI's), tricyclic antidepressants, and antiepileptics show low efficacy5while opioids1,6have abuse liability and are lethal when overdosed. This highlights an urgent need to develop a safer and more effective therapeutics for chronic pain.

[0176] The cannabinoid receptor type 1 (CB1) is a non-opioid target of particular interestas it is expressed throughout the pain neuraxis, and CB1 agonists are analgesic in several different animal pain models7,8. Several synthetic cannabinoid receptor agonists (SCRAs) that potently and efficaciously target CB1 receptors have recently been discovered2,9.

[0177] There are two key obstacles to using SCRAs to treat chronic pain: central nervoussystem (CNS) side-effects10–12and tolerance13,14. Activation of CB1 receptors in the CNS results in undesired side effects including psychoactivity in humans, as well as catalepsy and hypothermia in rodents15. To circumvent these issues, peripherally-selective CB1 agonists have been developed16,17such as CB1318–20and PRNMI21. However, these agonists still exhibit signs of central CB1 engagement at functionally relevant doses; there is a need to substantially enhance the peripheral selectivity. A second key obstacle is tolerance, which limits the long-term effectiveness of SCRAs13,14,22as well as peripheral CB1 agonists described in the literature18,23. Prior work suggests tolerance can be mediated by receptor interactions with β-arrestins24–28.25However, most SCRAs strongly stimulate the β-arrestin pathway.29

[0178] We sought to leverage the high potency and efficacy of SCRAs as analgesics whileminimizing side effects and tolerance by rationally designing a new generation of CB1 agonists that are peripherally restricted and that minimize β-arrestin-2 signaling. First, we reasoned that increasing ligand charge would decrease CNS penetrance. Yet, in the available molecular structures of CB1, there was little space in the binding pocket to accommodate such a modification or expansion. Remarkably, using molecular dynamics simulations, we uncovered a cryptic pocket30,31that spontaneously opens beneath the conventional CB1 ligand binding site to reveal the conserved, negatively charged D1632.50residue, which has been shown to modulate biased signaling in other 38 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO receptors3,32. By positioning a positively charged functional group on the agonist to interact with this newly uncovered site, we hypothesized that we could efficiently achieve both design aims while maintaining high potency and efficacy.

[0179] Using the highly potent FUB33 as the template ligand, we designed a series of novel CB1ligands that extend into the cryptic pocket through an alkyl linker with a terminal guanidino group. The lead ligand VIP36 displayed significantly reduced β-arrestin-2 recruitment relative to FUB while maintaining high affinity and G-protein-mediated efficacy. Cryo-electron microscopy (cryoEM) studies, and SAR analysis illuminated the molecular mechanism underlying these next generation agonists and revealed previously unknown CB1 conformational states with unique signaling properties.

[0180] Importantly, VIP36 showed potent peripheral CB1 receptor-dependent analgesia whileshowing limited analgesic tolerance. Signs of central CB1 receptor engagement such as hypothermia, catalepsy, sedation and tail flick antinoccieption were only evident at 100x the analgesic dose. In summary, we have used a variety of complementary techniques to rationally design a therapeutically desirable ligand for CB1 with potential for treating chronic pain safely and effectively. In the process, these molecules have served as powerful tools to uncover elusive mechanisms of cannabinoid receptor biased signaling which potentially can be extrapolated to other GPCRs more broadly. Computational ligand design

[0181] To develop a peripherally restricted agonist with minimal arrestin signaling, we sought tointroduce a charged functional group targeting the D2.50site of CB1 (Fig.1a). Our previous work onthe µ opioid receptor suggested that targeting this site within a GPCR core leads to reduced arrestinefficacy34,35. However, in all available structures of CB1, the presence of the bulky aromatic residues F2003.36and W3566.48(known as the toggle switch residues)36blocks access to D2.50from the orthosteric binding pocket37. Moreover, pharmacological experiments at CB1—in contrast to µOR and many other GPCRs—do not provide evidence of an Na+binding site adjacent to D2.50, raising further questions about the accessibility of D2.50(Extended Data Fig.1a).

[0182] To determine whether this site might be accessible in other receptor conformations, weperformed molecular dynamics simulations (MD) of CB1 bound to FUB. The simulations revealed the transient opening of a cryptic pocket that extends from the orthosteric binding pocket to the D2.50residue (Fig.1b). This previously unknown cryptic pocket opens rarely (8% of the simulation time) and necessitates the separation of the toggle switch residues, in particular a rotation of W3566.48(Fig. 1b). A ligand could potentially enter the cryptic pocket when it is transiently open and form favorable interactions that further stabilize it in the open conformation. The simulations therefore provided a 39 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO receptor conformation with an accessible D2.50site, supporting the feasibility of our approach and enabling further structure-guided design.

[0183] Using simulation frames revealing the cryptic pocket, we designed a family of ligandspredicted to extend into this site. Starting with the FUB scaffold, we incorporated a flexible linker ending in a positively charged head group (Fig.1c). Using docking and molecular modeling tools, we investigated several design variables: the linker length and identity, the attachment points on the scaffold, and the choice of charged head group. We selected the guanidine group as the head group because its high pKa would ensure a charged state at physiological pH, predicted to reduce CNS penetrance. We then docked multiple guanidine-containing compounds to the receptor structure identified in the simulation. The ligand (compound 8, VIP36) predicted to bind best while simultaneously forming a tight interaction with D2.50consisted of a four carbon O-alkyl linker connecting the guanidine group to the indazole ring of FUB (Fig.1c, Extended Data Fig.1b). Synthesis and in vitro characterization of next-generation CB1 ligands

[0184] We synthesized a broad range of FUB analogs with a guanidine group in the tail toconfirm the predictions from our structural model. The chemical structures of all novel ligands synthesized for this study are shown in Extended Data Fig.2 and synthetic scheme for the lead entity is shown in Extended Data Fig.2. Initially we explored various attachment points on the FUB scaffold. To this end, we attached four carbon length linkers to the 4, 5, 6 and 7 positions of the indazole ring through an ether linkage, resulting in compounds 8, 14, 21 and 27 (as shown in Extended Data Fig.3).

[0185] The ligands with 4-carbon linkers at different attachment points on the ring i.ecompounds 8, 14, 21, 27, were characterized for G-protein activity using the cAMP9,38assay in CB1 transfected HEK293Tcell lines. Among the substitutions, the highest efficacy wasobserved for G- protein signaling when the C6 position on FUB was diversified (8, VIP36) (Emax=106%, EC50=2.17 nM) (Extended Data Table 1). This was consistent with the computational model, as this position on FUB scaffold points toward the cryptic pocket (Fig.1b). We further evaluated the effect of the linker length at the C6 position with lengths ranging from 2- to 8-carbons. We assessed these variations using both G-protein (cAMP)38and BRET based β-arrestin-2 recruitment38,39assays. While the potencies in the cAMP assay were not substantially different between the linker lengths, the potency and efficacy of β-arrestin-2 recruitment were greatly reduced at n=4 i.e. VIP36, showing large separation between G-protein and β-arrestin-2 potency and efficacy, as well as optimal G-protein bias (Fig.2a, Fig.2b, cyan, Extended Data Table 1 and (Fig.5). This is consistent with a linker length necessary to engage the cryptic pocket (Fig.1c). FUB on the other hand was found to be a super- agonist (Fig.2b-a) robustly recruiting β-arrestin-2 (Emax=178% vs Emax=47% for VIP36). An in 40 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO vitro GTPase assay, further demonstrated that VIP36 acted as a full agonist of G-protein activation relative to FUB for Gi1(Extended Data Fig.4b) as observed in the cAMP as well (Extended Data Fig.4a) and appeared to be biased for Gi1and Gi3Gα-protein subtypes (Extended Data Fig.4c). Furthermore, VIP36 showed no detectable recruitment of β-arrestin-1 to CB1 (Extended Data Fig. 4d). VIP36 retained high affinity for the receptor in a radioligand competition assay (ki= 22 nM for VIP36 vs ki= 75 nM for FUB) (Extended Data Fig.4e). To test the importance of the linker for dampening arrestin recruitment, we synthesized two analogs based on FUB substituted for either -OCH3 (4) or -OH (5). Both the analogs showed lower G protein efficacy compared to VIP36, with 5robustly also recruiting β-arrestin-2 (Extended Data Table 1 and Extended Data Figure 2).

[0186] Our experimental data thus supports our computational prediction that VIP36 has theoptimal linker position and length to open the toggle switch residues, engage the cryptic pocket, and interact with D2.50, leading to vastly reduced arrestin efficacy compared to the parent FUB as well as the control CP955,40. Structural and computational characterization of VIP36-bound CB1

[0187] To investigate VIP36 interactions with CB1 and validate the proposed bindingmechanism, we determined a cryoEM structure of VIP36 bound to Gi1-coupled CB1 (Fig.2c, Extended Data Fig.5, SI Table S1) at a nominal resolution of 2.86 Å. A conserved set of interactions with the FUB portion of the agonist are present in the two structures, including direct packing of the second extracellular loop (ECL2) via F268ECL2and a π-π interaction with the key toggle switch residue F2003.36(Fig.2d). The FUB moiety in VIP36 overlays well with the previously determined FUB-bound CB1 structure (PDB: 6N4B)36(Fig.2e). The well-defined cryoEM density for the guanidinium group in VIP36 shows that it is positioned in the predicted cryptic pocket (Fig. 1b). Though the density for VIP36 is slightly discontinuous between the FUB moiety and the charged tail, the chosen pose fits the density well, is in line with previous structural work on FUB alone and issupported by MD simulations (Extended Data Figure 7a). The "conventional active" conformationof the toggle switch residue W3566.48, as observed in the FUB structure, is incompatible with the guanidinium linker in VIP36 (Fig.2d, clash). To accommodate the linker, W3566.48moves outward to the conformation predicted from initial MD simulation studies (Fig.1b, Fig.2d). This movement leads to an "active-open" conformation of W3566.48and an outward shift of TM3 (V2043.40) to accommodate this new rotamer state. The guanidinium group on VIP36 does not directly contact D2.50(Fig.2d) in the cryoEM structure. Rather, it is involved in cation-π interactions with the toggle 41 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO switch residues (4.0 and 4.4 Å to W3566.48and F2003.36, respectively) and forms a strong hydrogen bond with N3897.45(3 Å) (Fig.2d). D2.50remains ~6-7 Å removed from the guanidinium group precluding a typical salt bridge interaction (Fig.2d), at least in the single conformational state observed from cryoEM. This is a notable departure from our previous work with µOR, where bitopicligands designed to interact with D2.50, showed clear ionic interactions34.

[0188] To probe these interactions further and the possible role of buried waters, we conductedMD simulations using our cryo-EM structure of VIP36-CB1 embedded in a hydrated lipid bilayer. VIP36 remained stable in the same general binding pose as in the cryoEM structure (Extended Data Figure 7a). However, multiple distinct conformations of the guanidinium group and linker are observed via MD simulations; VIP36 most frequently samples longer-range, water-mediated interactions with D2.50(similar to the position seen in the cryo-EM structure) while also sometimes directly engaging D2.50via short-range (< 3.5Å) charge-charge interactions (Extended Data Fig.6a). Mechanism of G-protein Bias through Cryptic Pocket Engagement

[0189] To unravel the precise molecular mechanism underlying the G-protein bias of VIP36, weexplored the effects of altering the ligand head group (Fig.2f). Interestingly, changing the charge or polarity in the ligand tail had drastic effects on arrestin recruitment. Removing the charge, byreplacing the guanidino group of VIP36 with either plain carbon chains ((42), (43)) or introducingphenyl rings ((44), (45)), led to high arrestin recruitment (Fig.2f). Both of these groups are predicted to keep the cryptic pocket open through steric affects (and π-π stacking with the toggle switch residues in the case of the phenyl rings), suggesting cryptic pocket opening alone is not sufficient for reduced arrestin recruitment. While positively charged amine analogs ((46), (47)) retained low arrestin efficacy, polar analogs like -CN (48), -OH (49) and even -COOH (50) showed >100% arrestin efficacy (Fig.2f and Extended Data Table 1). A bioisotere of guanidine group like squarimide (52) showed higher arrestin efficacy compared to VIP36 suggesting that a positive charge in the tail or greater basicity of the functional group at tail end was essential for maintaining diminished arrestin recruitment. The most dramatic difference in activity was seen with the urea head group (VIP2.33) (53) which substituted the imine nitrogen in VIP36 with an oxygen. The compound reverted arrestin efficacy to a level similar to that observed for FUB (VIP2.33 Emax=200%, FUB Emax=178%, VIP36 Emax=47%) (Extended Data Table 1). These results were consistent across multiple assays and conditions (BRET, TANGO, Extended Data Table 1-2).39 38The previously designed, peripherally targeted CB1 agonist CB13 shows high efficacy at β-arrestin-2 (Extended Data Table 1-2). VIP2.33 retained high efficacy at β-arrestin-1 pathway, similar to FUB (Extended data Fig.4d). Taken together, the SAR developed by substituting the polar and charged guanidino group with other neutral 42 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO but polar residues suggests that positive charge in the tail group reduces arrestin efficacy and can be a mechanism for reduced arrestin recruitment seen with VIP36.

[0190] A single atom substitution between the guanidinium group of VIP36 and the urea groupof VIP2.33 results in a ~4-fold increase in arrestin recruitment (Fig.2f), resulting in an arrestin profile similar to that of the original FUB template (Fig.3a, 2a, 2b). To identify the precise molecular mechanism of this dramatic change in signaling response, we determined the cryoEM structure of VIP2.33-bound CB1 (Extended Data Fig.5, SI Data Table S2). Though, the FUB moiety in VIP36 and VIP2.33 overlay well (Fig.2g), some key differences were observed. Unlike with VIP36, the density of the linker of VIP2.33 is no longer visible (Fig.2g, right panel), suggesting a lack of a single conformation for the linker and the urea moiety. Similarly, while F2003.36displays well- defined density in the structures of both VIP36 and VIP2.33-bound CB1, W3566.48is less ordered in the presence of VIP2.33 (Fig.2g). Interestingly, previous work suggested that differing conformations of W3566.48might be a mechanism for biased signaling at CB1.26

[0191] Because our cryoEM studies suggested that VIP2.33-bound CB1 displays extensiveconformational sampling of both the ligand tail and the toggle switch, we used MD simulations to obtain, a dynamic view of the relationship between local interactions and biased signaling in CB1. We compared the molecular interactions and effects of our G protein–biased lead (VIP36) and the arrestin-biased analog (VIP2.33) with those observed for FUB.

[0192] Most notably, VIP36 interacted more strongly with the D2.50 side chain than didVIP2.33 (Fig. 3c). This is consistent with the fact the guanidino group of VIP36 can form an ionicinteraction with the D2.50sidechain, whereas the urea group of VIP2.33 engages it more weakly through occasional hydrogen bonding. In contrast to the differing interactions at the base of the cryptic pocket, the FUB scaffolds of VIP36 and VIP2.33 adopted similar poses, and both ligands maintained the separation of toggle switch residues (Extended Data Fig.6c). Thus, mere opening of the cryptic pocket would not be a sufficient mechanism to explain the unique biased signaling profile of VIP36. Furthermore, both VIP36 and VIP2.33 resulted in similar conformations of W3566.48(Extended Data Fig.6d), which suggests that possibleinteractions between VIP36 and W3566.48arealso not driving G-protein bias.

[0193] How do the differing ligand-protein interactions within the cryptic pocket ultimately leadto biased signaling? We found that with VIP36 bound, the neighboring transmembrane helix 7 (TM7) is stabilized in one conformation, whereas with VIP2.33 bound, TM7 preferentially adopts an alternative, rotated conformation (Fig.3d-e). This is notable because TM7 connects to the intracellular, transducer binding site and because TM7 conformation has been shown to modulate biased signaling in other GPCRs.40,41In particular, the alternative TM7 conformation seen with 43 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO VIP2.33 bound has been specifically associated with arrestin-biased signaling at other GPCRs. Our simulations suggest that VIP36 interacts both directly with D2.50and TM7 (Extended Data Figure 7c), creating a network of contacts that strongly influence TM7 conformation. The importance of a positively charged functional group (whether guanidinium or amine) for G-protein bias in the SAR studies (Extended Data Table 1) supports that the ionic interaction observed between the ligand and D2.50is critical to forming this network of interactions. Thus, the simulations and SAR together suggest a structural mechanism by which the interactions between the ligand, D2.50, and TM7 ultimately control the intracellular receptor conformation to elicit signaling bias.

[0194] In addition to differences observed in TM7 from simulation, we were interested inprobing the dynamics of the pivotal intracellular region of TM6, another region previously shown to demonstrate significant bias-dependent changes in conformation in the angiotensin II type 1 receptor.42To do this, we site-specifically introduced the environmentally sensitive fluorophore monobromobimane (bimane) at the intracellular end of TM6 (residue 336) (Extended Data Fig.6b). Indeed, our fluorescence experiments show that VIP36 and VIP2.33 have divergent effects on the conformational ensemble of the intracellular region of TM6. FUB binding results in a decrease in fluorescence and right-shift in lambda max, consistent with an outward movement and resultant solvent exposure of TM6. VIP36 binding results in an even further down- and right-shift in fluorescence intensity while VIP2.33 reverts the profile to a more apo-like spectrum (Extended Data Fig.6b). The divergent behavior between VIP36 and VIP2.33 in these bimane experiments is consistent with the two ligands stabilizing distinct sets of intracellular conformations that differentially engage Giand arrestin. VIP36 produces analgesia that is dependent on peripheral CB1 receptors

[0195] Before testing our lead CB1 bitopic VIP36 in vivo, we first carried out in vitro ADMEassays. These include evaluating plasma and brain binding, plasma stability and membrane permeability using PAMPA (parallel artificial membrane permeation assays) and MDCK (Madin- Darby canine kidney) (SI Table S3). VIP36 showed high plasma stability in both mouse and human species (SI Table S3a). In PAMPA, an assay which mimics gut wall permeability, the drug showed poor permeability compared to established controls. The poor passive diffusion could be due to the high polarity and the highly basic guanidine group. In MDCK assays which mimic BBB, VIP36 was found to have high efflux ratio similar to quinidine suggestive of a possibility that it can be a substrate for P-gp (SI Table S3b). We next conducted pharmacokinetic assays (PK) to measure the compound's distribution in plasma and brain tissues. These assays involved VIP36, the previously reported peripherally restricted cannabinoid agonist CB1343and FUB2(Fig.4a and Extended Data Fig.8a- 44 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO d). VIP36 was detected at higher concentrations in plasma than the brain with an unbound brain-to-plasma partition coefficient (Kp,uu,brain) of 0.0012 indicating peripheral selectivity (Fig. 4a) and (SITable S3c). FUB was found to be brain penetrant with a coefficient of 2.95 while CB13 showed a coefficient of 0.016. Thereby, VIP36 was more than 2400-fold more peripherally restricted over the parent template (FUB), and about 13-fold more restricted than CB13. VIP36 PK was also looked at using the IV route given the known variability associated with the IP route and drug again showed high peripheral selectivity in this assay as well (Extended Data Fig.8d) The peripheral selectivity seen is consistent as well with high efflux seen in the MDCK assays in vitro, which will effectively eliminate CNS exposure. No intact drug was however found in PK in either plasma or brain using the PO route consistent with PAMPA assay results.

[0196] We next sought to evaluate VIP36's analgesic efficacy using three distinct in vivo painmodels: The Complete Freund's adjuvant (CFA) model of inflammatory pain, the SNI (spared nerve injury) model of neuropathic pain and the chronic nitroglycerin (NTG) model of headache pain55.

