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. 2019 Nov 26;10(12):1641–1647. doi: 10.1021/acsmedchemlett.9b00406

Discovery of Axelopran (TD-1211): A Peripherally Restricted μ-Opioid Receptor Antagonist

Daniel D Long 1,*, Scott R Armstrong 1, David T Beattie 1, Christina B Campbell 1, Timothy J Church 1, Pierre-Jean Colson 1, Sean M Dalziel 1, John R Jacobsen 1, Lan Jiang 1, Glenmar P Obedencio 1, Miroslav Rapta 1, Daisuke Saito 1, Ioanna Stergiades 1, Pamela R Tsuruda 1, Priscilla M Van Dyke 1, Ross G Vickery 1
PMCID: PMC6912869  PMID: 31857840

Abstract

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The effects of opioids in the central nervous system (CNS) provide significant benefit in the treatment of pain but can also lead to physical dependence and addiction, which has contributed to a growing opioid epidemic in the United States. Gastrointestinal dysfunction is an additional serious consequence of opioid use, and this can be treated with a localized drug distribution of a non-CNS penetrant, peripherally restricted opioid receptor antagonist. Herein, we describe the application of Theravance’s multivalent approach to drug discovery coupled with a physicochemical property design strategy by which the N-substituted-endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenyl carboxamide series of μ-opioid receptor antagonists was optimized to afford the orally absorbed, non-CNS penetrant, Phase 3 ready clinical compound axelopran (TD-1211) 19i as a potential treatment for opioid-induced constipation.

Keywords: μ-Opioid receptor antagonists, multivalent approach, peripherally restricted, opioid-induced constipation

Analgesic opioids such as morphine remain a mainstay of therapeutic intervention to treat chronic malignant and nonmalignant pain as a result of their agonism of the G-protein coupled μ-opioid receptor, principally within the central nervous system (CNS).1,2 However, despite their effectiveness, opioids are associated with a number of centrally mediated side effects, including severe physical dependence and addiction, which in recent years has been highlighted as the cause of a growing opioid epidemic in the United States (US).3 An additional serious consequence of both therapeutic and illicit opioid use is the induction of a predominantly peripherally mediated gastrointestinal (GI) dysmotility, termed opioid bowel dysfunction (OBD).4,5 The most attractive approach to combat the often debilitating effects of OBD in patients is by employing a localized drug distribution of a non-CNS penetrant, peripherally restricted opioid receptor antagonist which has no impact on beneficial agonist analgesia but which can selectively alleviate the GI dysfunction.6 Four peripherally selective opioid receptor antagonists have been approved by the FDA for the treatment of opioid-related bowel dysfunction, Figure 1.

Figure 1.

Figure 1

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Peripherally restricted opioid receptor antagonists.

Alvimopan (Entereg) (1) was approved in the US in 2008 as an oral treatment to accelerate upper and lower gastrointestinal recovery following partial large or small bowel resection surgery with primary anastomosis in a hospital setting.7 Methyl naltrexone (Relistor) (2), approved for subcutaneous dosing (2008)8 and as an oral formulation (2016),9 naloxegol (Movantik) (3),10 approved in 2014 as an oral formulation, and most recently naldemedine (Symproic) (4)11 approved in 2017 as an oral agent, are all indicated for the treatment of opioid-induced constipation (OIC) for adult patients with chronic noncancer pain. In addition, the non-opioid ligand Amitiza (lubiprostone), a Cl-channel activator, was approved in 2013 for the treatment of constipation caused by opioids in patients with chronic, noncancer pain.12

We previously reported the discovery of the N-substituted-endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenyl carboxamide series A of μ-opioid receptor antagonists, Figure 2.13 Herein, we describe the design strategy by which this series was optimized to afford the clinical compound axelopran (TD-1211) 19i, Figure 2, which balances predictable and moderate oral bioavailability with a high degree of peripheral selectivity. The pharmacological profile and optimized synthesis of 19i are additionally described.

