human CB2 receptor ( Ki = 45.5 nM ); human CB1 receptor ( Ki = 48.3 nM ); rat CB1 receptor ( Ki = 55.4 nM )
Cannabinoid CB1 receptor (rat CB1: Kᵢ = 55.4 nM; human CB1: Kᵢ = 48.3 nM), Cannabinoid CB2 receptor (human CB2: Kᵢ = 45.5 nM) [1]
- Human CB1 (hCB1) receptor (EC₅₀ = 46 ± 5 nM, efficacy = 39 ± 11% in cAMP assay) [2]

3-[2-Cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 59-3074) is a novel, selective cannabinoid CB1/CB2 receptor ligand (Ki = 55.4, 48.3, and 45.5 nM at rat and human cannabinoid CB1 and human CB2 receptors, respectively), with partial agonist properties at these receptors in guanosine 5-[γ35S]-thiophosphate triethyl-ammonium salt ([35S]GTPγS) binding assays[1].

---

1. In guanosine 5-[γ³⁵S]-thiophosphate triethyl-ammonium salt ([³⁵S]GTPγS) binding assays, BAY 59-3074 exhibited partial agonist properties at cannabinoid CB1 and CB2 receptors [1]
2. In a functional cAMP assay using Chinese Hamster Ovary (CHO) cells stably expressing hCB1 receptor, BAY 59-3074 showed an EC₅₀ of 46 ± 5 nM and an efficacy of 39 ± 11% (relative to the full agonist CP55940) [2]
3. Molecular docking, induced fit, and molecular dynamics (MD) studies revealed that BAY 59-3074 binds to the orthosteric binding site of hCB1, with its sulfonyl region interacting with T197³·³³ and W279⁵·⁴³ via water-bridged hydrogen bonds, and π-stacking with aromatic residues such as F268^ECL2 and F170²·⁵⁷ [2]

BAY 59-3074 (oral administration; 0.3–3 mg/kg; daily; for 2 weeks; male Wistar rats) Rat models of chronic neuropathic and inflammatory pain show improved antihyperalgesic and antiallodynic effects against thermal or mechanical stimuli with treatment.

---

BAY 59-3074 (0.3-3 mg/kg, p.o.) induced antihyperalgesic and antiallodynic effects against thermal or mechanical stimuli in rat models of chronic neuropathic (chronic constriction injury, spared nerve injury, tibial nerve injury, and spinal nerve ligation models) and inflammatory pain (carrageenan and complete Freund's adjuvant models). Antiallodynic efficacy of BAY 59-3074 (1 mg/kg, p.o.) in the spared nerve injury model was maintained after 2 weeks of daily administration. However, tolerance developed rapidly (within 5 days) for cannabinoid-related side effects, which occur at doses above 1 mg/kg (e.g., hypothermia). Uptitration from 1 to 32 mg/kg p.o. (doubling of daily dose every 4th day) prevented the occurrence of such side effects, whereas antihyperalgesic and antiallodynic efficacy was maintained/increased. No withdrawal symptoms were seen after abrupt withdrawal following 14 daily applications of 1 to 10 mg/kg p.o. It is concluded that BAY 59-3074 may offer a valuable therapeutic approach to treat diverse chronic pain conditions.[1]

---

BAY 59-3074 [3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butane-sulfonate] is a structurally novel cannabinoid CB1/CB2 receptor partial agonist with analgesic properties. The present study was performed to confirm its receptor binding profile in a highly sensitive in vivo assay. Rats (n=10) learned to discriminate BAY 59-3074 (0.5 mg/kg, p.o., t-1 h) from vehicle in a fixed-ratio: 10, food-reinforced two-lever procedure after a median number of 28 training sessions. BAY 59-3074 generalized dose-dependently (ED(50): 0.081 mg/kg, p.o.) and the cue was detectable between 0.25 and 4 h after administration. The selective cannabinoid CB1 receptor antagonist SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] blocked the discriminative effects of BAY 59-3074 (ID50: 1.79 mg/kg, i.p.). Complete generalization was also obtained after i.p. administration of BAY 59-3074 (ED50 value: 0.41 mg/kg), and the reference cannabinoids BAY 38-7271 [(-)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-butanesulfonate, 0.011 mg/kg], CP 55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol, 0.013 mg/kg], HU-210 [(-)-11-OH-Delta8-tetrahydrocannabinol dimethylheptyl, 0.022 mg/kg], WIN 55,212-2 [(R)-4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo [3,2,1-ij] quinolin-6-one, 0.41 mg/kg] and (-)-Delta9-tetrahydrocannabinol (0.41 mg/kg). Non-cannabinoids with analgesic properties, such as morphine, amitriptyline, carbamazepine, gabapentin and baclofen, did not generalize to the cue. It is concluded that the discriminative stimulus effects of BAY 59-3074 are specifically mediated by cannabinoid CB1 receptor activation [3].