[0197] Injection of CFA into the hind paw resulted in mechanical allodynia. VIP36administration reversed CFA-induced mechanical allodynia in a dose- and time-dependent manner (Fig.6a), however the high degree of variability in this model reduces confidence in the calculated ED50(Fig.6b). In the neuropathic pain model, VIP36 administration also reversed SNI-induced mechanical allodynia (Fig 4b) in a dose (0.03, 0.1, 0.3, 1, 3 mg / kg I.P.) and time dependent manner (Fig.6c). The ED50of VIP36 was 0.22 mg / kg (95% confidence interval: 0.01-12.64 mg / kg) (Fig.4b) and the peak effect occurred at 1 hour post injection. Repeated nitroglycerin injections, an established chronic migraine model44, produced cephalic allodynia, which was also attenuated by administrationof VIP36(8) (1 mg / kg I.P.; (Fig. 4c). In this model, VIP36 appears quite effective at suppressingchronic NTG-induced cephalic allodynia as a post-treatment. Interestingly, in a different migraine model, another peripherally restricted CB1 ligand45was only effective as a pre-treatment but not as a post-treatment. Combined, our data demonstrate that acute administration of VIP36 shows analgesic activity in models of inflammatory pain, neuropathic pain and migraine pain. The SNI model results in a long-lasting allodynia which is amenable to repeated dosing studies. Therefore, we chose this model as our primary endpoint for evaluating VIP36's analgesic activity.

[0198] We next evaluated CB1 subtype selectivity as well as selectivity at other targets. VIP36's(1 mg / kg I.P.) anti-allodynic efficacy was blocked by the peripheral CB1 antagonist AM6545 (10 mg / kg I.P.) but not the global CB2 antagonist, AM630 (3 mg / kg I.P.)(Fig 4E). Neither antagonist produced changes in mechanical thresholds alone. Similar results were obtained in the CFA assay (Fig.6d). These data indicate that VIP36's anti-allodynic effects are dependent on peripheral CB1 receptors (Fig.4d). Functional selectivity for CB1 over CB2 receptors was also confirmed in-cell 45 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO cAMP assays and biochemical GTP turnover assays (Extended Data Fig.8ef). Our structure of VIP36-bound CB1 provides a rationale for this observed subtype selectivity. While CB1 has an aliphatic residue in TM5 (L2765.51) that accommodates the extreme outward movement needed for the W3566.48to occupy the "active-open" conformation of the toggle switch, CB2 has a bulky aromatic in this position (F2875.51) that acts to sterically block formation of the cryptic pocket (Extended Data Fig.8g). The selectivity over a broad range of targets was tested in binding assays (~45 CNS targets) as well as functional assays (320 targets) through PDSP-NIMH screening46(Fig. 7-8). and cardioalerts like hERG (Fig.8d) and 5HT2A (Fig.8e). Other off targets identified included MOR (Fig.7a-b) and SSTR4 (Fig.8a-c). Follow up assays revealed VIP36 had reasonable to high selectivity for CB1 over all targets probed (See Fig.7a-b, Fig.8a-e). VIP36 shows limited tolerance and does not engage central CB1 receptors at therapeutic doses

[0199] Cannabinoid agonists, including those that are peripherally selective, produce efficacy inmodels of pain initially, but form tolerance to the anti-allodynic effects over repeated dosing12,18. We hypothesized that the decreased arrestin recruitment observed for VIP36 (Fig.2b) would reduce the development of analgesic tolerance following repeated dosing. SNI mice were administered VIP36 (1 mg / kg, IP) twice daily over 9 days. VIP36 reversed SNI-induced allodynia, as before, and this effect was sustained through all 9 days of testing while FUB started showing signs of tolerance by day 3 (Fig.4e and Fig.6e). In contrast, CB13 induced significant tolerance over repeated dosing, with an approximately 50% reduction in effect18Such tolerance effects47–55have also been well-documented for cannabinoid agonists, thus highlighting the unique functional effects of VIP36.

[0200] Engagement of central CB1 receptors produces antinociception as well as so-called"tetrad" behaviors which include hypothermia, altered locomotor activity, catalepsy and thermal antinociception using the tail flick assay.56VIP36 was administered at supratherapeutic doses (3, 10, 20, 30 mg / kg, I.P.) and evaluated for tetrad behaviors to test for the functional engagement of central CB1 receptors. VIP36 induced catalepsy, hypothermia and thermal antinociception in a tail flick assay only at doses >100 times the ED50,specifically at 30 mg / kg, IP. (Fig.4f, 4i). Hypothermia emerged at slightly lower doses (20 and 30 mg / kg dose; Fig.4g). Locomotor changes were tested at 3 mg / kg (I.P., 15x ED50dose) and no alterations were observed (Fig.4h). The lack of sedation separates VIP36 from another promising peripherally restricted agonist, PrNMI, which showed mild sedation at analgesic doses.21,45,57,58These data indicate VIP36 is not engaging central CB1 receptors at therapeutic doses. 46 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0201] We sought to directly compare the in vivo pharmacology of VIP36 to the parentcompound FUB. In SNI mice FUB produced a dose dependent anti-allodynia affect with an ED50of 0.01 mg / kg (Fig.4b). FUB produced significant catalepsy at 0.1 mg / kg (Fig.4f), and hypothermia at 0.03 mg / kg (Fig.4g). and antinociception (Fig.4i) Anti-allodynic and anti-nociceptive responses to FUB are confounded by the cataleptic actions seen at doses of 0.1 mg / kg and above. This explains the observation that FUB resulted in von Frey thresholds well above baseline at 0.3 mg / kg, consistent with a sedative-like effect (Fig 4b). Consistent with this notion in two separate experiments, FUB significantly decreased locomotion (0.01 mg / kg, analgesic ED50as well as at 0.03 mg / kg) and 0.1 mg / kg (Fig.4h and Fig.6f), showing no separation between the analgesia and central CB1 receptor engagement. These data indicate that, unlike FUB, VIP36 produces efficacy across several different pain models without engaging central CB1 receptors. DISCUSSION

[0202] CB1 receptors represent a viable target for developing non-opioid analgesics.Unfortunately, activation of CB1 produces unwanted centrally mediated effects such as psychoactivity and potentially arrestin-mediated effects such as tolerance which limit its therapeutic window.56Previous efforts to develop peripherally restricted CB1 agonists have been hindered by their tendency to penetrate the central nervous system at doses close to their analgesic thresholds. This underscores the need for novel molecules with enhanced peripheral selectivity. Here, we set out to design a peripherally selective CB1 agonist with reduced β-arrestin-2 recruitment to test the hypothesis if we can consolidate beneficial analgesic effects of highly potency SCRAs while avoiding adverse effects mediated by central activity and arrestin recruitment.

[0203] Our early computational studies identified a cryptic pocket and suggested we might beable to exploit it in order to access the conserved D2.50residue. Computational ligand design and extensive medicinal chemistry optimization, led to the identification of VIP36, which contains a charged guanidino group to both limit central penetration and engage the D2.50site, limiting arrestin recruitment. In contrast to the parent FUB, VIP36 had greatly reduced arrestin efficacy but maintained high G-protein activation. CryoEM studies of VIP36 bound CB1 / Gi-protein complex showed that the ligand was indeed able to stabilize the cryptic pocket, confirming our computational models. Through a systematic structure-based medicinal chemistry approach, we were able to demonstrate that interactions of the ligand tail with D2.50drive bias for G-protein over arrestin in the cannabinoid receptor system.

[0204] VIP36 produced analgesic actions in models of inflammatory, neuropathic and migrainepain. Strikingly, VIP36 maintained anti-allodynic efficacy throughout all 9 days (18 total injections) 47 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO of dosing in the SNI model. This contrasts to both FUB and our previous report using CB13, which developed tolerance in an inflammatory pain model18. The limited tolerance is likely due to the decreased arrestin recruitment of VIP36 (Emax=47%) compared to CB13 (Emax=175%) and FUB (Emax=178%) (Extended Data Table.1),24,25. Importantly, the extent of peripheral restriction seen for VIP36 results in at least a 100-fold separation between analgesic effects and the onset of adverse effects like hypothermia. FUB, our parent template, though highly potent as an analgesic, showed sedation at analgesic ED50doses, limiting its therapeutic value.

[0205] In summary, we demonstrate the rational design of a peripherally restricted CB1-targeting analgesic. This analgesic (VIP36) exhibits efficacy across a variety of peripheral pain models while showing limited tolerance and demonstrates a more favorable side-effect profile relative to typical SCRAs. Our study also provides extensive insight into a mechanism of biased signaling at CB1, which is likely shared by other GPCRs. Our findings not only contribute to the development of CB1-targeted therapeutics but also enhance the broader understanding of GPCR ligand design and can enable the rational design of functionally and / or peripherally selective ligands at other GPCRs. REFERENCES

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[0264] Chemistry Reagents purchased from Sigma-Aldrich Chemicals, Fisher scientific,Alfa Aesar, TCI, Ambeed Inc, AA blocks, were used without further purification. While performing synthesis, reaction mixtures were purified by silica gel flash chromatography on E. Merck 230–400 mesh silica gel 60 using a Teledyne ISCO CombiFlash Rf instrument with UV detection at 280 and 254 nm. RediSep Rf silica gel normal phase columns were used with a gradient range of 0–100% MeOH in DCM and EtOAc in hexane. Reported yields are isolated yields upon purification of each intermediate. Final clean (purity ≥95%, LC-MS Agilent 1100 Series LC / MSD) compounds were used for the study. NMR spectra were collected using Varian 400 MHz and 500 MHz NMR instrument at the NMR facility of Washington University School of Medicine in St. Louis collected via the Bruker Topspin Software (Bruker Topspin 3.5 pI 6). Chemical shifts are reported in parts per million (ppm) 52 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO relative to residual solvent peaks at the nearest 0.01 for proton and 0.1 for carbon (CDCl31H: 7.26,13C: 77.1; and CD3OD 1H: 3.31, 13C: 49.0). Peak multiplicity is reported as the NMR spectra were processed with MestreNova software 14.1.1-245751, namely s – singlet, d – doublet, t – triplet, q – quartet, m – multiplet for examples. Coupling constant (J) values are expressed in Hz. Mass spectra were obtained at the St. Louis College of Pharmacy using the Agilent 1100 Series LC / MSD by electrospray (ESI) ionization with a gradient elution program (Ascentis Express Peptide C18 column, acetonitrile / water 5 / 95 / 95 / 5, 5 min, 0.05% formic acid) and UV detection (214 nM / 254 nM). High resolution mass spectra were obtained using a Bruker 10 T APEX -Qe FTICR-MS and the accurate masses are reported for the molecular ion [M+H]+. Detail experiments and characterization of the new compounds are included in the supporting information section.

[0265] We synthesized the library of ligands based on the FUB template aiming to targetD2.50 residue present in cryptic pocket in allosteric sodium binding pocket.021053 / WO WSTL021053.WO

[0266] General procedure A: xy-1H-indazole-3-carboxylate (2): Toa stirred solution of 6-methoxy-1H-indazole-3-carboxylic acid 1 (0.5 g, 2.59 mmol) in MeOH (20 mL), was added TMSCl (0.64 mL, 5.18 mmol) and the reaction was stirred at 52 °C for 12h. After completion of the reaction, solvent was evaporated off, obtained residue subjected to flash column chromatography, product was eluted with EtOAc:hexane (1:1) yielded compound 2 (0.45 g, 84%).1H NMR (500 MHz, CDCl3) δ 8.08 (d, 1H, H-4), 7.05 – 6.96 (m, 2H, H-7,5), 4.05 (s, 3H, Ar-OCH3), 3.90 (s, 3H, OCH3).

[0267] General procedureenzyl)-6-methoxy-1H-indazole-3-carboxylate (3) : To a stirred solution of compound 2 (0.4 g, 1.95 mmol) in THF (20 mL),were added Cs2CO3 (0.95 g, 2.92 mmol), 4-fluorobenzylbromide (0.27 mL, 2.34 mmol), resulting suspension was stirred at 54 °C for 12h. After completion of reaction, it was diluted with H2O (20 mL) and extracted with EtOAc (20 mL×3), combined organic layers were dried over anhydrous Na2SO4,and solvents were evaporated off under reduced pressure. The crude product obtained was purified by flash column chromatography using EtOAc:hexane (2:8) yielded product 3 (0.38 g, 62%).1H NMR (500 MHz, CDCl3) δ 8.08 (d, J = 10.0 Hz, 1H, H- 4), 7.22 – 7.16 (m, 2H, o-Ar), 7.02 – 6.94 (m, 3H, H-5, m-Ar), 6.62 (d, J = 2.5 Hz, 1H, H-7), 5.60 (s, 2H, CH2-Ar), 4.03 (s, 3H, Ar-OCH3), 3.80 (s, 3H, OCH3). Other isomer was eluted as mixture with 4-fluorobenzylbromide.

[0268] General procedure C: Muorobenzyl)-6-methoxy-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (4) : The methyl ester 3 (0.4 g, 1.27 mmol) was dissolved in THF:H2O (6 mL, 4:1) at 0 °C, LiOH (2N, 7 mL, 6.36 mmol) was 54 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO added and the reaction mixture was stirred at rt for overnight. After completion of the reaction, it was acidified to pH (3-4) with 1N HCl and extracted with EtOAc (20 mL×3), the organic layer was washed with brine and dried over anhydrous Na2SO4.Solvents were evaporated off under reduced pressure, and obtained crude acid was used in the next step without further purification.

[0269] The crude acid was dissolved in DMF (5 mL), and HATU (0.73 g, 1.91 mmol),(S)-tert-leucine methyl ester (0.35 g, 1.91 mmol), Et3N (0.54 mL, 3.82 mmol) were added sequentially, and the reaction was stirred under argon atmosphere for 24h. Reaction mixture was diluted with ice cold H2O (20 mL) and extracted with EtOAc (20 mL×3), combined organic layer were washed with water (2×10 mL) and brine (10 mL). The organic solvents were dried over anhydrous Na2SO4 and evaporated off under reduced pressure, obtained crude residue was purified by flash column chromatography using EtOAc:hexane (2:8) to give a compound 4 (0.32 g, 60%).1H NMR (500 MHz, CDCl3) δ 8.16 (J = 5.0 Hz, 1H, H-4 ), 7.55 (d, J = 10.0 Hz, 1H, NH), 7.20 – 7.09 (m, 3H, o-Ar, H-5), 7.01 – 6.95 (m, 2H, m-Ar), 6.57 (d, J = 2.0 Hz, 1H, H-7), 5.49 (s, 2H, CH2-Ar), 4.71 (d, J = 10.0 Hz, 1H, H'-(C-tBu)), 3.77 (s, 3H, Ar-OCH3), 3.74 (s, 3H, OCH3), 1.08 (s, 9H, (CH3)3).

[0270] General procedure D:-fluorobenzyl)-6-hydroxy-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (5): Compound 4 (1 g, 2.34 mmol) wasdissolved in CH2Cl2 (10 mL) at 0 °C, BBr3 (7 mL, 7.02 mmol) was added dropwise and resulting reaction mixture was stirred at rt for 3h. Reaction was quenched with ice cold water (10 mL) and organic layer was separated, aqueous layer was extracted with EtOAc (20 mL×3) and the combined organic solvents were dried over anhydrous Na2SO4 followed by evaporation in vacuum gave crude residue which was purified by flash chromatography using EtOAc:hexane (3:7) to give compound 5 (0.6 g, 61%).1H NMR (400 MHz, CDCl3) δ 8.14 (d,J = 8.0 Hz, 1H, H-4), 7.56 (d, J = 8.0 Hz, 1H, H- NH), 7.15 (d, J = 8.0, 2H, o-Ar), 7.01 –6.94 (m, 2H, m-Ar), 6.82 (d, J = 8.8, 1H, H-5), 6.64 (s, , 1H, H-7), 5.87 (s, 1H, OH), 5.44 (s, 2H, CH2-Ar), 4.69 (d, J = 12.0 Hz, 1H, H’-(C-tBu), 3.75 (s, 3H,-OCH3), 1.08 (s, 9H, (CH3)3). 55 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0271] General procedure E: ((tert-butoxycarbonyl)amino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (6): The above compound 5 (7 mg, 0.016 mmol) was dissolved in THF (2 mL), tert-butyl (4- bromobutyl)carbamate (4 mg, 0.019 mmol), TBAI (6 mg, 0.016) and Cs2CO3 (8 mg, 0.024) were added successively at room temperature and the reaction mixture was stirred at 53 °C for 8h. After completion of the reaction, solvent was evaporated off and residue was purified by flash column chromatography using EtOAc:hexane (3:7) gave compound 6 (7.5 mg, 80%).1H NMR (500 MHz, CDCl3) δ 8.19 (d, J = 10.0 Hz, 1H, H-4), 7.52 (d, J = 10.0 Hz, 1H, NH), 7.20 –7.15 (m, 2H, o-Ar), 7.05 –6.95 (m, 2H, m-Ar,), 6.91-6.81 (m, H-5), 6.58 (d, J = 3.0 Hz, 1H, H-7) 5.53 (s, 2H, CH2-Ar), 4.72 (d, J = 10.0 Hz, 1H, H’-(C-tBu)), 3.95 (t, J = 5.0 Hz, 2H, OCH2), 3.77 (s, 3H, OCH3), 3.23 – 3.15 (m, 2H, -NCH2), 1.86 – 1.71 (m, 2H, CH2), 1.71 – 1.63(m, 2H, ‘CH2), 1.45 (s, 9H, (Boc-(CH3)3,)), 1.09 (s, 9H, (CH3)3).

[0272] General procedure F:2,3-bis(tert-butoxycarbonyl)guanidino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3- dimethylbutanoate (7): Compound 6 (7.4 mg, 0.012 mmol) was dissolved in CH2Cl2 (2 mL) to it TFA (0.2 mL) was added and the reaction was stirred at rt for 2h. Solvent was evaporated off under vacuum and obtained residue was dissolved in MeOH:CH2Cl2 (3 ml 2:1), to this Et3N (0.03 mL, 0.36 mmol), N,N′-Di-Boc-1H-pyrazole-1-carboxamidine were added sequentially, resulting reaction mixture was stirred at rt for 12h. Solvent was evaporated off and residue was dissolved in EtOAc (10 mL) and water (5 mL), organic layer 56 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO was separated and aqueous layer was extracted with EtOAc (10 mL×2), combined organic layer were washed with brine (5 mL) and dried over anhydrous Na2SO4 followed by evaporation in vacuum gave crude residue which was purified by flash chromatography using EtOAc:hexane (3:7) yielded compound 7 (7.5 mg, 86%).1H NMR (400 MHz, CDCl3) δ 11.50 (s, 1H, NH Boc), 8.37 (s, 1H, NH), 8.18 (d, J = 12.0 Hz, 1H, H-4), 7.52 (d, J = 12.0 Hz, 1H, H-NH), 7.17 (d, J = 12.0, 2H, o-Ar ), 7.01 (t, J = 8.0 Hz, 2H, m-Ar ), 6.91 (bs, 1H, H-5), 6.57 (d, J = 1.2 Hz, 1H, H-7), 5.52 (s, 2H, CH2-Ar), 4.71 (d, J = 8.0 Hz, 1H, H’-(C- tBu) ), 3.95 (t, J = 5.6 Hz, 2H, OCH2), 3.76 (s, 3H, OCH3), 3.49 (q, J = 4.0 Hz, 2H, CH2-N), 1.88 – 1.82 (m, 2H, CH2), 1.81– 1.74 (m, 2H,‘CH2), 1.50 (s, 9H, (Boc-(CH3)3), 1.48 (s, 9H, (Boc-(CH3)3), 1.08 (s, 9H, (CH3)3).