Figure 2.

Figure 2

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Theravance μ-opioid receptor antagonist core, clinical compound, and general pharmacophore.

At the time of this work, it was known that oral dosing of both alvimopan 1 and methyl naltrexone (MNTX) 2 resulted in minimal CNS exposure, likely a result of the zwitterionic character14,15 and charged quaternary amine,16 respectively, which presumably significantly reduce passive cellular permeability. However, this feature was additionally reflected in the poor oral bioavailability of both of these compounds, alvimopan 1 %F: 617 and methyl naltrexone 2 %F: < 1.18 Alternate clinical precedent for an orally absorbed, non-CNS penetrant profile is provided by the second generation of nonsedating antihistamines such as fexofenadine (Allegra) as well as the antidiarrheal μ-opioid receptor agonist loperamide (Imodium). In both cases, the lack of blood–brain barrier penetration of the compound was determined empirically and was latterly attributed to their being a substrate for the P-glycoprotein efflux transporter (P-gp).19,20 Our approach toward a predictable and balanced orally absorbed, non-CNS penetrant profile considered the overall physicochemical properties of the compounds. Many analyses of the optimal physicochemical properties required for oral absorption have been reported since Lipinski’s seminal report,21 but fewer have focused on the subset of CNS active compounds, although this has recently been addressed.22 In 2006, Hitchcock et al. analyzed the top 25 CNS drugs and proposed a set of guidelines for CNS penetration, focused on values for total polar surface area (tPSA) (<70A2), number of hydrogen bond donors (HBD) (0–2), lipophilicity clogDpH 7.4 (2–4), and molecular weight (MW) (<450 Da).23 It appears logical that inverting these guidelines within a set of limits related to oral absorption might provide compounds with the desired orally absorbed, non-CNS penetrant profile. As such, we proposed a target range of properties, as indicated in Table 1.

Table 1. In Vitro Screening Data.

Compound na R1a R2a μb pKi δb pKi κb pKi μc IA RLM t1/2 (min) DLM t1/2 (min) HLM t1/2 (min) Caco-2 Kp (1 × 10–6cm/s) tPSA (Å2) HBD (#) clogD (pH 7.4) MW (Da)
target range of properties                       70–120 2–4 0–2 450–550
naltrexone       9.8 8.2 9.3 12 >90c     84 70 2 0.8 341
MNTX 2       8.1 6.3 7.7 19 >90 >90 >90 2 67 2 –0.1 356
alvimopan 1       9.5 8.3 8.3 –7 >90 >90 >90 3 90 3 0.1 424
ADL 08-0011 5       9.5 7.8 7.6 –8 >90 >90 >90 5 61 2 0.7 367
6       10 8.9 9.1 –21 6c       23 1 2.7 323
7       6.2 5.5   4 >90c     <1 61 2 –0.7 277
18 1 c-hexylmethyl- H 9.6 8.2 9.8 12 42d       58 2 1.0 369
19a Ac 10 8.8 9.9 –1 74 16 63   67 1 1.2 412
19b tBu(CO)- 9.9 8.7 9.8 3 12 2 9   67 1 2.1 454
19c CF3C(O)- 9.1 8 9 9 3d       67 1 2.0 466
19d MeO(CO)- 9.9 8.6 9.7 7 2 2 7   76 1 1.6 428
19e iPrNHC(O)- 9.7 8.9 9.8 21 20 14   36 79 2 1.8 455
19f MeO2S- 9.6 8.1 9.1 1 13 2 19   84 1 1.2 448
19g Me2NSO2- 9.7 8.7 9.7 0 8 5 9   87 1 1.2 477
19h HOCH2C(O)- 10 8.4 9.9 –3 77 18 79 40 87 2 0.7 428
19i 2S,3-dihydroxypropionate- 9.8 8.8 9.9 –3 87 27 90 5 107 3 0.5 458
19j MeO2SCH2C(O)- 10 8.9 10 0 50 28 90 18 101 1 0.9 490
19k 1H-tetrazole-5-CH2(CO)- 9.4 8.5 8.8 0 90 90 90 <1 121 2 –0.1 480
19l 2-furylC(O)- 10 8.9 10 1 10 2 17 - 80 1 1.8 464
19h 1 c-hexylmethyl- HOCH2C(O)- 10 8.4 9.9 –3 77 18 79 40 87 2 0.7 428
24a Bn 10 8.3 9.7 –3 85 90 90 21 87 2 0.5 421
24b phenethyl- 9.2 7.7 9.1 –2 34 61 90 16 87 2 0.6 436
24c phenpropyl- 9.1 7.5 9.3 –13 32d     26 87 2 0.8 450
24d c-pentylmethyl- 9.6 7.8 9.5 4 90 14 90 12 87 2 0.5 414
24e c-butylmethyl- 8.8 7.5 9.2 13 90 90 90 14 87 2 0.3 399
24f n-hexyl- 9.1 7.2 8.5 –9 71 11 90 45 87 2 0.5 416
24g 4,4-diF-c-hexylmethyl- 9.9 7.9 9.6 –5 90 90 90 14 87 2 1.1 464
29 4-THPmethyl- 7.9 6.7 8.2 0 90 90 90 <1 96 2 0 430
32 2-ethyl-n-butyl- 9.8 7.9 9.9 9 90 75 90 32 87 2 0.6 416
32 1 2-ethyl-n-butyl- HOCH2C(O)- 9.8 7.9 9.9 9 90 75 90 32 87 2 0.6 416
33 2 10 8.5 9.7 3 89 90 90 20 87 2 0.7 430
25 3 9.2 7.7   –1 90 90 21   87 2 0.8 444
26 4 9.2 7.7   1 28 19 12   87 2 1.1 458
27 5 9 7.6   –2 26 9 9   87 2 1.4 472
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a