---

1. In rat models of chronic neuropathic pain (chronic constriction injury [CCI], spared nerve injury [SNI], tibial nerve injury, spinal nerve ligation) and inflammatory pain (carrageenan, complete Freund's adjuvant), BAY 59-3074 (0.3-3 mg/kg, p.o.) induced dose-dependent antihyperalgesic and antiallodynic effects against thermal and mechanical stimuli [1]
2. In the SNI model, the antiallodynic efficacy of BAY 59-3074 (1 mg/kg, p.o.) was maintained after 2 weeks of daily administration; tolerance to cannabinoid-related side effects (e.g., hypothermia, occurring at doses >1 mg/kg) developed rapidly within 5 days, while uptitration from 1 to 32 mg/kg p.o. (doubling the daily dose every 4 days) prevented side effects and maintained/increased antihyperalgesic/antiallodynic efficacy [1]
3. No withdrawal symptoms were observed after abrupt withdrawal following 14 days of daily administration of BAY 59-3074 (1-10 mg/kg, p.o.) in rats [1]
4. In the rat hot plate assay, the analgesic and hypothermic effects of BAY 59-3074 were blocked by the CB1 antagonist SR 141716A; in a drug discrimination procedure, the cue induced by BAY 59-3074 was also blocked by SR 141716A [1]
5. In a rat drug discrimination study, rats trained to discriminate BAY 59-3074 (0.5 mg/kg, p.o.) from vehicle showed dose-dependent generalization to BAY 59-3074 (ED₅₀ = 0.081 mg/kg, p.o.; ED₅₀ = 0.41 mg/kg, i.p.); the discriminative stimulus effects were blocked by SR 141716A (ID₅₀ = 1.79 mg/kg, i.p.) [3]
6. Complete generalization to BAY 59-3074 was observed with reference cannabinoids (BAY 38-7271, CP 55,940, HU-210, WIN 55,212-2, (-)-Δ⁹-THC), while non-cannabinoid analgesics (morphine, amitriptyline, carbamazepine, gabapentin, baclofen) did not generalize to its cue [3]
7. In C57BL6 mice administered 3 mg/kg of BAY 59-3074 via intraperitoneal (IP) injection, the maximum brain concentration (Cmax) was ~341 ng/mL, with a brain:plasma Cmax ratio of 0.40 [2]

Radioligand Displacement Assay [2]
Further characterization of 21 was performed using radioligand displacement of [3H]CP55940 and equilibrium dissociation constant (Ki) value was determined as described previously. Data reported are average values from 3 measurements with <30% standard error.

---

1. Receptor binding assay for Ki determination: Membrane preparations from tissues/cells expressing rat CB1, human CB1, and human CB2 receptors were incubated with BAY 59-3074 and a radiolabeled cannabinoid receptor ligand. The binding affinity (Kᵢ) of BAY 59-3074 for each receptor subtype was calculated based on the displacement of the radioligand [1]
2. [³⁵S]GTPγS binding assay for agonist activity: Membrane preparations containing CB1/CB2 receptors were incubated with BAY 59-3074, GDP, and [³⁵S]GTPγS. The incorporation of [³⁵S]GTPγS into the membranes was measured to evaluate the partial agonist activity of BAY 59-3074 at CB1 and CB2 receptors [1]
3. Radioligand displacement assay for hCB1 binding affinity: Membranes from CHO cells overexpressing hCB1 were incubated with [³H]CP55940 and serial dilutions of BAY 59-3074. The equilibrium dissociation constant (Ki) was determined by measuring the displacement of the radiolabeled ligand [2]