[0273] General procedure G:fluorobenzyl)-6-(4-guanidinobutoxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate VIP36 (8): To a stirred solution ofcompound 7 (7 mg, 0.0096 mmol) in CH2Cl2 (2 mL) at 0 °C, was added TFA (0.5 mL) andthe resulting reaction mixture was stirred at rt for 18h. After completion of the reaction solvent was evaporated off and diluted with water (2 mL) and neutralized with NaOH (1N) solution, aqueous layer was extracted with EtOAc (10 mL×2). The organic layer dried over anhydrous Na2SO4 followed by evaporation in vacuum gave crude residue which was purified by flash chromatography using MeOH:DCM (2:8) yielded compound 8 (4 mg, 78%).1H NMR (400 MHz, CD3OD) δ 8.04 (d, J = 12.0 Hz, 1H, H-4), 7.31 – 7.25 (m, 2H, o- Ar), 7.06 (m, 2H, m-Ar), 6.98 – 6.90 (m, 2H, H-7, 5), 5.66 (s, 2H, CH2Ar), 4.57 (bs, 1H, H’- (C-tBu ), 4.06 (t, J = 8.0 Hz, 2H, CH2-O), 3.76 (s, 3H, OCH3), 3.26 (t, J = 6.9 Hz, 2H, CH2N), 1.89 – 1.75 (m, 4H, CH2, ‘CH2), 1.07 (s, 9H,(CH3)3).13C NMR (100 MHz, CD3OD) δ 171.2 (C=O), 162.9 (CO=NH), 159.3 (C=N, C6), 142.2 (Ar-F), 136.6 (C-3), 129.0 (Ci-Ar), 128.9 (C-4), 122.43 (Cm-Ar), 117.5 (Cm-Ar), 115.22 (C7), 115.0 (C9), 91.1 (C5), 67.4 (C- 57 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO O), 59.9 (C--tBu), 51.8 (OCH3), 40.8 (C-NH2), 34.3 (C-(CH3)3), 25.9 (CH3)3), 25.6(CH2), 25.4(‘CH2). HRMS calcd for C27H35FN6O4+H+, 527.2776; found, 527.2775.

[0274] Methyl 7-methoxy-1H-indazole-3-carboxylate (9): Following generalprocedure A, compound 7-methoxy-1H-indazole-3-carboxylic acid (0.5 g, 2.60 mmol),TMSCl (0.66 mL, 5.20 mmol) afforded product 9 (0.4 g, 75%).1H NMR (500 MHz, CDCl3) δ 7.77 (dd, J = 8.2, 2.8 Hz, 1H, H-4), 7.21 (t, J = 7.8 Hz, 1H, H-5), 6.77 (d, J = 7.7 Hz, 1H, H-6), 4.03 (s, 3H, CH3OAr), 3.97 (s, 3H, CH3O). O OMe

[0275] Methyl 1-(4-fluole-3-carboxylate (10):Following general procedure B, compound 18 (0.8 g, 3.86 mmol), Cs2CO3 (1.88 g, 5.79mmol), 4-flurobenzyl bromide (0.92 mL, 7.53 mmol) afforded the product 10 (0.8 g, 65%) and 10’(0.3 g, 24%).10:1H NMR (500 MHz, CDCl3) δ 7.68 (d, J = 8.2 Hz, 1H, H-4), 7.11(m, , 3H, H-5, o-Ar), 6.85 (t, J = 8.7 Hz, 2H, m-Ar), 6.64 (d, J = 7.7 Hz, 1H, H-6), 5.77 (s,2H, CH2Ar), 3.93 (s, 3H, CH3OAr ), 3.82 (s, 3H, OMe).

[0276] 10’: 1H NMR (500 MHz, CDCl3) δ 7.55 (d, J = 8.8 Hz, 1H, H-4), 7.36 (s, 1H, H-5), 7.24 – 7.16 (m, 1H, o-Ar), 7.02 – 6.91 (m, 2H, m-Ar), 6.64 (d, J = 8.3 Hz, 1H, H-6), 6.08 (s, 2H, CH2Ar), 4.05 (s, 3H, CH3OAr), 3.99 (s, 3H, OMe).58 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0277] Methyl(S)-2-(1-(4-fluorobenzyl)-7-methoxy-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (11): Following general procedure C, compound 10 (0.1 g, 0.31 mmol),LiOH (2N, 1.5 mL, 3.1 mmol, HATU (0.091g, 0.24 mmol), Et3N (0.068 mL, 0.48 mmol), (S)-tert-leucine methyl ester (0.036 g, 0.2 mmol), afforded the product 11 (0.025 g, 31%).1H NMR (500 MHz, CDCl3) δ 7.91 (dd, J = 10.0, 0.5 Hz, 1H, H-4), 7.54 (d, J = 10.0 Hz, 1H, H- NH), 7.25 – 7.21 (m, 2H, o-Ar), 7.15 (dd, J = 10.0, 5.0 Hz, 1H, H-5), 6.98 (d, J = 10.0 Hz, 2H, m-Ar), 6.74 (dd, J = 10.0, 0.5 Hz, 1H, H-6), 5.81 (s, 2H, CH2Ar), 4.73 (d, J = 10.0 Hz, 1H, H’-(C-tBu), 3.93 (s, 3H, CH3OAr), 3.77 (s, 3H, OMe), 1.09 (s, 9H, (CH3)3).

[0278] Methyl(S)-2-(1-(4-fluor oxy-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (12): Following general procedure D, compound 11 (0.05 g, 0.11 mmol), BBr3(0.2 mL, 0.19 mmol) afforded the product 12 (0.015 g, 32%).1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.2 Hz, 1H, H-4), 7.61 (d, J = 9.5 Hz, 1H, H-NH), 7.30 – 7.20 (m, 2H, o-Ar), 6.95 (m, 3H, m-Ar, H-5), 6.72 (d, J = 7.3 Hz, 1H, H-6), 5.85 – 5.69 (m, 2H, CH2Ar), 4.70 (d, J = 9.5 Hz, 1H, H’-(C-tBu), 3.74 (s, 3H, OCH3), 1.08 (s, 9H, (CH3)3).

[0279] Methyl (S)-2-(7-(4-((teramino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (13): Following general procedure E, compound 12 (0.022 g, 0.053 mmol), tert-butyl (4-bromobutyl)carbamate (0.020 g, 0.079 mmol), Cs2CO3 (0.026 g, 0.079 mmol) TBAI (0.02 g, 0.053 mmol) afforded the product 13 (0.023 g, 74%).1H NMR (500 MHz, CDCl3) δ 7.91 (d, J = 8.2 Hz, 1H, H-4), 7.52 (d, J = 9.7 Hz, 1H, H-NH), 7.18 – 7.09 (m, 3H, o-Ar, H-5), 6.97 (d, J = 8.7 Hz, 2H, m-Ar), 6.71 (d, J = 7.7 Hz, 1H, H-6), 5.84 (s, 2H, CH2Ar), 4.72 (d, J = 9.7 Hz, 1H, H’-(C-tBu), 4.08 (t, J = 6.3 59 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Hz, 2H, CH2O), 3.75 (s, 3H, CH3O), 3.14 (d, J = 6.2 Hz, 2H, ‘CH2N), 1.87 – 1.75 (m, 2H, ‘CH2), 1.58 – 1.51 (m, 2H, “CH2O), 1.45 (s, 9H, (Boc-(CH3)3), 1.08 (s, 9H, (C-tBu)).

[0280] Methyl(S,Z)-2-(7-(4-(2,3 arbonyl)guanidino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (14): Following general procedure F, compound 13(0.015 g, 0.025 mmol), TFA (0.2 mL), N,N′-Di-Boc-1H-pyrazole- 1-carboxamidine (0.023 g, 0.077 mmol), Et3N (0.02 mL, 0.15 mmol) afforded the product 14 (0.010 g, 56%). The obtained product was carried forward to the next step.

[0281] Methyl(S)-2-(1-(4-fluoranidinobutoxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (15): Following general procedure G, compound 14(9 mg, 0.012 mmol), TFA (0.2 mL) afforded the product 15 (4.5 mg, 71%).1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 9.0 Hz, 1H, NH), 7.77 (d, J = 8.2 Hz, 1H, H-4), 7.34 – 7.26 (m, 1H, H-NH), 7.19 – 7.13 (m, 2H, o-Ar ), 7.03 (t, J = 8.7 Hz, 2H, m-Ar), 6.90 (d, J = 7.6 Hz, 1H, H-6), 5.93 (s, 2H, NH”, NH”), 5.65 (d, J = 8.0 Hz, 1H, CH2Ar), 4.57 (d, J = 2.7 Hz, 1H, H’-(C-tBu)), 4.17 (t, J = 6.2 Hz, 2H, CH2O ), 3.76 (s, 3H, CH3O), 3.19 (t, J = 7.1 Hz, 2H, H2N), 1.92 – 1.81 (m, 2H, ‘CH2), 1.66 (dt, J = 14.8, 7.6 Hz, 2H, “CH2), 1.07 (s, 9H,(C-tBu).13C NMR (100 MHz, CD3OD) δ 171.6, (C=O)), 163.4 (CO=NH), 162.6 (C=N), 145.3 (C3) , 136.8 (C4, C8,), 115.1 ((i-Ar-)C9,), 114.9 ((o-Ar-), 113.6 (m-Ar-) 106.9 (C7), 90.3 (C5), 67.5 (C-OMe), 59.9 (C-O), 54.7 (C-Ar),40.7 (C-NH), 34.3 (‘C), 25.9 (C”), 25.6 (C-tBu), 25.3 C’”, HRMS calcd for C27H35FN6O4H+, 527.2776; found, 527.2773. 60 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0282] Methyl(S)-2-(5-metho arboxamido)-3,3-dimethylbutanoate(16): Followinggeneral procedure C, compound 5-methoxy-1H-indazole-3-carboxylic acid (0.4 g, 2.08 mmol), HATU (1.2 g, 3.12 mmol), Et3N (0.88 mL,6.24 mmol), (S)-tert-leucine methyl ester (0.58 g, 3.12 mmol), afforded the product 16 (0.32 g, 48%).1H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 8.8 Hz, 1H, H-7), 7.59 (s, 1H, H-4), 7.31 (d, J = 8.6 Hz, 1H, H-6), 6.96 (d, J = 8.9 Hz, 1H, H

[0283] N), 4.69 (d, J = 9.4 Hz, 1H, H’-(C-tBu) ), 3.75 (s, 6H, ArOMe, MeO), 1.05 (s,9H, (-tBu(H) ).

[0284] Methyl(S)-2-(1-(4-fluoxy-1H-indazole-3-carboxamido)-3,3-dimethyl butanoate (17): Following general procedure B, compound 16 (0.12 g, 0.37 mmol), Cs2CO3(0.18 g, 0.56 mmol), 4-fluorobenzyl bromide (0.08 mL, 0.56 mmol) afforded the product 17 (0.13 g, 65%).1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 1.8 Hz, 1H, H-7), 7.53 (d, J = 9.6 Hz, 1H, H-6), 7.13 (m, 3H, , H-C4, o-Ar ), 7.01 – 6.88 (m, 3H, m-Ar, NH’), 5.49 (s, 2H, CH2Ar), 4.69 (d, J = 9.6 Hz, 1H, H’-(C-tBu)), 3.80 (s, 3H, ArOMe ), 3.72 (s, 3H,-OMe), 1.06 (s, 9H, (-tBu(H))).

[0285] Methyl(S)-2-(1-(4-fluy-1H-indazole-3-carboxamido)-3,3-dimethyl butanoate (18): Following general procedure D, compound 17 (0.12 g, 0.28 mmol), BBr3 (0.8 mL, 0.8 mmol) afforded the product 18 (0.076 g, 66%).1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H, H-NH), 7.68 (d, J = 9.9 Hz, 1H, H-7), 7.13 – 7.03 (m, 3H, H-4, 61 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO oAr), 6.98 (d, J = 9.0 Hz, 1H, H-6), 6.90 (t, J = 8.5 Hz, 2H, H-o-Ar), 5.45 (s, 2H, H-m-Ar), 4.60 (d, J = 10.0 Hz, 1H, H’-(C-tBu)), 3.69 (s, 3H, -OMe ), 1.02 (s, 9H, tBu(H).

[0286] Methyl (S)-2-(5-(4- o)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (19): Following general procedure E, compound 18 (0.02 g, 0.048 mmol), tert-butyl (4-bromobutyl)carbamate (0.018 g, 0.072 mmol), Cs2CO3 (0.023 g, 0.072 mmol), TBAI (0.017 g, 0.048 mmol) afforded the product 19 (0.023 g, 74%).1H NMR (400 MHz, CDCl3) δ 7.64 – 7.59 (m, 1H, H-7), 7.47 (d, J = 9.7 Hz, 1H, H-6), 7.10 (m,3H, H-4, o-Ar), 7.00 – 6.89 (m, 3H, NH, m-Ar), 5.48 (s, 2H, CH2Ar), 4.64 (d, J = 9.6 Hz, 1H, H’-(C-tBu)), 3.95 (t, J = 6.2 Hz, 2H, CH2O), 3.70 (s, 3H, OCH3), 3.42 (s, 1H, NH), 3.12 (d, J = 6.1 Hz, 2H, H’-(NH2)), 1.75 (dt, J = 14.5, 6.3 Hz, 2H, ‘CH2), 1.59 (d, J = 7.6 Hz, 2H, H’-(“CH2)), 1.37 (s, 9H, H- Boc-(CH3)3), 1.02 (s, 9H, tBu(H).

[0287] Methyl(S,Z)-2-(5-l)guanidino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (20): Following general procedure F, compound 19 (0.015 g, 0.025 mmol), TFA (0.5 mL), N,N′-Di-Boc-1H-pyrazole- 1-carboxamidine (0.012 g, 0.038 mmol), Et3N (0.01 mL, 0.075 mmol) afforded the product 20 (9 mg, 50%).1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H, NH), 7.67 (m, 1H, H-7), 7.53 (d, J = 9.6 Hz, 1H, HN), 7.21 – 7.12 (m, 3H, H-4 m-Ar), 7.03 – 6.97 (m, 3H, H-6, o-Ar), 5.55 (s, 2H, CH2Ar), 4.71 (d, J = 9.6 Hz, 1H, H’-(C-tBu)), 4.03 (t, J = 6.2 Hz, 2H, CH2O), 3.76 (s, 3H, OCH3), 3.49 (q, J = 6.8 Hz, 2H, H’-(NH)), 1.84 (d, J = 7.7 Hz, 2H, H-‘CH2), 1.80 – 1.72 (m, 2H, H’-(“CH2)), 1.49 (s, 9H, H-Boc-(CH3)3), 1.48 (s, 9H, ‘H-Boc-(CH3)3), 1.09 (s, 9H, tBu(H)) 62 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0288] Methyl(S)-2-(1-(4-f inobutoxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (21): Following general procedure G, compound 20(8 mg, 0.011 mmol), TFA (0.5 mL) afforded the product 21 (4.1 mg, 73%).1H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H, H-7), 7.60 (d, J = 9.4 Hz, 1H, H-6), 7.53 (d, J = 2.3 Hz, 1H, H- 4), 7.19 – 7.15 (m, 3H, H-NH, m-Ar), 7.00 – 6.92 (m, 3H, NH, o-Ar), 5.53 (s, 2H, CH2Ar), 4.61 (d, J = 9.4 Hz, 1H, H’-(C-tBu)), 3.94 (t, J = 5.9 Hz, 2H, CH2O), 3.75 (s, 3H, OCH3), 3.24 (d, J = 6.2 Hz, 2H, H’-(NH)), 1.80 – 1.71 (m, 4H H-‘CH2, “CH2), 1.08 (s, 9H, tBu(H))).13C NMR (100 MHz, CDCl3) δ 171.9 (C=O), 162.8 (C=O)-NH), 157.5 (C=N), 155.3 (C-O), 136.6 (C3)), 135.8 (C8), 131.5 ((i-Ar), 129.0 (o-Ar), 128.9 (o-Ar), 123.9 (m-Ar), 120.0 (C9), 115.9 (C6), 110.8 (C7), 106.7 (C4), 67.6 (CH2-O), 59.9 (CH2-O), 53.2 (C-(C-CH3)3), 51.9 (CH3O, CH2Ar), 41.1 (CH2N), 34.9 (C-(CH3)3), 29.7 (‘CH2), 26.7 (CH3)3),), 25.7 (“CH2). HRMS calcd for C27H35FN6O4 H+, 527.2775; found, 527.2775.

[0289] Methyl 4-bromle-3-carboxylate (21):Following general procedure A, compound 4-bromo-1H-indazole-3-carboxylic acid (1.0 g,4.15 mmol), TMSCl (1.02 mL, 8.29 mmol) afforded methyl ester which was used in nextstep. Following general procedure B, methyl ester (0.95 g, 3.72 mmol), Cs2CO3 (2.4 g, 7.44 mmol), 4-fluorobenzyl bromide (0.52 mL, 4.46 mmol) afforded the product 21 (0.77 g, 57%) and 21’ (144 mg, 11%). Minor 21’:1H NMR (500 MHz, CDCl3): δ 7.76 (dd, J = 8.7, 0.7 Hz, 1H, H-7), 7.47 (dd, J = 7.2, 0.8 Hz, 1H, H-5), 7.34 – 7.28 (m, 2H, H-(o-Ar), 7.19 (dd, J = 8.6, 7.2 Hz, 1H, H-6), 7.04 – 6.95 (m, 2H, , m-Ar ), 5.83 (s, 2H, CH2-Ar), 3.98 (s, 3H, H-OMe).Major 21: 1H NMR (500 MHz, CDCl3): δ ppm 7.48 (dd, J = 7.4, 0.8 Hz, 1H, H-7), 7.30 (dd,J = 8.5, 0.8 Hz, 1H, H-5), 7.18 (m, , H-(o-Ar, 6)), 7.03 – 6.94 (m, 2H, m-Ar ), 5.62 (s, 2H, CH2-Ar), 4.04 (s, 3H, H-OMe). 63 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0290] Methyl 1-(4-fluorobenz tramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole-3-carboxylate(22): Toa stirred solution of compound 21 (0.25 g, 0.69 mmol) in 1.4-dioxane (5 mL) was added Bis(pinacolato)diboron (0.35 g, 1.38 mmol) and reaction was bubbled with argon for 10 min. KOAc (0.2 g, 2.07 mmol), and Pd(dppf)Cl2CH2Cl2(57 mg, 0.096 mmol) were added sequentially and reaction was stirred at 80 °C for 10 h. After completion of reaction, reaction mixture was diluted with water (5 mL) and extracted with EtOAc (10 mL×3), combined organic layer washed with brine (5 mL) and dried over anhydrous Na2SO4. Solvents evaporated off and obtained residue was purified by flash column chromatography using (20% EtOAc:hexane) 22 (0.25 g, 88%).1H NMR (500 MHz, CDCl3) δ 7.46 (dd, J = 6.0, 1.7 Hz, 1H, H-7), 7.37 – 7.30 (m, 2H, H-6, 5 ), 7.14 – 7.06 (m, 2H, H-o-Ar), 6.95 (m, 2H, H(o-Ar)), 5.64 (s, 2H, CH2-Ar), 4.02 (s, 3H, H-(OMe), 1.47 (s, 12H, H-(CH3)4). O OHOMeF

[0291] Methyl 1-(4-fluorobenzH-indazole-3-carboxylate (23): To astirred solution of compound 22 (0.45 g, 1.09 mmol) in THF: H2O (1:1, 10 mL) was added NaBO3.‘’•‘’4H2O (0.5 g, 3.29 mmol) and reaction was stirred at rt for 2 hrs. After completion of reaction, reaction mixture was quenched with sat. NH4Cl (5 mL) and extracted with EtOAc (10 mL×3), combined organic layer washed with brine (5 mL) and dried over anhydrous Na2SO4. Solvents were evaporated off and obtained residue was purified by flash column chromatography using (20% EtOAc:hexane) 23 (0.28 g, 85%).1H NMR (500 MHz, CDCl3) δ 10.49 (s, 1H, H -OH), 7.31 – 7.24 (m, 1H, H-7), 7.23 – 7.15 (m, 2H, H(6, 5)), 7.02 – 6.94 (m, 2H, H(o-Ar), 6.82 (d, J = 8.2 Hz, 1H,m-Ar), 6.73 – 6.66 (m, 1H, m-Ar), 5.59 (s, 2H, H-(CH2-Ar), 4.09 (s, 3H, H-(OMe)). 64 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0292] Methyl(S)-2-(1-(4-fluor oxy-1H-indazole-3-carboxamido)-3,3-dimethyl butanoate (24): Followinggeneral procedure C, compound 23 (80 mg, 0.26mmol), LiOH.H2O (1.3 mL, 1 M in H2O, 1.3 mmol) afforded the acid , which was used innext step without further purification and treated with HATU (0.2 g, 0.52 mmol), Et3N (0.11 mL, 0.78 mmol), (S)-tert-leucine methyl ester (72 mg, 0.39 mmol) afforded the product 24(40 mg, 37%). 1H NMR (40 MHz, CDCl3) δ 11.63 (s, 1H, H-(OH), 7.79 (d, J = 9.7 Hz, 1H,H-7), 7.30 – 7.17 (m, 3H, H-6,5, NH), 7.08 – 6.94 (m, 2H, H-(o-Ar), 6.76 (d, J = 8.4 Hz, 1H, H-(m-Ar), 6.65 (d, J = 7.7 Hz, 1H, H-(m-Ar)), 5.53 (s, 2H, H-(CH2Ar)), 4.71 (d, J = 9.7 Hz, 1H, H-(C-(CH3)3), 3.79 (s, 3H, H(O-CH3)), 1.09 (s, 9H, H-(CH3)3).