n, R1, and R2 for compounds 18 and 19a–l can be found in Scheme 2, for compounds 24a–g, 25–27, Scheme 3; for compound 29, Scheme 4; for compounds 32–33, Scheme 5.

b

μ-, δ-, and κ-opioid receptor binding pKi values were determined using a [3H]-DPN radioligand binding assay with membranes prepared from CHO-K1 cells stably transfected with human recombinant μ- and δ-opioid receptor and guinea pig κ-opioid receptor.

c

Functional activities were determined using GTP binding assays with [35S]-GTPγS in membranes prepared from CHO-K1 cells stably transfected with the human recombinant μ-opioid receptor: the maximum compound-evoked response (minus basal) is expressed as a percentage of the maximum response evoked by DAMGO.

d

Tested in assay at a concentration of 15 μM; all other compounds were tested at a concentration of 3 μM.

Variation of the N-substituent (R) of the novel μ-opioid receptor antagonist series A, Figure 2, was considered the most appropriate position to explore and optimize the overall physicochemical properties in tandem with potency, consistent with the application of Theravance’s multivalent approach to drug discovery.13 The core A is considered the primary binding group and the N-substituent a handle by which secondary binding interactions and physicochemical properties can be modulated. Some early work on the nature of the N-substituent of the reported active metabolite (ADL 08-0011, 5) of alvimopan 1 was instructive, Figure 3.24

Figure 3.

Figure 3

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Alvimopan metabolite and its constituent parts.