cAMP Accumulation Assay [2]
The cAMP assays were performed in Chinese Hamster Ovary (CHO) cells stably expressing the human CB1 receptor (hCB1) cultured under standard cell culture conditions (37 °C, 5% carbon dioxide, DMEM media with 1% Penicillin/Streptomycin and 400 µg/mL G418) using the Lance™ assay kit and manufacturer’s instructions were closely followed . In brief, stimulation buffer containing 1X Hank’s Balanced Salt Solution (HBSS), 5 mM HEPES, 0.1% BSA stabilizer, and 0.5 mM final IBMX was prepared and titrated to pH 7.4 at rt. Serial dilutions of the test compounds and 300 nM forskolin, both prepared at 4× the desired final concentration in stimulation buffer, were added to a 96-well white ½ area microplate. The CHO-hCB1 cells were lifted with a non-enzymatic solution, and 4000 cells were added to each well. After incubating for 30 min at room temperature, Eu-cAMP tracer and uLIGHT-anti-cAMP working solutions were added per the manufacturer’s instructions. After incubation for 1 h, the TR-FRET signal (ex 337 nm, em 620 and 650 nm) was read on a CLARIO star multimode plate reader. Data were analyzed using Prism software. Nonlinear regression analysis was performed to fit data and obtain maximum response (Emax), EC50, correlation coefficient (r2), and other parameters. All experiments were performed in duplicate 2–3 times to ensure reproducibility and data are reported as mean ± standard error of mean unless noted otherwise.

---

1. cAMP accumulation assay in CHO-hCB1 cells: CHO cells stably expressing hCB1 were cultured under standard conditions (37 °C, 5% CO₂, DMEM with 1% Penicillin/Streptomycin and 400 µg/mL G418). The cells were lifted with a non-enzymatic solution and seeded into 96-well plates (4000 cells/well). Serial dilutions of BAY 59-3074 and forskolin (300 nM) were added, and the cells were incubated for 30 min at room temperature. Eu-cAMP tracer and uLIGHT-anti-cAMP working solutions were added, and the TR-FRET signal (ex 337 nm, em 620/650 nm) was detected to measure cAMP levels and evaluate the agonist activity of BAY 59-3074 [2]

Male Wistar rats (160-250 g)
\n0.3 mg/kg, 1 mg/kg, and 3 mg/kg
\nOral administration; daily; for 2 weeks.

---

\n In rats, generalization of BAY 59-3074 to the cue induced by the cannabinoid CB(1) receptor agonist (-)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 38-7271) in a drug discrimination procedure, as well as its hypothermic and analgesic effects in a hot plate assay, were blocked by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). BAY 59-3074 (0.3-3 mg/kg, p.o.) induced antihyperalgesic and antiallodynic effects against thermal or mechanical stimuli in rat models of chronic neuropathic (chronic constriction injury, spared nerve injury, tibial nerve injury, and spinal nerve ligation models) and inflammatory pain (carrageenan and complete Freund's adjuvant models). Antiallodynic efficacy of BAY 59-3074 (1 mg/kg, p.o.) in the spared nerve injury model was maintained after 2 weeks of daily administration. However, tolerance developed rapidly (within 5 days) for cannabinoid-related side effects, which occur at doses above 1 mg/kg (e.g., hypothermia). Uptitration from 1 to 32 mg/kg p.o. (doubling of daily dose every 4th day) prevented the occurrence of such side effects, whereas antihyperalgesic and antiallodynic efficacy was maintained/increased. No withdrawal symptoms were seen after abrupt withdrawal following 14 daily applications of 1 to 10 mg/kg p.o. It is concluded that BAY 59-3074 may offer a valuable therapeutic approach to treat diverse chronic pain conditions.[1]

---

\nPharmacokinetic Testing [2]
\nFemale C57BL/6 mice were bred in house and used at ~10 weeks of age for pharmacokinetic (PK) testing. Three animals were tested per time point. Doses were formulated in 2% NMP in canola oil, and all compounds were delivered at 3 mg/kg by intraperitoneal injection (IP). Tissues were taken at 0.5, 1, 2, and 4 h post dose. Animals were subjected to whole body perfusion using saline prior to tissue collection. Brain samples were homogenized with 50:50 ethanol:water (1:5, v/v). Forty µL of the homogenate, 10 µL of acetonitrile, and 150 µL of 100 ng/mL reserpine in acetonitrile containing 0.1% formic acid were vortexed and centrifuged. Plasma samples were diluted with 10 µL of acetonitrile, 150 µL of 100 ng/mL reserpine in acetonitrile containing 0.1% formic acid, vortexed and centrifuged. Samples were subjected to LC/MS/MS analysis. Standards were prepared in blank samples and used for calibration curves. Chromatography was performed using a Phenomenex Luna C18 column.