[0293] Methyl (S)-2-(4-(4-((tino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (25): Following general procedure E, compound 24 (39 mg, 0.09 mmol), tert-butyl (4-bromobutyl)carbamate(0.035 g, 0.14 mmol), Cs2CO3 (0.075 g, 0.23 mmol), TBAI (3 mg, 0.009 mmol) afforded the product 25 (0.044 g, 80%).1H NMR (500 MHz, CD3OD): δ ppm 8.89 (d, J = 10 Hz, 1H, NH)), 7.32 –7.15 (m, 3H, H-(7, 6, 5), 6.96 (m, 2H, o-Ar ), 6.90 (d, J = 10 Hz, 1H, m-Ar), 6.63 (d, J = 10 Hz, 1H, ‘m- Ar), 5.60 (s, 2H, (CH2Ar), 5.11 (s, 1H, ‘NH), 4.93 (d, J = 10 Hz, 1H, H-(C-(CH3)3), 4.45 – 4.30 (m, 2H, (OCH2), 3.75 (s, 3H, OCH3)), 3.25 – 3.12 (m, 2H, CH2N), 2.04 – 1.90 (m, 2H, ‘CH2), 1.70 – 1.55 (m, 2H, (“CH2), 1.42 (s, 9H, H-(‘CH3)3), 1.07 (s, 9H, H-(CH3)3). 65 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0294] Methyl(S,E)-2-(4-(4-( onyl)guanidino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate(26): Following general procedure F, compound 25 (0.044 g, 0.08 mmol), TFA (0.4 mL), N,N′-Di-Boc-1H-pyrazole- 1-carboxamidine (0.037 g, 0.12 mmol), Et3N (0.06 mL, 0.48 mmol) afforded the product 26 (0.039 g, 71%).1H NMR (500 MHz, CD3OD): δ ppm 11.50 (s, 1H, ‘NH), 9.00 (d, J = 10 Hz, 1H, “NH), 8.35 (bs, 1H, NH), 7.30 – 7.15 (m, 3H, H-(7,6,5)), 6.97 (m, 2H, o-Ar), 6.90 (d, J = 10 Hz, 1H, m-Ar), 6.63 (d, J = 10 Hz, 1H, m-Ar), 5.61 (s, 2H, (CH2Ar)), 4.94 (d, J = 10 Hz, 1H, H-(C-(CH3)3), 4.65 – 4.35 (m, 2H, (OCH2), 3.79 (s, 3H, OCH3), 3.55 – 3.45 (m, 2H, CH2N), 2.041.95 (m, 2H, ‘CH2) 1.85 – 1.70 (m, 2H, (“CH2), 1.49 (s, 9H, H(‘CH3)3), 1.48 (s, 9H, H- (“CH3)3), 1.07 (s, 9H, H-CH3)3).

[0295] Methyl (S)-2-(1-(4-fluanidinobutoxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (27): Following general procedure G, compound 26(0.039 g, 0.06 mmol), TFA (0.4 mL) afforded the product 27 (0.025 g, 89%).1H NMR (400 MHz, CD3OD): δ ppm 7.37 (d, J = 8 Hz, 1H, H-7), 7.30 (m, 2H, H-6, 5), 7.25 – 7.10 (m, 1 H, H-o-Ar), 7.02 (m, 2H, H- m-Ar, H-o-Ar), 6.81 (d, J = 8 Hz, 1H, m-Ar), 5.65 (bs, 2H, CH2Ar), 4.72 (s, 1H, H-(C-(CH3)3),), 4.50 – 4.35 (m, 2H, (OCH2 ), 3.77 (s, 3H, OCH3), 3.24 (t, J = 8 Hz, 2H, CH2N), 2.05 – 1.90 (m, 2H, ‘CH2), 1.85 – 1.74 (m, 2H, “CH2), 1.08 (s, 9H, H-CH3)3).13C NMR (100 MHz, CD3OD): δ ppm 171.7 (C=O), 163.7 (C=O-(NH)), 162.4 (C=N), 161.2 (C6), 157.2 (C-F), 151.3 (C3), 143.0 (C8), 137.1 (C6), 132.3 (i-Ar), 132.2 (C7), 129.2 ((o-Ar),), 129.1 (o-Ar), 128.5 (C4), 127.7 (C9), 115.2 (m-Ar), 115.0 (m-Ar), 103.1 (C5), 68.0 (OCH2), 60.6 (C(C-CH3)3), 52.1 (OMe), 51.0 (CH2Ar), 40.7 (CH2N), 34.7 (C- 66 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO CH3)3), 25.7 (CH3)), 25.1(CH2). HRMS calcd for C27H35FN6O4- H+, 527.2776; found, 527.2784.

[0296] Methyl (S)-2-(6-(2-((te mino)ethoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (28): Following general procedure E, compound 5 (0.03 g, 0.072 mmol), tert-butyl (2-bromoethyl)carbamate (0.024 g, 0.10 mmol), Cs2CO3 (0.07 g, 0.21 mmol), TBAI (0.025 g, 0.072 mmol) afforded the alkylated product 28 (0.028 g, 70%).1H NMR (500 MHz, CDCl3) δ 8.20 (d, J = 8.9 Hz, 1H, H-4), 7.53 (d, J = 9.7 Hz, 1H, H-NH), 7.19 (m, 2H, o-Ar), 7.01 – 6.90 (m, 4H, H-5), 5.52 (s, 2H, H- CH2Ar), 4.72 (d, J = 9.7 Hz, 1H, H-(C-(CH3)3), 4.00 (t, J = 5.2 Hz, 2H, OCH2), 3.76 (s, 3H, OCH3), 3.54 (q, J = 5.5 Hz, 2H, CH2N), 1.45 (s, 9H, H(tBu(CO)), 1.09 (s, 9H, (CH3)3).

[0297] Methyl (S,Z)-2-(6-(2-(2rbonyl)guanidino)ethoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (29): Following general procedure F, compound 28 (0.028 g, 0.050 mmol), TFA (0.5 mL), N,N′-Di-Boc-1H-pyrazole- 1-carboxamidine (0.023 g, 0.075 mmol), Et3N (0.025 mL, 0.15 mmol) afforded the product 29 (0.03 g, 85%).1H NMR (500 MHz, CDCl3) δ 11.48 (s, 1H, NH), 8.74 (t, J = 5.6 Hz, 1H, ‘NH), 8.21 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.7 Hz, 1H, H-NH), 7.20 – 7.16 (m, 2H, o- Ar), 7.01 (m, 2H, m-Ar), 6.95 – 6.82 (m,1H, H-5), 6.62 (s, 1H, H-7), 5.52 (s, 2H, H- CH2Ar), 4.71 (d, J = 9.6 Hz, 1H, H-(C-(CH3)3), 4.07 (t, J = 5.2 Hz, 2H, OCH2), 3.85 (q, J = 5.3 Hz, 2H, NCH2), 3.76 (s, 3H, OCH3), 1.51 (s, 9H, H(tBu(CO))), 1.48 (s, 9H, ‘H(tBu(CO),), 1.09 (s, 9H, (CH3)3). 67 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0298] Methyl(S)-2-(1-(4-flu nidinoethoxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (30): Following general procedure G, compound 29(0.03 g, 0.042 mmol), TFA (0.5 mL) afforded the product 30 (8 mg, 40%).1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H, H-NH, 8.01 (d, J = 8.9 Hz, 1H, H-4), 7.58 (s, H-NH), 7.14 (dd, J = 8.4, 5.3 Hz, 2H, o-Ar ), 6.95 – 6.79 (m, 3H, H-5, m-Ar), 6.72 (d, J = 8.9 Hz, 1H, ‘NH), 6.57 (s, 1H, H-7), 5.45 (s, 2H, CH2Ar), 4.59 (d, J = 9.4 Hz, 1H, H-(C-(CH3)3), 4.00 (t, J = 4.4 Hz, 2H, OCH2), 3.73 (s, 3H, OCH3), 3.59 – 3.49 (m, 2H, OCH2), 1.07 (s, 9H,(CH3)3).13C NMR (100 MHz, CDCl3) δ 172.9 (C=O), 162.5 (C=O(NH)), 158.5 (C=N, C6), 157.7 (C-F), 141.7 (C3), 136.8 (C8), 131.4 (i-Ar), 129.0 (o-Ar), 128.9 (-o-Ar), 123.2 (-m-Ar), 118.3 (m-Ar), 115.3 (C4), 115.9 (C7), 115.6 (C9), , 91.8 (C5), 67.6(OCH2), 60.0 C(C-CH3)3), 51.9(OMe), 41.2 (CH2N), 34.8 (C-CH3)3), 26.6 (CH3)3). HRMS calcd for C25H31FN6O4H+, 499.2463; found, 499.2462.

[0299] Methyl (S)-2-(6-(3-((termino)propoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (31): Following general procedure E, compound 5 (0.015 g, 0.077 mmol), tert-butyl (3-bromopropyl)carbamate (0.027 g, 0.11 mmol), Cs2CO3 (0.038 g, 0.11 mmol), TBAI (0.028 g, 0.077 mmol) afforded the alkylated product 31 (0.036 g, 81%).1H NMR (500 MHz, CDCl3) δ 8.19 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.7 Hz, 1H, H-NH), 7.17 (dd, J = 8.6, 5.3 Hz, 2H, o-Ar ), 7.01 (m, 2H, m-Ar), 6.89 (m, 1H, H-5), 6.59 (s, 1H, H-7), 5.51 (s, 2H, CH2Ar), 4.71 (d, J = 9.7 Hz, 1H, H-(C- (CH3)3), 3.99 (t, J = 6.0 Hz, 2H, OCH2), 3.76 (s, 3H. OCH3), 3.38 – 3.19 (m, 2H, H-C-N), 2.02 – 1.94 (m, 2H, CH2), 1.43 (s, 9H, H(tBu(CO)), 1.09 (s, 9H,(CH3)3). 68 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0300] Methyl(S,Z)-2-(6-(3-( bonyl)guanidino)propoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (32): Following general procedure F, compound 31 (0.032 g, 0.056 mmol), TFA (0.5 mL), N,N′-Di-Boc-1H-pyrazole- 1-carboxamidine (0.026 g, 0.084 mmol), Et3N (0.02 mL, 0.16 mmol) afforded the product 32 (0.036 g, 92%).1H NMR (500 MHz, CDCl3) δ 11.51 (s, 1H, NH), 8.68 (t, J = 4.9 Hz, 1H ‘NH), 8.19 (d, J = 8.9 Hz, 1H, H-4), 7.61 (d, J = 2.1 Hz, 2H, H-NH), 7.53 (d, J = 9.6 Hz, 1H, H-NH), 7.20 – 7.13 (m, 2H, o-Ar ), 7.08 – 6.99 (m, 2H, m-Ar), 6.60 (m, 1H, H-5), 6.34 (m, 1H, H-7), 5.52 (s, 2H, OCH2), 4.71 (d, J = 9.6 Hz, 1H, H-(C-(CH3)3), 4.02 (t, J = 5.8 Hz, 2H, OCH2), 3.76 (s, 3H, OCH3), 3.64 (q, J = 6.2 Hz, 2H, NH2), 2.08 (q, J = 6.1 Hz, 2H, CH2), 1.50 –1.48 (m, 18H, (H(tBu(CO)2), 1.09 (s, 9H, (CH3)3 ).

[0301] Methyl (S)-2-(1-(4-fluanidinopropoxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (33): Following general procedure G, compound 32(0.032 g, 0.044 mmol), TFA (0.5 mL) afforded the product 33 (0.018 g, 81%).1H NMR (500 MHz, CD3OD) δ 8.06 (d, J = 8.9 Hz, 1H, H-4), 7.29 (dd, J = 8.7, 5.4 Hz, 2H, o-Ar), 7.07 (m, 2H, m-Ar), 7.02 – 6.96 (m, 2H, H-7, 5), 5.67 (s, 2H, ArCH2), 4.58 (s, 1H, H-(C-(CH3)3), 4.11 (t, J = 5.8 Hz, 2H, OCH2), 3.77 (s, 3H, OCH3), 3.42 (t, J = 6.8 Hz, 2H, NH2), 2.09 (d, J = 6.4 Hz, 2H, H-CH2), 1.08 (s, 9H, (CH3)3).13C NMR (100 MHz, CD3OD) δ 171.6 (C=O), 162.7 (C=O(NH)), 159.0 (C=N), 157.3 (C6, Ar-F), 142.3 (C3), 136.6 (C8), 132.5 (iAr), 132.4 (C7), 129.0 (o-Ar), 128.9 (o-Ar), 122.5 (C4), 117.6 (C7), 115.2 (m-Ar), 115.0 (m-Ar), 91.4 (C5), 65.0 (OCH2), 59.94 (CH), 59.92 (Ar-CH2), 51.8 (OCH3), 38.23 (CNH), 28.1 (CH2), 25.6 (CH3)3). HRMS calcd for C26H33FN6O4 H+, 513.2620; found, 513.2616. 69 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0302] Methyl(S,Z)-2-(6-((5-( rbonyl)guanidino)pentyl)oxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (34): Following a procedure general procedure E, compound 5 (0.025 g, 0.054 mmol), alkyliodo compound (0.37 g, 0.082 mmol) and Cs2CO3(0.026 g, 0.082 mmol) afforded the product 34 (0.017 g, 44%).1H NMR (500 MHz, CDCl3) δ 8.34 (s, 1H, NH), 8.17 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.6 Hz, 1H, H-NH), 7.20 – 7.12 (m, 2H, o-Ar), 7.01 (m, 2H, m-Ar), 6.89 (m, H-5), 6.57 (d, J = 1.7 Hz, 1H, H-7), 5.51 (s, 2H, CH2-Ar), 4.71 (d, J = 9.7 Hz, 1H, H-(C-(CH3)3), 3.91 (t, J = 6.3 Hz, 2H, OCH2), 3.75 (s, 3H, OCH3), 3.43 (q, J = 7.0 Hz, 2H, C-NH), 1.80 (p, J = 6.6 Hz, 2H, CH2), 1.65 – 1.61 (m, 2H, CH2), 1.55 – 1.52 (m, 2H, “CH2), 1.49 (s, 18H, (H(tBu(CO)2), 1.08 (s, 9H, (CH3)3 ).

[0303] Methyl (S)-2-(1-(4-flanidinopentyl)oxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (35): Following general procedure G, compound 34 (0.015g, 0.020 mmol), TFA (0.5 mL) afforded the compound 35 (7 mg, 70%).1H NMR (500 MHz, CD3OD) δ 8.03 (d, J = 8.9 Hz, 1H, H-NH), 7.83 (d, J = 9.4 Hz, 1H, H-4), 7.29 (m, 2H, o-Ar), 7.06 (m, 3H, m-Ar, H-5), 6.95 (d, J = 1.9 Hz, 1H, H-7), 5.66 (s, 2H, CH2Ar), 4.58 (d, J = 9.4 Hz, 1H, H-(C-(CH3)3), 4.03 (t, J = 6.2 Hz, 2H, CH2O), 3.77 (s, 3H, OCH3), 3.21 (t, J = 7.0 Hz, 2H, H-NH), 1.85 (dt, J = 13.7, 6.3 Hz, 2H, CH2), 1.66 (q, J = 7.2 Hz, 2H, ‘CH2), 1.60 – 1.55 (m, 2H, “CH2), 1.08 (s, 9H, (CH3)3).13C NMR (100 MHz, CD3OD) δ 171.6 70 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO (C=O), 162.8 (C=O(NH)), 159.4 (C=N, C6), 142.4(C3), 136.6(C8), 132.5(i-Ar),129.0(o-Ar), 128.9(o-Ar), 122.4(C9), 117.4(C4), 115.3 (C7), 115.2 (m-Ar), 115.0 (m-Ar), 91.2 (C5), 67.7 (OCH2), 59.9 (CH), 51.8 (OMe, CH2Ar), 41.0 (C-NH), 34.3 (CH)(CH3)3, 28.4 (CH2), 28.3 (‘CH2), 25.6 (CH3)3, 23.0 (“CH2). HRMS calcd for C28H37FN6O4 H+541.2933; found 541.2935.

[0304] Methyl(S)-2-(6-((6-((ter amino)hexyl)oxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (36): Following general procedure E, compound 5 (0.04 g, 0.096 mmol), tert-butyl (6-bromohexyl)carbamate (0.04 g, 0.14 mmol), Cs2CO3 (0.09 g, 0.093 g, 0.28 mmol), TBAI (0.035 g, 0.096 mmol) afforded the compound 36 (0.04 g, 68%).1H NMR (500 MHz, CDCl3) δ 8.19 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.7 Hz, 1H, H-NH), 7.21 – 7.14 (m, 2H, o-Ar), 7.07 – 6.97 (m, 3H, m-Ar, 5), 6.57 (d, J = 2.1 Hz, 1H, H-7), 5.52 (s, 2H, CH2-Ar), 4.72 (d, J = 9.6 Hz, 1H, H-(C-(CH3)3), 3.92 (t, J = 6.4 Hz, 2H. CH2O), 3.76 (s, 3H, OCH3), 3.12 (d, J = 7.7 Hz, 2H, CH2N), 1.82 – 1.74 (m, 2H, CH2), 1.54 – 1.48 (m, 4H, ‘CH2-“CH2), 1.44 (s, 9H, H(tBu(CO), 1.38 (d, J = 6.8 Hz, 2H, (CH2), 1.09 (s, 9H, (CH3)3).

[0305] Methyl(S,Z)-2-(6-((6-(2rbonyl)guanidino)hexyl)oxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (37): Following general procedure F, compound 36 (0.024 g, 0.039 mmol), TFA (0.5 mL), N,N′-Di-Boc-1H-pyrazole- 71 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO 1-carboxamidine (0.018 g, 0.058 mmol), Et3N (0.02 mL, 0.017 mmol) afforded the product 37 (0.016 g, 55%).1H NMR (500 MHz, CDCl3) δ 8.53 (s, 1H, NH), 8.18 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.6 Hz, 1H, H-NH), 7.23 – 7.13 (m, 2H, o-Ar), 7.01 (m, 3H, m-Ar, H-NH), 6.90 (s, J = 7.4 Hz, 1H, H-5), 6.58 (m, 1H, H-7), 5.52 (s, 2H, CH2Ar), 4.71 (d, J = 9.6 Hz, 1H, H-(C-(CH3)3), 3.92 (s, 2H, OCH2), 3.76 (s, 3H, OCH3), 3.52 – 3.38 (m, 2H, H (C-N), 1.78 (d, J = 6.8 Hz, 2H, CH2), 1.67 – 1.57 (m, 2H, ‘CH2), 1.50 (s, 22H, (CH2)4 H(tBu(CO)2), 1.09 (s, 9H, (CH3)3).

[0306] Methyl(S)-2-(1-(4-fluoanidinohexyl)oxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (38): Following general procedure G, compound 37 (10 mg, 0.013 mmol), TFA (0.5 mL) afforded product 38 (4 mg, 57%).1H NMR (500 MHz, CDCl3) δ 7.99 (d, J = 8.9 Hz, 1H, H-4), 7.47 (d, J = 9.5 Hz, 1H, H-NH), 7.09 – 7.06 (m, 2H, o-Ar), 6.90 (m, 3H, o-Ar, H-7), 6.78 (d, J = 7.9 Hz, 1H, H-5), 6.50 (d, J = 2.0 Hz, 1H, H-7), 5.40 (s, 2H, OCH2), 4.55 (d, J = 9.5 Hz, 1H, H-(C-(CH3)3), 3.77 (t, J = 6.3 Hz, 2H, OCH2), 3.66 (s, 3H, OCH3), 3.01 (d, J = 10.6 Hz, 2H, HC-N ), 1.61 (t, J = 7.2 Hz, 2H, CH2), 1.46 (t, J = 7.4 Hz, 2H, CH2), 1.33 – 1.24 (m, 4H, -(CH2)2, 0.99 (s, 9H, (CH3)3).13C NMR (125 MHz, CDCl3) δ 162.6 (C=O), 159.2 (C=O(NH)), 157.2 (C=N, C6), 142.2 (C-3), 136.9 (C8), 131.7 (iAr), 128.9 (o-Ar), 123.0 (o-Ar), 117.7 (C4), 115.8 (o-Ar), 115.6 (o-Ar), 115.2 (C7, C9), 91.4 (C5), 68.0 (OCH2), 59.9 (CH), 52.5 (OMe), 51.9 (CH2Ar), 41.3 (CH2N), 34.9 (C(CH3)3), 28.7 (CH2), 28.3 (‘CH2), 26.6 ((CH3)3), 26.1 (‘CH2), 25.5 (“CH2). HRMS calcd for C29H39FN6O4 H+, 555.3089; found, 555.3089. 72 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0307] Methyl (S)-2-(6-((8-((te amino)octyl)oxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (39): Following general procedure E, compound 5 (0.024 g, 0.057 mmol), tert-butyl (8-bromooctyl)carbamate (0.026 g, 0.086 mmol), Cs2CO3(0.028 g, 0.086 mmol), TBAI (0.02 g, 0.057 mmol) afforded the product 39 (0.02 g, 72%).1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.6 Hz, 1H, H-NH), 7.22 – 7.15 (m, 2H, o-Ar), 7.00 – 6.96 (m, 2H, m-Ar, H-5), 6.56 (s, 1H, H- 7), 5.51 (s, 2H, -OCH2), 4.71 (d, J = 9.6 Hz, 1H, H-(C-(CH3)3), 4.51 (s, 1H, NH), 3.90 (t, J = 6.3 Hz, 2H, OCH2), 3.75 (s, 3H, OCH3), 3.15 – 3.05 (m, 2H, CH2-N), 1.82 – 1.71 (m, 2H, CH2), 1.43 (m, 13H, (CH2)2 ,H(tBu(CO)), 1.32 (s, 6H, (CH2)3), 1.08 (s, 9H, (CH3)3).

[0308] Methyl (S,Z)-2-(6-((8-(arbonyl)guanidino)octyl)oxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (40): Following general procedure F, compound 39(0.02 g, 0.031 mmol), TFA (0.2 mL), N,N′-Di-Boc-1H-pyrazole-1- carboxamidine (0.014 g, 0.046 mmol), Et3N (0.01 mL, 0.093 mmol) afforded the product 40 (0.018 g, 75%).1H NMR (400 MHz, CDCl3) δ 11.51 (s, 1H, H-HN), 8.32 (s, 1H, H-‘NH), 8.22 – 8.12 (m, 1H, H-4), 7.52 (d, J = 9.6 Hz, 1H, H-NH), 7.16 -7.02 (m, 2H, o-Ar), 7.00 (m, 2H, m-Ar ), 6.90 (d, J = 9.2 Hz, 1H, H-5), 6.57 (s, 1H, H-7), 5.51 (s, 2H, OCH2), 4.70 (d, J = 9.7 Hz, 1H, H-(C-(CH3)3), 3.90 (t, J = 6.3 Hz, 2H, OCH2), 3.75 (s, 3H, OCH3), 3.40 (q, J = 73 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO 6.5 Hz, 2H, N-CH2), 1.76 (p, J = 6.8 Hz, 2H, CH2), 1.56 (t, J = 7.1 Hz, 2H, ‘CH2), 1.50 – 1.47 (m, 18H, H(tBu(CO)2), 1.43 (d, J = 8.8 Hz, 2H, ”CH2 ), 1.33 (d, J = 8.3 Hz, 6H, “’CH2), 1.08 (s, 9H, (CH3)3).

[0309] Methyl (S)-2-(1-(4-flu anidinooctyl)oxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (41): Following general procedure G, compound 40(0.018 g, 0.023 mmol), TFA (0.5 mL) afforded the product 41 (6 mg, 46%).1H NMR (500 MHz, CDCl3) δ 8.04 (d, J = 8.9 Hz, 1H, H-4), 7.54 – 7.45 (m, 2H, o-Ar), 7.14 – 7.06 (m, 3H, m-Ar, H-5), 6.95 – 6.89 (m, 2H, NH2,), 6.81 (dd, J = 8.9, 2.0 Hz, 1H, H-7), 6.51 (d, J = 2.0 Hz, 1H, ‘NH), 5.43 (s, 2H, OCH2), 4.58 (d, J = 9.5 Hz, 1H, H-(C-(CH3)3), 3.82 (t, J = 6.4 Hz, 2H, OCH2), 3.68 (s, 3H, OCH3), 3.03 – 2.96 (m, 2H, H-(C- NH2), 1.65 (t, J = 7.3 Hz, 2H, CH2), 1.45 (d, J = 7.2 Hz, 2H, ‘CH2), 1.35 (d, J = 13.9 Hz, 2H, (“CH2)), 1.21 (m, 6H, (CH2)3), 1.01 (s, 9H, CH3)3).13C NMR (125 MHz, CDCl3) δ 172.1 (C=O), 159.2 (C=O(NH)), 157.4 (C=N,C6), 141.8(C3), 137.0 (C8), 132.2 (i-Ar), 129.8 (o-Ar)), 118.9 (C4), 116.6 (C9), 115.8 (m-Ar), 115.6 (m-Ar), 115.2 (C7), 91.4 (C5), 68.0 (OCH2), 59.8 (-C(C-CH3)3), 52.6 (OCH3), 51.9 (CH2Ar), 41.5 (CH2-N), 34.9 (C-(CH3)3, 28.9 (‘CH2), 28.4 (“CH2), 26.7 (CH3)3, 26.4 (CH2)2, 25.8 (CH2)2. HRMS calcd for C31H43FN6O4H+, 583.3402; found, 583.3401.74 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0310] Methyl(S)-2-(1-(4-fluorobenzyl)-6-(hexyloxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (42): Following general procedure E, compound 5 (0.015 g, 0.036 mmol), 6-bromohexane (0.032 g, 0.20 mmol), Cs2CO3(0.023 g, 0.12 mmol), TBAI (0.014 g, 0.036 mmol) afforded the alkylated product 42 (0.012 g, 66%).1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.0 Hz, 1H, H-4), 7.52 (d, J = 8.0 Hz, 1H, H-NH), 7.17 (m, 2H, o-Ar), 7.01 - 6.91 (m , 3H, m-Ar, H5), 6.57 (m, 1H, H-7), 5.52 (s, 2H, CH2-Ar), 4.72 (d, J = 8.0 Hz, 1H, H-C(CH3)3), 3.92 (t, J = 8.0 Hz, 2H CH2-O), 3.76 (s, 3H, CH3-O), 1.77 (p, J = 6.7 Hz, 2H, CH2), 1.54 – 1.42 (m, 2H, ‘CH2), 1.33 (m, 4H, (‘‘CH2)2), 1.09 (s, 9H, CH3)3), 0.94 – 0.87 (m, 3H, CH3).13C NMR (100 MHz, CDCl3) δ 172.1 (C=O), 162.3 (C=O(NH), 159.2 (C6), 142.1 (C3), 137.3 (32), 131.8 (iAr),), 128.8 (o-Ar), 128.8 (o-Ar), 123.6 (C4), 117.9 (C9), 115.9 (m- Ar), 115.6 ((m-Ar),), 115.0 (C7), 91.2 (C5), 68.3 (OCH2), 59.5 ((-C(C-CH3)3), 52.6(OCH3), 51.8 (CH2Ar), 35.0 (C-(CH3)3), 31.6 (CH2), 29.1 (‘CH2), 26.7 ((CH3)3), 25.7 (“CH2), 22.6 (“’CH2), 14.0 (CH3). HRMS calcd for C28H36FN3O4 Na+, 520.2582; found, 520.2585.

[0311] Methyl(S)-2-(1-(4-fluoroxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (43): Following general procedure E, compound 5 (0.015 g, 0.036 mmol), 8-bromooctane (0.042 g, 0.20 mmol), Cs2CO3(0.039 g, 0.12 mmol), TBAI (0.014 g, 0.036 mmol) afforded the alkylated product 43 (0.010 g, 53%).1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.7 Hz, 1H, H-NH), 7.17 (m, 2H, o-Ar), 7.01 (m, , 2H, m-Ar), 6.91 (dd, J = 8.9, 2.0 Hz, 1H, H-5), 6.57 (d, J = 2.0 Hz, 1H, H-7), 5.52 (s, 2H, CH2-Ar), 4.72 (d, J = 9.7 Hz, 1H, H-(C-(CH3)3), 3.92 (t, J = 6.5 Hz, 2H, CH2-O), 3.76 (s, 3H, CH3O), 1.77 (p, J = 6.8 Hz, 2H, CH2), 1.45 (t, J = 7.7 Hz, 2H, ‘CH2), 1.35 – 1.25 (m, 8H, (CH2)4), 1.09 (s, 9H, (CH3)3), 0.89 (t, J = 6.7 Hz, 3H, CH3).13C NMR (100 MHz, CDCl3) δ 173.8 (C=O), 162.3 (C=O(NH)), 159.2 (C6), 142.1 (C3), 136.8 (C8), 131.8 (iAr), 128.8 (o- Ar), 128.7 (o-Ar), 123.6 (C4), 117.9 (C9), 115.9 (o-Ar), 115.6 (o-Ar), 114.3 (C7), 91.2 (C5), 68.3 (OCH2), 59.5 ((-C(C-CH3)3), 52.6 (O-CH3), 51.8(CH2Ar), 35.0 (C-(CH3)3), 31.8 (CH2), 75 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO 29.3 (‘CH2), 29.2 (‘CH2), 29.1 (“CH2), 26.7 (CH3)3), 26.0 (“’CH2), 22.6 (“”CH2), 14.1 (CH3). HRMS calcd for C30H40FN3O4Na+, 548.2895; found, 548.2896.

[0312] Methyl(S)-2-(6-(benzyl enzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (44): Following general procedure E, compound 5 (0.012 g, 0.029 mmol), benzyl bromide (0.014 g, 0.087 mmol), Cs2CO3(0.019 g, 0.058 mmol), TBAI (0.010 g, 0.029 mmol) afforded the alkylated product 44 (7 mg, 48%).1H NMR (500 MHz, CDCl3) δ 8.21 (d, J = 8.9 Hz, 1H, H-4), 7.51 (d, J = 9.7 Hz, 1H, H-NH), 7.41 – 7.32 (m, 5H, H-Ar), 7.14 (ddd, J = 8.0, 5.4, 2.7 Hz, 2H, p-Ar, 7), 7.03 – 6.95 (m, 3H, m-Ar’, H-5), 6.64 (d, J = 1.2 Hz, 1H, NH), 5.49 (s, 2H, CH2Ar), 5.06 (s, 2H, CH2(O)Ar ), 4.72 (d, J = 9.3 Hz, 1H, H- C(CH3)3), 3.76 (s, 3H, OCH3), 1.09 (s, 9H, (CH3)3).13C NMR (125 MHz, CDCl3) δ 172.1 (C=O), 162.2 (C=O(NH)), 158.7 (C6), 141.9 (C3), 137.4 (C8), 136.4 (i-Ar’), 128.9 (i-Ar), 128.8 (p-Ar’), 128.6 (o-Ar), 128.1 (m-Ar’), 127.4 (m-Ar’), 123.8 (C4), 118.3(C9), 115.9 (m- Ar), 115.7 (m-Ar), 115.1(C7), 92.2 (C5), 70.3 (CH2-OAr), 59.5 (C(C-CH3)3), 52.8 (O-CH3), 51.8 (ArCH2), 35.0 (C-(CH3)3), 26.7 (CH3)3). HRMS calcd for C29H30FN3O4Na+, 526.211; found, 526.2114.

[0313] Methyl(S)-2-(1-(4-fluorethoxy-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (45): Following general procedure E, compound 5 (10 mg, 0.024 mmol), (2-bromoethyl)benzene (13 mg, 0.072 mmol), Cs2CO3(0.023 g, 0.072 mmol), TBAI (9 mg, 0.024 mmol) afforded a product 45 (5 mg, 41%).1H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.6 Hz, 1H, H-NH), 7.37 – 7.22 (m, 6H, Ar’), 7.14 (m, , 2H, m-Ar), 7.00 (m, , 2H, o-Ar’), 6.92 (s, 1H, H-5), 6.57 (d, J = 2.0 Hz, 1H, NH), 5.49 (s, 2H, ArCH2), 4.71 (d, J = 9.7 Hz, 1H, H-(C(CH3)3), 4.14 (t, J = 7.1 Hz, 2H, OCH2), 3.76 (s, 3H, 76 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO OCH3), 3.10 (t, J = 7.1 Hz, 2H, ArCH2), 1.08 (s, 9H, (CH3)3).13C NMR (100 MHz, CDCl3) δ 172.1 (C=O), 162.2 (C=O(NH)), 158.9 (C6), 142.0 (C3), 137.9 (C8), 137.3 (iAr’), 131.7 (i- Ar), 129.0 (pAr), 128.8 (o-Ar), 128.7 (o-Ar), 128.5 (m-Ar’), 126.6 (o-Ar), 123.7 (C4), 118.1 (C9), 115.7 (m-Ar), 115.0 (C7), 91.3 (C5), 69.0 (CH2-OAr), 59.5 (C(C-CH3)3), 52.6 (O-CH3), 51.9 (ArCH2), 35.7 (CH2-Ar), 35.0 (C-(CH3)3, 26.7 ((CH3)3). HRMS calcd for C30H32FN3O4 H+, 518.2449; found 518.2449.

[0314] Methyl(S)-2-(6-(4-ami orobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (46): Following general procedure F, compound 6 (0.015 g, 0.025 mmol), TFA (0.5 mL), afforded the product 46 (5 mg, 41%).1H NMR (500 MHz, CDCl3) δ 8.14 (d, J = 8.8 Hz, 1H, H-4), 7.53 (d, J = 10.2 Hz, 1H, H-NH), 7.16 (s, 2H, o-Ar), 6.98 (s, 2H, m-Ar ), 6.84 (s, 1H, H-5), 6.56 (s, 1H, H-7), 5.50 (d, J = 25.3 Hz, 2H, CH2Ar), 4.69 (d, J = 9.9 Hz, 1H, H-C(CH3)3 ), 3.93 (d, J = 24.0 Hz, 2H, CH2O), 3.75 (s, 3H, CH3O), 3.10-3.01(bs, 2H, (CH2N)) 1.82 (m, 4H, (CH2)2), 1.08 (s, 9H, (CH3)3. HRMS calcd for C26H33FN4O4 H+, 485.2558; found 485.2559.

[0315] Methyl(S)-2-(6-((6-ami-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (47): To the stirred solution of compound 36 wasadded TFA (0.5 mL) and reaction was stirred at rt for 12h. Solvent was evaporated off, diluted with H2O (2 mL) and neutralized with NaOH (1N, 2 mL), extracted with EtOAc 77 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO (3×10 mL). The organic layer dried over anhydrous Na2SO4and solvent was evaporated off, obtained crude residue was purified by flash chromatography using (DCM:MeOH, 10:1) to give a product 47 (12 mg, 52%).1H NMR (500 MHz, CDCl3) δ 8.14 (d, J = 8.9 Hz, 1H, H-4), 7.52 (d, J = 9.6 Hz, 1H, H-NH), 7.21 – 7.10 (m, 2H, o-Ar), 7.04 – 6.93 (m, 2H, o-Ar), 6.86 - 6.55 (m, 2H, H-7,5), 5.48 (s, 2H, -OCH2), 4.69 (d, J = 9.6 Hz, 1H, H-(C(CH3)3), 3.87 (t, J = 6.3 Hz, 2H, O-CH2), 3.74 (s, 3H, OCH3), 2.89 (t, J = 7.6 Hz, 2H, CH2N ), 1.78 – 1.62 (m, 4H, (CH2)2), 1.50 –1.33 (m, 4H, -(CH2)2, 1.08 (s, 9H, (CH3)3).13C NMR (125 MHz, CDCl3) δ 172.1 (C=O), 162.3 (C=O(NH), 159.1 (C6), 142.1 (C3), 137.3 (C8), 131.8 (iAr), 129.0 (o- Ar), 128.8 (o-Ar), 123.5 (C4), 118.0 (C9), 115.8 (m-Ar), 115.6 (m-Ar), 115.0 (C7), 91.3 (C5), 67.9 (CH2O), 59.6 (C(C-CH3)3), 52.6 (OCH3), 51.8 (CH2Ar), 39.9(CH2N), 35.0 (C-CH3)3), 28.7 (CH2), 26.7 (CH3)3), 26.6 (CH2), 26.0 (“CH2), 25.5 (“’CH2). HRMS calcd for C28H37FN4O4H+, 513.2871; found, 513.2871.