---

\n1. Rat chronic pain model protocol (neuropathic pain: CCI, SNI, tibial nerve injury, spinal nerve ligation; inflammatory pain: carrageenan, CFA): Rats were subjected to surgical procedures to induce chronic neuropathic pain or injected with carrageenan/CFA to induce inflammatory pain. BAY 59-3074 was administered orally at doses of 0.3-3 mg/kg, and thermal/mechanical hyperalgesia/allodynia were assessed at specific time points. For the SNI model, BAY 59-3074 (1 mg/kg, p.o.) was administered daily for 2 weeks to evaluate tolerance; for dose uptitration studies, the dose was doubled every 4 days from 1 to 32 mg/kg p.o. [1]
\n2. Rat drug discrimination assay (literature 1 and 3): Rats were trained to discriminate BAY 59-3074 (0.5 mg/kg, p.o., 1 h before testing) from vehicle in a fixed-ratio 10, food-reinforced two-lever procedure. After training, rats were tested with different doses of BAY 59-3074 (p.o./i.p.) or other cannabinoids, and the lever selection was recorded to assess generalization. The CB1 antagonist SR 141716A was administered to block the discriminative effects of BAY 59-3074 [1]
\n3. Rat hot plate assay: Rats were placed on a hot plate (fixed temperature), and the latency to paw licking/jumping was measured to evaluate analgesic effects. BAY 59-3074 was administered, and body temperature was monitored to assess hypothermic effects; SR 141716A was co-administered to confirm CB1-mediated effects [1]
\n4. Mouse pharmacokinetic (PK) assay: Female C57BL/6 mice (~10 weeks old) were administered BAY 59-3074 at 3 mg/kg via IP injection (formulated in 2% NMP in canola oil). Tissues (brain/plasma) were collected at 0.5, 1, 2, and 4 h post-dose. Brains were homogenized with 50:50 ethanol:water, and plasma/brain homogenates were processed with acetonitrile and reserpine internal standard, followed by LC/MS/MS analysis [2]

1. In C57BL6 mice injected intraperitoneally with 3 mg/kg BAY 59-3074, the maximum concentration in the brain (Cmax) was approximately 341 ng/mL, the brain/plasma Cmax ratio was 0.40, and the compound gradually accumulated in the brain during the test time points [2].

1. In rats, oral doses of BAY 59-3074 exceeding 1 mg/kg caused cannabinoid-related side effects (e.g., hypothermia), and tolerance developed rapidly within 5 days of daily administration [1]. 2. Increasing the dose of BAY 59-3074 from 1 mg/kg to 32 mg/kg (doubling the daily dose every 4 days) prevented the occurrence of side effects (e.g., hypothermia) [1].

[1]. 3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 59-3074): a novelcannabinoid Cb1/Cb2 receptor partial agonist with antihyperalgesic and antiallodynic effects. J Pharmacol Exp Ther. 2004 Aug;310(2):620-32.

[2]. Structure–Activity Relationship Development Efforts towards Peripherally Selective Analogs of the Cannabinoid Receptor Partial Agonist BAY 59-3074. Molecules. 2022 Sep 2;27(17):5672.

[3]. Discriminative stimulus effects of the structurally novel cannabinoid CB1/CB2 receptor partial agonist BAY 59-3074 in the rat. Eur J Pharmacol. 2004 Nov 28;505(1-3):127-33.