[0316] Methyl(S)-2-(6-((5-cy-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (48): Following general procedure E, compound 5 (0.02 g, 0.046 mmol), 6-bromohexanenitrile (0.041 g, 0.23 mmol), Cs2CO3 (0.047 g, 0.14 mmol), TBAI (25 mg, 0.069 mmol) afforded the alkylated product 48 (15 mg, 65%).1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.9 Hz, 1H, H-4), 7.52 (s ,1H, H-NH), 7.17 (m, 2H, o-Ar), 7.01 (m, , 2H, m-Ar), 6.89 (d J=, 1H, 2.1 Hz, 1H, H-5), 6.56 (d, J = 2.0 Hz, 1H, H-7), 5.52 (s, 2H, CH2Ar), 4.70 (d, J = 9.6 Hz, 1H,H-C(CH3)3), 3.93 (t, J = 6.2 Hz, 2H, OCH2), 3.75 (s, 3H, OCH3), 2.38 (t, J = 6.9 Hz, 2H, H-CN), 1.86 – 1.77(m, 2H, H-(CH2)), 1.75 – 1.70 (m, 2H, (‘CH2)), 1.68 – 1.61 (m, 2H, (“CH2)), 1.08 (s, 9H, (CH3)3).13C NMR (100 MHz, CDCl3) δ 172.1 (C=O), 162.2 (C=O(NH), 158.9 (C6), 142.0 (C3), 137.3 (C8), 131.7 (iAr), 128.8 (o-Ar), 128.7 (o-Ar), 123.6 (C4), 119.5 (CN), 118.1 (C9), 115.8 (m-Ar), 115.6 (m-Ar), 114.8 (C7), 91.2 (C5), 67.6 (CH2O), 59.5 (C(C-CH3)3), 52.6 (OCH3), 51.8 (CH2Ar), 35.0 (C-CH3)3), 28.3 (CH2), 26.6 (CH3)3), 25.3 (‘CH2) 25.1 (“CH2), 17.1 (‘CH2(CN)). HRMS calcd for C28H33FN4O4 Na+, 531.2378; found, 531.2378. 78 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0317] Methyl (S)-2-(1-(4-flu droxyhexyl)oxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (49): Following general procedure E, compound 5 (0.03 g, 0.072 mmol), 6-bromohexan-1-ol (0.052 g, 0.29 mmol), Cs2CO3 (0.07 g, 0.21 mmol), TBAI (0.026 g, 0.072 mmol) afforded the alkylated product 49 (0.032 g, 88%).1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.9 Hz, 1H, H-4), 7.52 (s, 1H, H-NH), 7.22 – 7.14 (m, 2H, o-Ar), 7.00 (m, 2H, m-Ar), 6.90 (d, J = 8.9.0 Hz, 1H, H-5), 6.57 (d, J = 2.1 Hz, 1H, H-7), 5.51 (s, 2H, CH2Ar ), 4.71 (d, J = 9.5 Hz, 1H, H-C(CH3)3), 3.92 (t, J = 6.5 Hz, 2H, OCH2), 3.75 (s, 3H, OCH3), 3.65 (t, J = 6.6 Hz, 2H, ‘OCH2), 1.84 – 1.74 (m, 2H, CH2), 1.59 (p, J = 6.8 Hz, 2H, ‘CH2), 1.52–1.37 (m, 4H, (CH2)2)), 1.08 (s, 9H, CH3)3)).13C NMR (100 MHz, CDCl3) δ 172.1 (C=O), 162.2 (C=O(NH), 159.2 (C6, CF), 142.1 (C3), 137.3 (C8), 131.8 (iAr), 131.7 (iAr), 128.8 (o-Ar), 128.7(o-Ar), 123.6 (C4), 118.0 (C9), 115.8 (m-Ar), 115.6 (m-Ar), 115.0 (C7), 91.2 (C4), 68.1 (CH2O), 62.8 (‘CH2O), 59.5 (C(C-CH3)3), 52.6 (OCH3), 51.8 (CH2Ar), 35.0 (C-CH3)3), 32.6 (CH2), 29.1 (‘CH2), 26.7 ((CH3)3), 25.9 (CH2), 25.5(“CH2). HRMS calcd for C28H36FN3O5 H+, 514.2711; found, 514.2710.

[0318] (S)-6-((1-(4-fluorobenz-3,3-dimethyl-1-oxobutan-2-yl)carbamoyl)-1H-indazol-6-yl)oxy)hexanoic acid (50): To the stirred solution of alcohol 49 (0.02 g, 0.038 mmol) in CH2Cl2was added Dess-Martin periodinane (0.049 g, 0.11 mmol) and the reaction mixture was stirred at rt for 3h. Reaction was quenched by addition of diethyl-ether (10 mL) and saturated Na2S2O3(10 mL) and stirred for 10 min,it was extracted 79 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO with diethyl ether (10 mL×2), the combined organic layer dried over anhydrous Na2SO4.Solvent was evaporated off to give a crude aldehyde which was used in the next reaction without any purification. The crude aldehyde was dissolved in THF:H2O (2 mL, 1:1), to this 2-methyl-2-butene (0.04 mL, 0.38 mmol), NaH2PO4 (0.021 g, 0.15 mmol) and NaClO2 ( 0.01 g, 0.11 mmol) were added sequentially, reaction mixture was stirred at 0 °C for 1.5 h. After completion of the reaction, it was acidified using (1N HCl), extracted with EtOAc (3×10 mL). The combined organic layer dried over anhydrous Na2SO4, solvent evaporated off, obtained residue was purified by flash chromatography using (EtOAC:hexane, 8:2) to give acid 50 (7 mg, 35%).1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.9 Hz, 1H, H-4), 7.53 (d, J = 9.7 Hz, 1H, H-NH), 7.17 (m, 2H, o-Ar), 7.06 – 6.97 (m, 2H, m-Ar), 6.90 (dd, J = 9.0, 2.0 Hz, 1H, H-5), 6.56 (s ,H-7), 5.52 (s, 2H, CH2Ar), 4.71 (d, J = 9.5 Hz, 1H, H-C(CH3)3), 3.92 (t, J = 6.4 Hz, 2H, OCH2), 3.76 (s, 3H, OCH3), 2.39 (t, J = 7.3 Hz, 2H, (CH2), 1.84 – 1.76 (m, 2H, (‘CH2), 1.74 – 1.68 (m, 2H, ‘CH2), 1.55 (dd, J = 9.4, 6.2 Hz, 2H, “CH2), 1.08 (s, 9H, (CH3)3).13C NMR (100 MHz, CDCl3) δ173.9 (C=O(OH)), 172.1 (C=O), 162.3 (C=O(NH)), 159.1(C6, CF), 142.1 (C3), 137.3 (C8), 131.8 (iAr), 131.7 (iAr), 128.8 (o-Ar), 128.7 (o-Ar), 123.6 (C4), 118.0 (C9), 115.9 (o-Ar), 115.7 (o-Ar), 115.0 (C7), 91.2 (C5), 67.9 (CH2O), 59.5 (C(C-CH3)3), 52.6 (CH3I), 51.8 (CH2Ar), 35.0 (C-CH3)3), 33.6 (CH2(C=O)), 28.8(CH2), 26.7 (CH3)3, 25.6(‘CH2), 24.4. (“CH2) HRMS calcd for C28H34FN3O6 Na+, 550.2323; found, 550.2325.

[0319] Methyl(S)-2-(6-(4-((2amino)-3,4-dioxocyclobut-1-en-1-yl)amino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (51): To the stirred solution of compound 6 (0.025 g, 0.042 mmol) dissolved in CH2Cl2, was added TFA (0.5 mL) and reaction was stirred at rt for 12h. After completion of the reaction solvent was evaporated off and residue was dissolved in EtOH (3 mL), to it tert-butyl (2- amino-3,4-dioxocyclobut-1-en-1-yl)carbamate (0.013 g, 0.051 mmol) and Et3N (0.01 mL, 0.126 mmol) were added sequentially, reaction was stirred at rt for 18h. After completion of 80 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO solvent evaporated off and residue diluted with water (2 mL) and extracted with EtOAc (3×10 mL). The organic layer dried over anhydrous Na2SO4 and solvent evaporated off, obtained crude residue was purified by flash chromatography using (EtOAC:hexane 4:6) to give a product 51 (17 mg, 60%).1H NMR (500 MHz, CDCl3) δ 8.35 (s, 1H, H-NH), 8.19 (d, J = 8.9 Hz, 1H, H-4), 7.60 – 7.49 (m, 2H, o-Ar), 7.24 – 7.16 (m, 2H, m-Ar), 7.06 – 6.95 (m, 2H, H-7,5), 6.94 (s, 1H, H-N), 6.61 (s, 1H, “NH), 5.53 (s, 2H, CH2Ar), 4.71 (d, J = 9.7 Hz, 1H, H-C(CH3)3), 4.10 –3.96 (m, 2H, OCH2), 3.82 – 3.78 (m, 2H, CH2NH), 3.76 (s, 3H, OCH3), 1.93 –1.817 (m, 4H, (CH2)2, 1.50 (s, 9H, tBu(CO2)), 1.09 (s, 9H, (CH3)3).

[0320] Methyl(S)-2-(6-(4-((2but-1-en-1-yl)amino)butoxy)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (52): Following general procedure G, compound 51 (0.015 g, 0.020 mmol), TFA (0.5 mL) afforded the compound 52 (8 mg, 66%).1H NMR (400 MHz, CD3OD) δ 8.02 (d, J = 8.9 Hz, 1H, H-4), 7.35 – 7.23 (m, 2H, o-Ar,), 7.05 (m, 2H m-Ar,), 6.97 (m, J = 2.0 Hz, 1H, H-7), 6.91 (dd, J = 8.9, 2.0 Hz, 1H, H-5), 5.64 (s, 2H, (CH2)2), 4.57 (s, 1H, H-C(CH3)3), 4.05 (t, J = 5.9 Hz, 2H, (CH2), 3.75 (s, 3H, OCH3), 3.73 – 3.60 (m, 2H, OCH2), 1.95 – 1.76 (m, 4H, (CH2)2), 1.07 (s, 9H, (CH3)3).13C NMR (100 MHz, CD3OD) δ 183.0 (C=O), 182.8 (‘C=O), 171.6 (“C=O), 162.8 (C=O(NH)), 159.3 ((C-O), 142.4 (C3), 136.6 (C8), 132.51 (iAr), 132.5 (iAr), 129.1 (o-Ar), 129.0 (o-Ar), 122.4 (C4), 117.4(C9), 115.2 (o-Ar), 115.0 (C7), 91.3 (C5), 67.4 (CH2O), 59.9 (C(C-CH3)3), 51.8 (OCH3), 51.0 (CH2Ar), 43.5 (CH2N), 34.3 (C-CH3)3), 27.6 (CH2)2, 25.6( CH3)3). HRMS calcd for C30H34FN5O6 Na+,602.23853; found 602.2386. 81 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0321] Methyl(S)-2-(1-(4-fluo idobutoxy)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate VIP2.33 (53): Compound 6 (0.04 g, 0.06 mmol) was dissolved in CH2Cl2(3 mL) to it TFA (0.3 mL) was added, and reaction was stirred at rt for overnight. Solvent was evaporated off and the crude residue dissolved in CH2Cl2 (3 mL), to it phenyl chloroformate (0.012 mL, 0.1 mmol), Et3N (0.025 mL) was added successively, reaction was allowed to stir at rt for overnight. Reaction mixture diluted with H2O (5 mL), and DCM 10 (mL), organic layer was separated, aqueous layer was extracted with DCM (10 mL×2), combined organic layers were dried over anhydrous Na2SO4and solvents were evaporated off, obtained crude residue was purified by flash column chromatography using (EtOAc:hexane 4:6). The product was dissolved in THF (2 mL), NH4OH (0.4 mL) was added, and the reaction mixture was stirred at 55oC for 24h. Solvent was evaporated off and crude residue was purified by flash chromatography using (EtOAc:hexane 4:6) yielded the product 53 (0.02 g, 65%).1H NMR (500 MHz, CDCl3) δ 8.13 (d, J = 8.9 Hz, 1H, H-4), 7.54 (d, J = 9.5 Hz, 1H, H-N), 7.22 – 7.12 (m, 2H, o-Ar), 7.05 – 6.94 (m, 2H, m-Ar ), 6.85 - 6.57 (m, 2H, H-5,7), 5.49 (s, 2H(CH2Ar)), 5.34 (s, 1H, H-NH), 4.73 (s, 2H, H-‘NH), 4.66 (d, J = 9.5 Hz, 1H, H-C(CH3)3), 3.88 (t, J = 6.2 Hz, 2H, CH2O), 3.75 (s, 3H, CH3O ), 3.27 – 3.16 (m, 2H, CH2N), 1.78 (dq, J = 8.5, 6.3 Hz, 2H, CH2), 1.70 – 1.59 (m, 2H, CH2), 1.08 (s, 9H, (CH3)3),13C NMR (126 MHz, CDCl3) δ 172.0 (C=O), 162.4 (C=O(NH)), 159.0 (C-O), 142.1 (C3), 137.1 (C8), 131.7 (iAr), 131.67 (iAr), 128.9 (m-Ar), 128.8 (m-Ar), 123.3 (C4), 117.9 ((o-Ar), 115.8 (C7), 115.6 (o-Ar), 115.0 (C7), 91.2 (C5), 67.7 (OCH2), 59.7 (C(C-CH3)3), 52.6 (OCH3), 51.8 (CH2Ar), 40.0 (CH2N), 34.9 (C-CH3)3), 26.8 (CH2), 26.6 (CH3)3), 26.4 (‘CH2). HRMS calcd for C27H34FN5O5 Na+550.2436; found, 550.2437. System setup for MD simulations

[0322] An active state CB1R structure with Fubinaca bound (PDB: 6N4B, the CB1R-Gprotein complex) was used for initial simulations (Figure 1). We also ran simulations using a 82 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO second CB1R receptor structure with Fubinaca bound for comparison (PDB: 8WU1, the CB1R-arrestin complex). Additional simulations used the cryo-EM structure of CB1R with VIP36 bound (Figure 3). For simulations with VIP2.33 bound, we modeled VIP2.33 using the structure of VIP36, changing only the ligand head-group. For all simulations, hydrogen atoms were added, and protein chain termini were capped with neutral acetyl and methylamide groups. The G-protein, arrestin, lipids, and stabilizing agents were removed where applicable. Prime (Schrodinger Inc suite of software tools (Glide, Prime), version 2019-1, ParamChem server (v1), Dabble (2.7.6), Dowser (1999 release)) was used to model in missing side-chains. Titratable residues were kept in their dominant protonation state at pH 7 except for D2.50and D3.49. In simulations with Fubinaca and VIP2.33, D2.50and D3.49, were protonated (neutral), as studies indicate that these conserved residues are protonated in active-state GPCRs and D2.50is buried within the hydrophobic core of the receptor1,2. In simulations with VIP36 bound, we reasoned that D2.50should be charged as it can form frequent ionic interactions with the nearby guanidinium group of VIP36, likely representing a much lower energy state than if both groups were neutral. This interaction cannot be formed by the urea group of VIP2.33. D3.49was maintained as neutral in all simulations (it does not interact with either ligand). Histidine residues were modeled as neutral, with a hydrogen atom bound to the epsilon nitrogen. The Dowser program was used to hydrate pockets within and around each structure3. Then the receptor was inserted into a pre-equilibrated palmitoyl-oleoyl- phosphatidylcholine (POPC) bilayer using Dabble4. Sodium and chloride ions were added to neutralize each system at a concentration of 150 mM. Approximate system dimensions were 80 Å x 90 Å x 90 Å with approximately 60,000 atoms. Simulation protocols

[0323] All simulations were run on a single Graphical Processing Unit (GPU) using theAmber20 Compute Unified Device Architecture (CUDA) version of particle-mesh Ewald molecular dynamics (PMEMD)5.We used the CHARMM36m parameter set for protein molecules, lipids, and ions, and the CHARMM TIP3P water model for waters6. Parameters for ligands were generated using the CHARMM General Force Field (CGenFF) with the ParamChem server7. Heating (to 310 K over 137.5 ps) and equilibration (28 ns with restraints on protein and ligand) steps were performed before production simulations8. Trajectory snapshots were saved every 200 ps. All simulations were at least 2 microseconds in length. 83 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Simulation analysis protocols

[0324] The AmberTools20 CPPTRAJ package8 was used to reimage trajectories, whileVisual Molecular Dynamics Python interface (1.9.3), (VMD 1.9.3)9, PyMOL (2.3.2) (Schrodinger Inc.), and Matplotlib Python package (3.6.2) were used for visualization and analysis.

[0325] In Figure 1b and Extended Data Figure 6, the reported distance used to quantifycryptic pocket opening is between sidechain atoms CD2 of F3.36and CH2 of W6.48(according to standard PDB atom typing). We additionally quantified cryptic pocket opening using simulations started from an alternative CB1R structure (PDB: 8WU1, the arrestin-CB1 complex), where we observed the cryptic pocket opening 12 ± 8% of the simulation time (mean ± s.e.m. calculated from 6 independent simulations) in comparison to 8 ± 3% from the initial simulations (PDB: 6N4B , G protein-CB1complex). The trajectory in Figure 1b, shows this value over the course of the simulation time, including both initial equilibration and production. In Figure 3c, the interaction distance is determined by minimum distance in simulations between heavy atoms of ligand head-group and sidechain of D2.50. In Figure 3e, we measured the distance between the Cɑ atoms of D2.50and P7.50. All histograms were computed from aggregating the measured values from at least 5 independent simulations, excluding equilibration. Drugs and chemicals for in vitro cell line and in vivo behavior assays

[0326] Complete Freund Adjuvant (Thermo Fisher, St. Louis, MO) was mixed withsaline in a 1:1 ratio prior to intraplantar injections. VIP36 was dissolved in a vehicle made of 10% DMSO (Sigma Aldrich, St. Louis, MO), 10% Koliophor (Sigma Aldrich, St. Louis, MO), and 80% saline and was administered intraperitoneal (I.P.) or per os (P.O). Nitroglycerin (NTG) was bought with a stock concentration of 5 mg / mL (30% alcohol, 30% propylene glycol, and 60% water). NTG was further diluted to 1 mg / mL concentration in saline the day of injection at a dose of 10 mg / kg (I.P.). AM6545 and AM630 (Caymen Chemical Company, Ann Arbor, MI) was dissolved in 20% DMSO, 8% ethanol, 8% Tween 80 (Thermo Fisher, St. Louis, MO) and 64% saline and administered I.P. AM630 was originally made into a DMSO stock solution of (25 mg / mL) and diluted the day of testing to the above vehicle. AM6545 underwent a similar process but required 45 minutes of sonication to fully dissolve. MDMB-Fubinaca (FUB) (Caymen Chemical Company, Ann 84 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Arbor, MI) was dissolved in 10% DMSO, 10% koliophor, 80% saline and administered I.P. All injections were given at 5 mL / kg unless otherwise stated. cAMP inhibition assay

[0327] To measure CB1 Gi-mediated cAMP inhibition, HEK293T (ATCC CRL-11268)cells were co-transfected using Lipofectamine 2000 (Invitrogen™ 11668019) with human Cannabinoid Receptor I (CB1) (Origene, SC111611) along with the biosensor pGloSensor™- 22F cAMP (Promega, E2301) in a ratio 1:1. The following day, transfected cells were plated into 96-well poly-D-lysine coated plate with DMEM supplemented with 1% dialyzed FBS at a density of 100,000 cells per well and incubated at 37ºC with 5% CO2 overnight. The next day, media was removed and 40 uL of freshly made assay buffer (20 mM HEPES, 1X HBSS, pH 7.4) was added to each well followed by 20 uL of drug solution (assay buffer, compounds, and 0.3% bovine serum album) for 15 min in the dark at room temperature.20ul of substrate buffer (assay buffer, 4 mM GloSensor™ cAMP Reagent (Promega), and 120uM Forskolin) was added per well and incubated at room temperature in the dark for another 15 min before reading. Luminescence intensity was quantified using a BioTek Synergy Neo Alpha plate reader. Results (relative luminescence units) were plotted as a function of drug concentration, normalized to % agonist control stimulation, and analyzed using ‘‘log(agonist) vs. response (three parameters)’’ in GraphPad Prism 10.1.2. Tango Arrestin recruitment assay

[0328] HTLA cells expressing TEV fused-β-Arrestin-2 were transfected with the CB1Tango construct using Lipofectamine 2000. The next day, 15,000 cells per well were plated in a 384-well poly-D-lysine coated plate along with DMEM supplemented with 1% dialyzed FBS and incubated overnight at 37ºC. The following day, the cells were stimulated overnight by the addition of compounds in assay buffer (20 mM HEPES, 1X HBSS, pH 7.4). After overnight stimulation, media and compounds were replaced by Substrate buffer (BrightGlo, Promega, 1:20 dilution in assay buffer). The plate was incubated for 20 min at room temperature in the dark before being counted using a BioTek Synergy Neo Alpha plate reader. Results (relative luminescence units) were plotted as a function of drug concentration, normalized to % agonist control stimulation, and analyzed using ‘‘log(agonist) vs. response (three parameters)’’ in GraphPad Prism 10.1.2. 85 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO BRET 1 (β-Arrestin recruitment)

[0329] To measure CB1-mediated β Arrestin recruitment, HEK293T cells were co-transfected using Lipofectamine 2000 with human CB1-Rluc8 along with Venus-tagged N- terminal β-arrestin(1 or 2) in a ratio 1:5. The following day, transfected cells were plated into 384-well Poly-D-lysine coated plate with DMEM supplemented with 1% dialyzed FBS at a density of 20,000 cells per well and incubated overnight at 37ºC. The next day, media was removed, and cells were rinsed with assay buffer (20 mM HEPES, 1X HBSS, pH 7.4), followed by the addition of the substrate buffer (assay buffer and 7.5 uM Coelentrazine h) for 5 min in the dark at room temperature. After incubation, the drug solution (assay buffer, compounds, and 0.3% bovine serum album) was added for another 5 min in the dark at room temperature. Plates were then immediately read for both luminescence at 485 nm and fluorescent eYFP emission at 530 nm using a BioTek Synergy Neo Alpha plate reader. Plates were read six times, and measurements from the 10 min read were used in all analyses. The ratio of eYFP / RLuc was calculated per well and was plotted as a function of drug concentration, it was normalized to % agonist control stimulation and analyzed using ‘‘log(agonist) vs. response (three parameters)’’ in GraphPad Prism 10.0. Bias analysis was carried out using the method proposed by Kenakin et al.10, that is, based on the Black and Leff operational method.12For this we followed the step-wise protocol described by Uprety et al. recently.11Gi1 MOR or Gq-5-HT2B (BRET) Assay.

[0330] To measure the Gαi1 protein mediated activation, human embryonic kidney(HEK293T) cells were cotransfected using a 1:1:1:1 DNA ratio of receptor: 5-HT2B / Gαi- Rluc:Gβ1:Gγ2-GFP2 or MOR / Gαi-Rluc:Gβ1:Gγ2-GFP2. Transfection was performed in OptiMEM using Transit 2020 at a 2:1 ratio of Transit:micrograms (μg) of DNA. After at least 18 h, 0.05% trypsin-EDTA was added to gently detach the cells, and subsequently, the cells were plated in a plating media (DMEM supplemented with 1% dialyzed FBS) at a density of 30^000–50^000 cells per well in poly-d-lysine-coated white and clear-bottom 96-well assay plates. The next day, a white adhesive bottom seal was applied, and the culture medium was carefully decanted and replaced by 60 μL of a drug buffer (1× Hank’s balanced salt solution (HBSS) and 20 mM HEPES, pH 7.4). Following assay buffer aspiration, cells were treated with 60 μL of freshly prepared coelenterazine 400a (5 μM) final concentration for Gαi1 activation. After 5 min of incubation, 30 μL of drug was individually added and incubated for 86 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO an additional 5 min. Subsequently, the plates were placed in an LB940 Mithras plate reader (Berthold Technologies) to measure BRET ratios by reading each well for 1 s. With respect to Gαi1 protein mediated activation, the BRET2 ratio was expressed as the ratio of the GFP2 emission to RLuc8 emission at 510 and 395 nm, respectively. The ratio of mVenus / RLuc was calculated per well, and the net BRET ratio was calculated by subtracting the mVenus / RLuc per well from the mVenus / RLuc ratio. The Graphpad Prism v10.1.2 software was used to plot the net BRET ratio versus the corresponding drug concentration. Bimane Fluorescence

[0331] A previously developed14 minimal cysteine CB1 version (with all the cysteineresidues, except C256 and C264 mutated to alanine) was used with a cysteine residue introduced at residue 336 (L6.28) on TM6. This residue was labeled with monobromobimane (bimane) by incubating 10 μM receptor with 10-molar excess of bimane at room temperature for one hour. Excess label was removed using size exclusion chromatography on a Superdex 20010 / 300 Increase column in 20 mM HEPES pH 7.5, 100 mM NaCl and 0.01% MNG / 0.001% CHS. Ligands at 10 μM was incubated with bimane-labeled CB1 at 0.1 μM for one hour at room temperature. Fluorescence data was collected with a FluorEssence v3.8 software on a Fluorolog instrument (Horiba) in photon-counting mode. Bimane fluorescence was measured by excitation at 370 nm with excitation and emission bandwidth passes of 4 nm. The emission spectra were recorded from 410 to 510 nm with 1 nm increment and 0.1 s integration time. GTP turnover assay

[0332] The GTP turnover assay was performed by using a modified protocol of theGTPase-GloTMassay (Promega) described previously14. CB1 at 1 μM in the absence of presence of ligands (10 μM ligand incubated for one hour at room temperature) was mixed with G-protein (1 μM) in 20 mM HEPES, pH 7.5, 50 mM NaCl, 0.01% L-MNG / 0.001% CHS, 100 μM TCEP, 10 μM GDP and 10 μM GTP and incubated at room temperature. GTPase-Glo-reagent was added to the sample after incubation for 60 minutes (Gi1-3) and 20 minutes for (Go). Luminescence was measured after the addition of detection reagent and incubation for 10 min at room temperature using a SpectraMax Paradigm plate reader. Radioligand Binding Experiments 87 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0333] Radioligand competition assays were carried out in membranes prepared fromSf9 insect cells expressing the human CB1 receptor. To reduce non-specific binding, 96-well plates were coated with a blocking buffer (20 mM HEPES, 100 mM NaCl, 0.05% BSA, pH 7.4). Incubation lasted at least one hour. Cold ligands were serially diluted using assay buffer (50 mM HEPES, 5 mM EDTA, 5 mM MgCl2, and 1% bovine serum albumin, pH 7.4). Membranes were diluted in the same assay buffer and incubated with the cold ligands and 10 nM [3H]-SR141716 for 1 hour on a shaker at room temperature. In the meantime, double thick 90 x 120 mm glass fibre Printed Filtermat B filters (Perkin Elmer) were soaked in 50 mM HEPES (pH 7.4) and 0.33% PEI. For conditions without sodium, NaCl and HEPES were substituted by KCl and Tris (pH 7.5) buffered by KOH, respectively. After incubation, the reaction was filtered using 20 mM HEPES (pH 7.4) and 100 mM NaCl via vacuum filtration on a MicroBeta Filtermat-96 cell harvester (Perkin Elmer). MicroBeta counter (Perkin Elmer) was used for radioactivity counting. Analysis was performed on GraphPad Prism 10 using nonlinear regression to determine the IC50and Ki values for each ligand.

[0334] Off target screening of 45 CNS receptors using binding assays: Bindingaffinities12, reported in SI fig 1a and 1d. were conducted by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP). Details of the methods and radioligands used for the binding assays are available on the NIMH-PDSP website at https: / / pdsp.unc.edu / pdspweb / content / UNC-CH%20Protocol%20Book.pdf.

[0335] Assessment of Off–target Activity Using PRESTO-Tango GPCR-ome13. Toidentify potential off–target activity of VIP36, we used the National Institutes of Mental Health Psychoactive Drug Screen Program. VIP36 was first tested for activity against 320 non-olfactory GPCRs using the PRESTO-Tango GPCRome screening β-arrestin2 recruitment assay at 3μM. The activity at each receptor was measured in quadruplicate. Screening of compounds was accomplished using previously described methods with several modifications (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4424118 / ). First, HTLA cells were plated in DMEM with 2% dialyzed FBS and 10 U / mL penicillin–streptomycin. Next, the cells were transfected using an in-plate PEI method (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4012321 / ). PRESTO-Tango receptor DNAs were resuspended in OptiMEM and hybridized with PEI prior to dilution and distribution into 384-well plates and subsequent addition to cells. After overnight incubation, drugs diluted in DMEM with 1% dialyzed FBS were added to cells without replacement of the medium. The remaining steps of the PRESTO-Tango protocol were followed as previously described. 88 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0336] hERG assay: Assay was performed using the protocol reported by Roth andcoworkers.14

[0337] Radioligand Binding Assays General Protocol: Briefly, membranes areprepared from either stably expressing cell lines or cells transfected with receptor of interest. Cells are washed, harvested, and lysed, and crude membrane fractions are then washed and pelleted. Receptor expression is quantified using Bradford assay and saturation binding with a reference radioligand in order to determine total protein and GPCR expression, respectively. Membranes are then stored at -80 ˚C prior to use in competition binding experiments. For competition binding, membranes are thawed then briefly triturated before being diluted and added to wellplates at a known final receptor concentration. Hot and cold ligands are then added and incubated with shaking. After incubation, the wells are then harvested onto filter mats soaked with 0.3% polyethyleneimine, which are then washed with buffer and dried. Scintillation reagent is then applied to the mats, which are then counted using a luminescence detector.

[0338] G-protein independent ^-arrestin recruitment: HTLA cells are transfectedwith GPCR tango constructs overnight and are plated in Poly-L-Lys (PLL) coated 384-well white clear-bottom cell culture plates at a density of 15,000 cells in 40 µl per well of DMEM with 1% dFBS. The cells are incubated for at least 6 hours (usually overnight) for them to recover before receiving drug stimulation. Drug stimulation solutions are prepared in filtered Tango assay buffer at 5x and added to cells (10 µl per well) for overnight. To measure antagonist activity, drug solutions are made at 6x of the final concentration and are preincubated with cells for 30 min before addition of 10 µl of and EC80 concentration of a reference agonist. The EC80 concentration is determined in separate preliminary dose- response assays. On the day of measurement, medium and drug solutions are removed and 20 µl per well of BrightGlo reagent (diluted by 20-fold with Tango assay buffer) is added. The plate is incubated for 20 minutes at room temperature in the dark before being counted on a luminescence counter. Purification of CB1

[0339] Human CB1 was expressed and purified as described previously15,16. The humanfull-length CB1 was tagged with N-terminal FLAG tag and C-terminal histidine tag. This CB1 construct was expressed in Spodoptera frugiperda Sf9 insect cells with the baculovirus method (Expression Systems). CB1 expressing insect cell pellets was solubilized with buffer 89 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO containing 1% lauryl maltose neopentyl glycol (L-MNG) and 0.1% cholesterol hemisuccinate (CHS) and purified by nickel-chelating Sepharose chromatography. The eluant from the Ni column was applied to a M1 anti-FLAG immunoaffinity resin. After washing to progressively decreasing concentration of L-MNG, the receptor was eluted in a buffer consisting of 20 mM HEPES pH 7.5, 150 mM NaCl, 0.05% L-MNG, 0.005% CHS, FLAG peptide and 5 mM EDTA. As the final purification step, CB1 was applied to a Superdex 200 10 / 300 gel filtration column (GE) in 20 mM HEPES pH 7.5, 150 mM NaCl, 0.02% L-MNG, 0.002% CHS. Ligand-free CB1 was concentrated to ∼500 µM and stored in -80 °C. Expression and purification of Gi / o heterotrimer

[0340] All heterotrimeric G-protein (Gi / o) follow similar protocols for expression andpurification. Heterotrimeric G protein was expressed and purified as previously described15,17. Trichuplusia ni (Hi5) cells were co-infected with human Gαi1 subunit virus and wild-type human β1γ2 (with histidine tagged β subunit) viruses. After cell pellet lysing with hypotonic buffer, G-protein was extracted in a buffer containing 1% sodium cholate and 0.05% n- dodecyl-β-D-maltoside (DDM, Anatrace). Detergent was exchanged from cholate / DDM to DDM on Ni Sepharose column. The Ni eluant was dialyzed overnight into 20 mM HEPES, pH 7.5, 100 mM sodium chloride, 0.1% DDM, 1 mM magnesium chloride, 100 μM TCEP and 10 μM GDP. During dialysis, Human rhinovirus 3C protease (3C protease) was added to cleave the His tag in the β subunit.

[0341] 3C protease was removed by Ni-chelating sepharose and the heterotrimetric G-protein was further purified with MonoQ 10 / 100 GL column (GE Healthcare). Protein was bound to the column and washed in buffer A (20 mM HEPES, pH 7.5, 50 mM sodium chloride, 1 mM magnesium chloride, 0.05% DDM, 100 μM TCEP, and 10 μM GDP). The protein was eluted

[0342] with a linear gradient of 0–50% buffer B (buffer A with 1 M NaCl). The collectedG protein was dialyzed into 20 mM HEPES, pH 7.5, 100 mM sodium chloride, 1 mM magnesium chloride,

[0343] 0.02% DDM, 100 μM TCEP, and 10 μM GDP. Protein was concentrated to about200 µM and flash frozen until further use. Purification of scFv16 90 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0344] Trichuplusia ni Hi5 insect cells was used to purify scFv16 as previouslydescribed15,17. The protein was expressed in the secreted form and was purified with a hexahistidine-tag. The supernatant from baculoviral infected cells was pH balanced and quenched with chelating agents and loaded onto Ni resin. After washing with 20 mM HEPES pH 7.5, 500 mM NaCl, and 20 mM imidazole, protein was eluted with 250 mM imidazole. Following dialysis with 3C protease into a buffer consisting of 20 mM HEPES pH 7.5 and 100 mM NaCl. scFv16 was further purified by size-exclusion chromatography on a Superdex 20016 / 60 column and the peak fraction was collected, concentrated and flash frozen. CB1-Gi1 complex formation and purification

[0345] Purified FLAG-tagged CB1 was incubated with excess VIP36 or VIP2.33 for ∼1 hour at room temperature. Simultaneously, Gi1 heterotrimer in DDM was incubated with 1% L-MNG / 0.1% CHS at 4 °C. The agonist-bound CB1 was incubated with a 1.25 molar excess of detergent exchanged Gi heterotrimer at room temperature for ∼ 3 hour. The complex sample was further incubated with apyrase for 1.5 hour on ice to stabilize a nucleotide-free complex. After the addition of 2 mM CaCl2, the complexes were purified on a M1 anti-FLAG column. After washing to remove excess G protein and reduce detergents, the complex was eluted in 20mM HEPES pH 7.5, 100mM NaCl, 0.01% L-MNG / 0.001% CHS, 0.0033% GDN / 0.00033% CHS, 10 µM VIP36 (or VIP2.33), 5 mM EDTA, and FLAG peptide. The complex was supplemented with 100 µM TCEP and incubated with 2 molar excess of scFv16 overnight at 4 °C. Size exclusion chromatography (Superdex 20010 / 300 Increase) was used to further purify the ligand-bound CB1-Gi-scFv16 complex. The complex in 20mM HEPES pH 7.5, 100mM NaCl, 10 µM VIP36 (or VIP2.33), 0.00075% L- MNG / 0.000075% CHS and 0.00025% GDN / 0.000025% CHS was concentrated to ∼12 mg / mL for electron microscopy studies. Cryo-EM data acquisition

[0346] Grids were prepared by applying 3 μL of purified CB1-Gi complex at 12 mg / mlto glow-discharged holey carbon gold grids (Quantifoil R1.2 / 1.3, 200 mesh). The grids were blotted using a Vitrobot Mark IV (FEI) with 3 s blotting time and blot force 3 at 100% humidity at room temperature and plunge-frozen in liquid ethane. A total of 9,822 movies for VIP36 and 5,832 movies for VIP2.33 were recorded on a Titan Krios electron microscope (Thermo Fisher Scientific-FEI) operating at 300 kV at a calibrated magnification of 96,000x 91 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO corresponding to a pixel size of 0.877 (VIP36) and 0.920 (VIP2.33) Å. Micrographs were recorded using a K3 Summit direct electron camera (Gatan Inc.) with an accumulated dose of ∼58 (VIP36) and ~44 (VIP2.33) electrons per Å2. Automatic data acquisition was done using SerialEM v3.9. Image processing and 3D reconstructions

[0347] Micrographs were subjected to beam-induced motion correctionusing MotionCor2 implemented in Relion v3.1. CTF parameters for each micrograph weredetermined by CTFFIND4. An initial set of 3,386,798 (VIP36) and 1,782,737 (VIP2.33) particle projections were extracted using semi-automated procedures and subjected to reference-free two-dimensional and multiple rounds of three-dimensional classification in Relion to remove low-resolution and otherwise poor-quality particles.295,299 (VIP36) and 161,137 (VIP2.33) final particles were reconstructed to a global nominal resolution of 2.86 Å (VIP36) and 3.03 Å (VIP2.33) at FSC of 0.143 using non-uniform refinement. Local resolution was estimated within Relion. Final data collection and processing numbers are presented in Table S1 and S2 and the processing workflow is presented in Extended Data. Fig.5. Model building and refinement

[0348] The initial template of CB1 was the MDMB-Fubinaca-bound CB1-Gi complexstructure (PDB: 6N4B). Phenix.elbow was used to generate Agonist coordinates and geometry restrains. Models were docked into the EM density map using UCSF Chimera. Coot was used for iterative model building and the final model was subjected to global refinement and minimization in real space using phenix.real_space_refine in Phenix. Model geometry was evaluated using Molprobity (online server). FSC curves were calculated between the resulting model and the half map used for refinement as well as between the resulting model and the other half map for cross-validation. The final refinement parameters are provided in SI Table S1. Table S1: CryoEM data collection, model refinement and validation of CB1 / VIP36 Gi1 / scFv16 Data Collection Voltage (kV) 300 92 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Magnification 105,000x Total electron dose (e- / Å2) 58 Defocus range (µm) -0.7 to -2.0 Calibrated pixel size (Å) 0.877 Micrographs collected 9,822 Data Processing Extracted particles 3,386,798 Particles used for final reconstruction 295,299 Final map resolution (Å, 0.143 FSC) 2.86 Map resolution range (Å) 2.5 – 4.5 Map sharpening B factor (Å2) 74.4 Model Content Initial models used (PDB code) 6N4B Total number of atoms 8512 No. of protein residues 1005 No. of ligands 1 Model Validation CC map vs. model (%) 0.81 RMSD Bond lengths (Å) / Bond angles (°) 0.007 / 0.995 Ramachandran plot statistics Favored (%) 91.82 Allowed (%) 8.09 Outliers (%) 0.00 Rotamer outliers (%) 0.00 C-beta deviations 0.00 Clash score 5.52 Table S2: CryoEM data collection, model refinement and validation of CB1 / VIP2.33 / Gi1 / scFv16 Data Collection Voltage (kV) 300 Magnification 96,000x 93 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Total electron dose (e- / Å2) 44 Defocus range (µm) -0.7 to -2.0 Calibrated pixel size (Å) 0.920 Micrographs collected 5,832 Data Processing Extracted particles 1,782,737 Particles used for final reconstruction 161,137 Final map resolution (Å, 0.143 FSC) 3.03 Map resolution range (Å) 2.5 – 4.5 Map sharpening B factor (Å2) 68.6 Model Content Initial models used (PDB code) 6N4B Total number of atoms 8482 No. of protein residues 1005 No. of ligands 1 Model Validation CC map vs. model (%) 0.75 RMSD Bond lengths (Å) / Bond angles (°) 0.005 / 0.645 Ramachandran plot statistics Favored (%) 94.34 Allowed (%) 5.66 Outliers (%) 0.00 Rotamer outliers (%) 0.00 C-beta deviations 0.00 Clash score 9.35 Pharmacokinetic Studies on FUB, VIP36 and CB13

[0349] Male C57Bl / 6J mice were dosed by intraperitoneal injection or IV at a volume of10 µl per gram body weight. Mice were anesthetized with isoflurane prior to the collection of blood and brain. VIP36 and CB13 were formulated in 10% DMSO / 10% Cremophor EL / 80% saline. MDMB-Fubinaca was formulated in 5% DMSO / 5% Cremophor EL / 90% saline. 94 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0350] Analytical Sample Preparations: 5µL plasma samples were directly loaded to a96-well Millipore Multiscreen Solvinert 0.45 micron low binding PTFE hydrophilic filter plate. Tissue samples were homogenized with water (x3 dilution) then 5 µL was loaded to the filter plate. All plasma / tissue samples were treated with 75 µL 90 / 10 acetonitrile / water with carbamazepine as the internal standard to extract the analyte and precipitate protein. The plates were agitated on ice for approximately ten minutes prior to centrifugation into a collection plate. Separate standard curves were prepared in blank mouse plasma and tissue homogenate and processed in parallel with the samples. The filtrate was directly analyzed by LC-MS / MS.. HPLC and MS / MS parameters are provided in the accompanying tables.