4,4,4-Trifluoro-1-butanesulfonic acid is an aromatic ether. Selective modulation of peripheral cannabinoid receptors (CBRs) has potential applications in the treatment of various diseases, including obesity, diabetes, liver disease, gastrointestinal disorders, and pain. While significant efforts have been devoted to developing selective antagonists or full agonists of CBRs, reports on the development of partial agonists are limited. Partial agonists targeting peripheral CBRs may possess desirable pharmacological properties without producing centrally mediated dissociation effects. Bayer has reported that BAY 59-3074, a partial agonist that penetrates the central nervous system and acts on both CB1 and CB2 receptors, has shown efficacy in rat models of neuropathic and inflammatory pain. In this report, we will present our efforts to synthesize analogs designed to improve peripheral selectivity while maintaining partial agonistic activity towards CB1. Our study ultimately identified a novel compound that is a partial agonist of the human CB1 (hCB1) receptor with significantly reduced brain exposure compared to BAY 59-3074. [2] The structure-activity relationship study of compound 1 (BAY 59-3074) will help in the development of compounds with better central nervous system penetration. Recent studies of the crystal structure of hCB1 and molecular docking with compound 1 have shown that the sulfonate linker is close to the polar region of the binding pocket and can therefore be replaced with a polar hydrogen bond donor linker to facilitate the development of peripherally selective analogs. Preliminary results confirm this, but controlling its potency level remains challenging. Potency and efficacy are influenced by the interaction of the linker, the linked group, and the core substitution mode. Although the structure-activity relationship of compound 1 for developing peripheral partial agonists has not been clarified, we identified two hCB1 partial agonists with improved physical properties. By changing the core substitution mode from meta to para and replacing the sulfonate linker with an amide group, we obtained benzylamide 24, which has a hydrogen bond donor and exhibited partial agonist activity in cAMP hCB1 assays. Replacing the linker group with a sulfonamide group and the alkyl chain with a cyclohexyl group yields sulfonamide 21, which also exhibits partial agonist activity in cAMP hCB1 assays. The physical properties of compound 21 are significantly favorable for its peripheral selectivity (TPSA = 91, cLogP = 4.2, hydrogen bond donor = 2). Pharmacokinetic studies confirm this, showing significantly lower brain concentrations than compound 1. Further structure-activity relationship studies based on compounds 21 and 24 hold promise for providing clues to obtaining compounds with the desired properties. The activity and efficacy of these compounds on hCB2 have not yet been verified, which will form the basis for future research and may include in vivo effect testing. [2]

---

3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 59-3074) is a novel selective cannabinoid CB(1)/CB(2) receptor ligand (K(i) of 55.4, 48.3 and 45.5 nM on rat and human cannabinoid CB(1) and human CB(2) receptors, respectively) and exhibits partial agonist properties on these receptors in a binding assay with guanosine 5-[γ(35)S]-triethylammonium thiophosphate ([(35)S]GTPγS)). In rats, the generalization of cues induced by the cannabinoid CB(1) receptor agonist (-)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 38-7271) in the drug identification procedure, as well as its cooling and analgesic effects in the hot plate test, can be blocked by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). [1]

---

1. BAY 59-3074 is a novel selective cannabinoid CB1/CB2 receptor partial agonist with anti-hyperalgesia and anti-abnormal pain effects, which may provide a valuable treatment for a variety of chronic pain diseases.[1]
2. BAY 59-3074 is a CB1/CB2 receptor partial agonist that can penetrate the blood-brain barrier; its structure is modified to develop peripheral selective analogs, thereby reducing its exposure in the brain.[2]
3. The discriminative stimulus effect of BAY 59-3074 in rats is specifically mediated by activation of cannabinoid CB1 receptors and does not generalize to non-cannabinoid analgesics.[3]

C18H13NO4F6S

453.35552

453.047

C, 59.99; H, 5.03; F, 4.52; N, 26.65; O, 3.81

406205-74-1

10479060

White to off-white solid powder

1.496g/cm3

490.569ºC at 760 mmHg

250.488ºC

1.521

6.501

0

11

7

30

709

0

O=S(CCCC(F)(F)F)(OC1=CC=CC(OC2=CC=CC(C(F)(F)F)=C2C#N)=C1)=O

LWUSZIVDPJPVBW-UHFFFAOYSA-N

InChI=1S/C18H13F6NO4S/c19-17(20,21)8-3-9-30(26,27)29-13-5-1-4-12(10-13)28-16-7-2-6-15(14(16)11-25)18(22,23)24/h1-2,4-7,10H,3,8-9H2

[3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl] 4,4,4-trifluorobutane-1-sulfonate

BAY59-3074; BAY-593074; BAY-59-3074; BAY 59-3074; BAY 593074; BAY-59-3074; 1-Butanesulfonic acid, 4,4,4-trifluoro-, 3-(2-cyano-3-(trifluoromethyl)phenoxy)phenyl ester; BAY-593074; 5FO5Z101GU; CHEMBL1354658; 3-(2-Cyano-3-(trifluoromethyl)phenoxy)phenyl 4,4,4-trifluorobutane-1-sulfonate; BAY593074

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~91 mg/mL (~200.7 mM)
Ethanol: ~91 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.51 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)