[0351] MDCK-MDR1 – The MDR1-MDCKI cell line was provided by the NationalInstitute of Health (Bethesda, MD).

[0352] Cell Culture: Cells were cultured in Dulbecco’s Modified Eagle Medium(DMEM) (Gibco, #11995-065) supplemented with 10% FBS (Gibco, #10082-147), 50 U / ml Penicillin-Streptomycin (Gibco, #15070-063), and 80 ng / ml colchicine (Sigma-Aldrich, #C9754) in a humidified atmosphere of 5% CO2 at 37 °C. Permeability Assay

[0353] Cells were seeded on Greiner Bio-One ThinCertTM inserts at a density of 50000cells / well. Testing was usually 4-7 days after plating, when the TEER value was > 250 Ohms / cm2. Prior to initiating the assay, the cells were equilibrated in Hank’s Balanced Salt Solution (HBSS) with 10 mM HEPES (Stemcell Technologies, #37150) and 0.02% BSA, pH 7.4 for 1 hour at 37 °C with 5% CO2 and 95% relative humidity. Test compound solutions for the donor chambers were prepared at 2 µM in the above buffer, containing 100 µM Lucifer Yellow (LY) (Sigma-Aldrich, #L0144) as the monolayer-integrity marker. The final DMSO concentration was 0.5 % for each test compound. To reduce non-specific binding, the HBSS buffer with 10 mM HEPES was supplemented with 0.5 % BSA in the receiver compartments. The permeability was examined in apical to basolateral (A-B) and basolateral to apical (B-A) directions. Samples were taken at the beginning of the incubation from the donor side, and after 90 minutes from the donor and receiver compartments and the concentration of drug was determined by LC-MS. LY fluorescence was measured at 430 / 535 nm using a BioTek Synergy Neo2 microplate reader (Agilent, Santa Clara, CA). LY transport of < 5 % was considered acceptable. Propranolol was used as a high permeability marker, nadolol for low permeability, and quinidine as positive control for MDR1. 95 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0354] Apparent permeability, Papp, was calculated using the equation:

[0355]

[0356] e permeability rate, C0 is the initial concentration in the donorcompartment, an s t e surface area of the insert (0.33cm2). The ratio of the calculated Papp values were used as an efflux ratio.

[0357] CB13 Instrument Settings LC (Shimadzu UFLC XR) conditions Compound CB13 I.S. e) GraTime (min) Mobile phase A (%) Mobile phase B (%) MSCompound CB13 I.S. e)96 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Ion Source Gas1 55 MDLC (Shimadzu UFLC XR) conditions Compound FUB I.S. e) GraTime (min) Mobile phase A (%) Mobile phase B (%) MSCompound FUB I.S. e)97 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Curtain GAS 35 VIPLC (Shimadzu UFLC XR) conditions Compound VIP36 I.S.e) GraTime (min) Mobile phase A (%) Mobile phase B (%) MSCompound VIP36 I.S.e)98 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Collision Gas 7 i Mic

[0358] Male and female C57BL / 6J mice (24–38 g, 8-12 weeks) were purchased fromJackson Laboratories (Bar Harbor, ME). All mice were maintained on a 12 hr light / dark cycle with Purina rodent chow and water available ad libitum and housed in groups of five until testing. These mice were kept at a constant temperature of 22^±^2^°C, and relative humidity was maintained at 40–50%. All animal studies reported adhere to the ARRIVE guidelines18. All procedures were preapproved by the Institutional Animal Care and Use Committee (Washington University) and conducted according to the 2011 NIH Guide for the Care and Use of Laboratory Animals. Mechanical Sensitivity Testing

[0359] Mechanical thresholds of the hindpaw was tested using von Frey hair filaments(Bending force ranging from 0.02 g to 2.56 g) in the up-and-down method.18A response to the hindpaw was defined as a raising, licking, or shaking of the stimulated paw. The first filament tested was 0.4 g and if there was no response a heavier filament (up) was used and if a response was generated a lighter filament (down) was used. Following the first response the mice were tested on 4 more times following the described pattern. Researchers were blinded to grouping throughout the experiment. Mice were tested in a behavior room, separate from the vivarium, with dimmed light and a while noise machine to limit sound interference. All mechanical tests were done between 08:00 and 17:00. Mice were habituated prior to testing on an elevated mesh platform and confined to a 10 x 10-cm acrylic holding container. 99 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO Following testing mice were counterbalanced so that each group had the same relative staring / post-SNI measurements. Spared Nerve Injury

[0360] Spared nerve injury (SNI) procedures were performed as described previously.21The three branches of the sciatic nerve were exposed by an incision and then separated from the biceps femoris muscle. The common peroneal and tibial branches were ligated with sutures and then cut distal to the ligation without interference of the sural nerve. Sham operations underwent a similar protocol with the incision and exposure of the nerve, but no ligation was made. Following surgery, regardless of sham or SNI mice, the skin was closed with staples that were removed post operate day 10 one incision was fully healed. To allow for full healing from surgery no behavioral testing began sooner than post operative day 7. Dose Response and Calculation of ED50

[0361] SNI Method. To evaluate efficacy in neuropathic pain, between-subjects doseresponse curves were conducted for VIP 36 and FUB in SNI mice. VIP36 (0.03, 0.1, 0.3, 1, or 3 mg / kg, I.P.) and FUB (0.003, 0.01, 0.03, 0.1, or 0.3 mg / kg, I.P.) were administered to mice day 10 after SNI surgery. Raw data for ED50 calculation were converted to % baseline threshold using the formula: (experimental value − post-SNI baseline) / (pre-SNI baseline − post-SNI baseline). ED50 was calculated using nonlinear regression through GraphPad Prism. Repeated Dosing Studies

[0362] To determine whether tolerance to VIP36 (1mg / kg) or MDMB-Fubinaca(0.1mg / kg) was induced, a repeated dosing paradigm was used. VIP36 or MDMB-Fubinaca was administered by I.P. injection twice daily - once in the morning around 09:00, and again around 17:00 each day 11 days in post-SNI studies. The antinociceptive testing occurred 1-2 hours after the morning dose every other day across the 11 days in the SNI model. The first dose of VIP36 or MDMB-Fubinaca was given on day 10 post-SNI. Both female and male mice were used in these studies. Nitroglycerin Model of Chronic Migraine 100 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO

[0363] Mice were administered nitroglycerine (NTG)19 or vehicle (saline) every otherday for 9 days. Cephalic mechanical thresholds were tested on days 1, 5, and 9. Test days a basal threshold was measured before mice received NTG that day. Following basal measures NTG was injected, and mice were subsequently tested 2 hours after for their post-treatment effect. On day 10 mice again had their baseline measurements taken and then were administered VIP36 1 mg / kg in 5 ml / kg at 0.2 mg / ml and injected IP, it was dissolved in 80% saline, 10% kolliphor, and 10% DMSO, or vehicle and tested 1 hr later.

[0364] Subtype selectivity: To determine CB1 selectivity, mice with SNI exhibitedhyperalgesia were administrated AM6303 mg / kg i.p, AM654510 mg / kg i.p and vehicle administered 30 min before VIP 1 mg / kg I.P. Tetrad

[0365] It has been previously demonstrated that centrally penetrating CB1 receptoragonist induce a set of behavioral effect known as the “tetrad”; catalepsy, locomotor ataxia, antinociception, and hypothermia.22We examined the triad in a time (0.5, 1, 2, 5, and 24 hr post treatment) and dose (VIP36): 3, 10, 20, 30 mg / kg; MDMB-Fubinaca: 0.01, 0.03, 0.1, 0.3 mg / kg) dependent manner with both VIP36 and the parent compound MDMB-Fubinaca. Day of testing separate cohorts of animals were dosed with VIP36, FUB, or Vehicle, then underwent a “triad” of behavioral measures in order; catalepsy, tail-flick antinociception, and rectal temperature at the listed time points. Separate cohort also had motor ataxia measured for VIP36 at 3mg / kg dose and MDMB-Fubinaca at 0.01 mg / kg, 0.03 mg / kg dose and 0.1 mg / kg dose. Catalepsy tests were performed by placing mice forepaws on a stationary bar approximately 3.5 cm off the ground and timing latency till first movement. A max cut off was set for 30 seconds. Tail flick antinociception was assessed by placing the distal part of the tail in 50°C water bath and timing for latency to respond. A max cut off 15 seconds was set to prevent tissue damage. Locomotor ataxia was assessed by injecting mice with compound or vehicle and immediately placing them in the locomotor box (VersaMax System from AccuScan Instruments, Inc. Columbus, Ohio, USA). Mice were in the box for 1 hour and measures of total distance travel for the full hour were recorded. Complete Freund Adjuvant Allodynia

[0366] Complete Freund Adjuvant is an established model of inflammation that inducesmechanical sensitization.20Mice had the pre-CFA baseline recorded than same day injected 101 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO with a 20-^L of 1:1 CFA:saline in a unilateral intraplanar injection into the right hind paw. The next day, approximately 19 hours after the CFA injection The post-CFA values for mechanical sensitivity were assessed. Dose Response and Calculation of ED50

[0367] VIP36 (0.1, 0.3, 1, or 3 mg / kg, I.P.) or vehicle was administered in separatecohorts of male and female mice following the days Post-CFA baseline. Mice were then subsequently tested 0.5, 1, 2, 5, and 24 hrs post VIP36 injection. Raw data for ED50 calculation were converted to % baseline through: (experimental value − post-CFA baseline) / (pre-CFA baseline − post-CFA baseline). ED50 was calculated using nonlinear regression through GraphPad Prism. The same process was used for FUB (0.01, 0.03, 0.1, or 0.3 mg / kg, I.P.) to generate ED50 and dose response curves for those studies.

[0368] Subtype selectivity: To determine CB1 selectivity, mice with CFA exhibitedhyperalgesia were administrated AM6303 mg / kg i.p, AM654510 mg / kg i.p and vehicle administered 30 min before VIP 1 mg / kg I.P. Table S3: ADME studies

[0369] y . . y IP36 was determinedin plasma and microsome in human and mice. b. Two assays were used to determine the permeability of VIP36 on MDCK and PAMPA assay. c. The unbound partition coefficient 102 CORE / 3510075.0144 / 200653156.1021053 / WO WSTL021053.WO was calculated (Cbrain / Cplasma). Compounds availabilities in mice were determined using the free fraction from protein binding and the free conc was calculated for plasma and brain.

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[0390] The dimensions and values disclosed herein are not to be understood as beingstrictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 wt.%” is intended to mean “about 40 wt.%”.

[0391] A number of implementations have been described. Nevertheless, it will beunderstood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims. 104 CORE / 3510075.0144 / 200653156.1

Claims

1. 021053 / WO WSTL021053.WO CLAIMS:

1. A compound of Formula 1, or a pharmaceutically acceptable salt thereof: OMe O 1) R1is hydrogen, alkyl, or substituted alkyl.

2. The compound of claim 1, wherein R1 is C1-C10 alkyl.

3. The compound of claim 1, wherein R1 is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl.

4. The compound of claim 1, wherein R1is methyl, n-hexyl, or n-octyl.

5. The compound of claim 1, wherein R1is substituted alkyl and the substituent is -NH- C(=N)-NH2, -NH-C(=O)-NH2, substituted cycloalkyl, substituted cycloalkenyl, aryl, amine, cyano, hydroxyl, or carboxyl.

6. The compound of claim 5, wherein R1 is substituted alkyl and the substituent is -NH- C(=N)-NH2 or -NH-C(=O)-NH2.

7. The compound of claim 5, wherein R1is substituted alkyl and the substituent is substituted cyclobutenyl.

8. The compound of claim 7, wherein R1 is substituted alkyl and the substituent is amino-dioxy cyclobutenyl. 105 CORE / 3510075.0144 / 200653156.1 021053 / WO WSTL021053.WO 9. The compound of claim 5, wherein R1is substituted alkyl and the substituent is aryl.

10. The compound of claim 9, wherein R1is substituted alkyl and the substituent is phenyl.

11. The compound of claim 5, wherein R1 is substituted alkyl and the substituent is -NH2.

12. The compound of claim 5, wherein R1is substituted alkyl and the substituent is cyano.

13. The compound of claim 5, wherein R1is substituted alkyl and the substituent is hydroxyl.

14. The compound of claim 5, wherein R1 is substituted alkyl and the substituent is carboxyl.

15. A compound of Formula 2, or a pharmaceutically acceptable salt thereof: 2) or substituted C1-C10 alkyl.

16. The compound of claim 15, wherein R1 is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl.

17. The compound of claim 15, wherein R1is methyl, n-hexyl, or n-octyl.

18. The compound of claim 15, wherein R1is substituted C1-C10alkyl and the substituent is -NH-C(=N)-NH2, -NH-C(=O)-NH2, substituted cycloalkyl, substituted cycloalkenyl, aryl, 106 CORE / 3510075.0144 / 200653156.1 021053 / WO WSTL021053.WO amine, cyano, hydroxyl, or carboxyl.

19. The compound of claim 18, wherein R1is substituted C3-C8alkyl and the substituent is -NH-C(=N)-NH2 or -NH-C(=O)-NH2.

20. The compound of claim 18, wherein R1 is substituted C3-C5 alkyl and the substituent is substituted cyclobutenyl.

21. The compound of claim 20, wherein R1 is substituted C3-C5 alkyl and the substituent is amino-dioxy cyclobutenyl.

22. The compound of claim 18, wherein R1is substituted C1-C3alkyl and the substituent is aryl.

23. The compound of claim 22, wherein R1 is substituted C1-C3 alkyl and the substituent is phenyl.

24. The compound of claim 18, wherein R1 is substituted C3-C8 alkyl and the substituent is -NH2.

25. The compound of claim 18, wherein R1is substituted C4-C6alkyl and the substituent is cyano.

26. The compound of claim 18, wherein R1 is substituted C3-C8 alkyl and the substituent is hydroxyl.

27. The compound of claim 18, wherein R1is substituted C4-C6alkyl and the substituent is carboxyl.

28. A compound of Formula 3, or a pharmaceutically acceptable salt thereof: 107 CORE / 3510075.0144 / 200653156.1 021053 / WO WSTL021053.WO 3) R3is hydrogen, -NH-C(=N)-NH2, -NH-C(=O)-NH2, amino-dioxy cyclobutenyl, phenyl, amine, cyano, hydroxyl, or carboxyl; and n is an integer of 0 to 8.

29. The compound of claim 28, wherein R3 is hydrogen and n is an integer of 1 to 8.

30. The compound of claim 28, wherein R3 is -NH-C(=N)-NH2 or -NH-C(=O)-NH2.

31. The compound of claim 28, wherein R3 is amino-dioxy cyclobutenyl or phenyl.

32. The compound of claim 28, wherein R3 is -NH2 or cyano.

33. The compound of claim 28, wherein R3 is hydroxyl or carboxyl.

34. The compound of any one of claims 30 to 33, wherein n is an integer of 1 to 8.

35. The compound of any one of claims 30 to 33, wherein n is an integer of 2 to 6.

36. The compound of any one of claims 30 to 33, wherein n is an integer of 3 to 6.

37. The compound of any one of claims 30 to 33, wherein n is an integer of 3 to 5.

38. The compound of any one of claims 30 to 33, wherein n is an integer of 4. 108 CORE / 3510075.0144 / 200653156.1 021053 / WO WSTL021053.WO 39. A compound of Formula 4, or a pharmaceutically acceptable salt thereof: 4) R4 is hydrogen, -NH-C(=N)-NH2, -NH-C(=O)-NH2, amino-dioxy cyclobutenyl, phenyl, amine, cyano, hydroxyl, or carboxyl; and n is an integer of 0 to 8.

40. The compound of claim 39, wherein R3is hydrogen and n is an integer of 1 to 8.

41. The compound of claim 39, wherein R3is -NH-C(=N)-NH2or -NH-C(=O)-NH2.

42. The compound of claim 39, wherein R3is amino-dioxy cyclobutenyl or phenyl.

43. The compound of claim 39, wherein R3is -NH2or cyano.

44. The compound of claim 39, wherein R3 is hydroxyl or carboxyl.

45. The compound of any one of claims 41 to 44, wherein n is an integer of 1 to 8.

46. The compound of any one of claims 41 to 44, wherein n is an integer of 2 to 6.

47. The compound of any one of claims 41 to 44, wherein n is an integer of 3 to 6.

48. The compound of any one of claims 41 to 44, wherein n is an integer of 3 to 5.

49. The compound of any one of claims 41 to 44, wherein n is an integer of 4. 109 CORE / 3510075.0144 / 200653156.1 021053 / WO WSTL021053.WO 50. The compound of claim 1, selected from: 110 CORE / 3510075.0144 / 200653156.1 021053 / WO WSTL021053.WO F , 111 CORE / 3510075.0144 / 200653156.1 021053 / WO WSTL021053.WO .

51. The compound of claim 1, selected from: .

52. A pharmaceutical composition comprising a compound of any one of claims 1 to 51 and a pharmaceutically acceptable carrier.

53. The pharmaceutical composition of claim 52, further comprising an additional active 112 CORE / 3510075.0144 / 200653156.1 021053 / WO WSTL021053.WO agent.

54. The pharmaceutical composition of claim 53 wherein the additional active agent is selected from the group consisting of an opioid analgesic, a non-steroidal anti-inflammatory drug, an anticonvulsant, an antidepressant, and a local anesthetic.

55. The pharmaceutical composition of any one of claims 52 to 54, wherein the pharmaceutically acceptable carrier is selected from the group consisting of a liquid carrier, a solid carrier, and a semi-solid carrier.

56. The pharmaceutical composition of claim 55, wherein the liquid carrier is selected from the group consisting of water, saline, and a pharmaceutically acceptable organic solvent.

57. The pharmaceutical composition of any one of claims 52 to 56, formulated for oral, parenteral, topical, or transdermal administration.

58. A method of treating pain in a subject in need thereof, comprising: administering to the subject an effective amount of a compound of any one of claims 1 to 51 or a pharmaceutical composition of any one of claims 52 to 57.

59. The method of claim 58, wherein the pain is selected from the group consisting of neuropathic pain, inflammatory pain, and chronic pain.

60. The method of claim 59, wherein the neuropathic pain is associated with a condition selected from the group consisting of diabetic neuropathy, postherpetic neuralgia, and chemotherapy-induced peripheral neuropathy.

61. The method of any one of claims 58 to 60, wherein the compound or pharmaceutical composition is administered orally, parenterally, topically, or transdermally.

62. The method of any one of claims 58 to 61, further comprising administering an additional active agent selected from the group consisting of an opioid analgesic, a non- steroidal anti-inflammatory drug, an anticonvulsant, an antidepressant, and a local anesthetic. 113 CORE / 3510075.0144 / 200653156.1

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