Comparing ADL 08-0011 5 to its constituent parts as the (CH2)3Ph compound 6 and propionic acid compound 7 highlighted that the lipophilic compound 6 had high binding affinity (pKi = 10.5), a low intrinsic activity (IA) consistent with an antagonist effect (IA = −21% of the maximum response achieved by DAMGO in a GTP binding assay) at the human recombinant μ-opioid receptor, and poor metabolic stability in rat liver microsomes (RLM) with a short half-life (t1/2 = 6 min), Table 1.25 In contrast, the hydrophilic, zwitterionic acid compound 7 exhibited weak binding affinity (pKi = 6.2), an antagonist profile (IA = 4%), and good RLM stability (t1/2 > 90 min). Combination of these features is achieved with ADL 08-0011 5, which is both a high affinity μ-opioid receptor antagonist (pKi = 9.6, IA = −8%) and a high RLM stability compound (t1/2 > 90 min). A similar result was obtained with the identical N-substituents applied to our previously reported endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenol series of μ-opioid receptor antagonists13 and led us to conclude that a two-component N-substituent consisting of a lipophilic and hydrophilic moiety could be tolerated, which attractively would allow us to effectively modulate the overall physicochemical properties. Many chemical approaches were attempted to leverage this insight, but most success was obtained with the general design of an N-alkyl linker to a branching N′-amine substituted with a lipophilic component (R1) and a polar, hydrophilic group (R2), Figure 2. A modular synthesis was designed to explore this concept, in which a set of aldehydes 12a–f, 13–15, and 16a and b were prepared, allowing for variation of the alkyl linker length (n) and the lipophilic group (R1), Scheme 1.

Scheme 1. Preparation of Aldehyde Intermediates 12a−f, 13−15, 16a−b.

Scheme 1

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Reagents and conditions: (i) For n = 1, R1 = cyclohexylmethyl-, phenethyl-, phenpropyl-, cyclobutylmethyl-, and n-hexyl-, and for n = 3, 4, and 5, R1 = 2-ethyl-nbutyl-, then R1Br, EtOH, 70-75°C, 38–94%; for n = 1, R1 = cyclopentylmethyl-, 4,4-diF-c-hexylmethyl-, then R1OMs, EtOH, 70–80°C, 64–77%; (ii) Boc2O, DCM, 0°C to RT, 43–100%; (iii) PySO3, DIPEA, DMSO, DCM, 0°C, 49–100%; (iv) for R1 = Bn, 4,4-diF-c-hexylmethyl-: AcOCH2COCl, DIPEA, DCM, 59–61%.

Reductive amination of aldehyde 12a with the previously described13endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenyl carboxamide series core 17 and subsequent Boc-deprotection afforded the representative intermediate 18 with an ethyl alkyl linker and N-cyclohexylmethyl as the lipophilic group (R1), from which a set of compounds 19a–l exploring the R2 group were prepared, Scheme 2.

Scheme 2. Preparation of Compounds 18 and 19a–l.

Scheme 2

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Reagents and conditions: (i) (a) 12a, NaBH(OAc)3, DCM, (b) TFA, DCM, 57% over 2 steps; (ii) (a) for R2 = Ac-, tBuC(O)-, CF3C(O)-, MeOC(O)-, MeO2S-, Me2NO2S-, AcOCH2C(O)-, 2-furylC(O)-, then R2Cl, DIPEA, DCM; iPrNCO, DIPEA, DMF; for R2 = 2,2-dimethyl-1,3-dioxolane-4S–C(O)-, MeO2SCH2C(O)-, 1H-tetrazole-5-CH2(CO)-, then R2OH, HATU, DIPEA, DMF (b) for R2 = AcOCH2CO-: LiOH·H2O, MeOH; for R2 = 2,2-dimethyl-1,3-dioxolane-4S–C(O)-: AcOH, H2O, 70°C.

All of the compounds 18 and 19a–l were high affinity μ-opioid receptor antagonists (pKi = 9.1–10, IA = −3 to 21%), Table 1. Although the acetamide 19a had reasonable human liver microsomes (HLM) stability (t1/2 = 63 min), the tert-butyl analogue 19b and trifluoromethyl compound 19c exhibited short half-lives in RLM, dog liver microsomes (DLM), and HLM (t1/2 ≤ 12 min). These amides 19a–c did not have target properties in our desired range with a tPSA of only 67 Å2 and 1 HBD. Poor LM stability was also a feature of the carbamate 19d, urea 19e, sulfonamide 19f, and sulfamate 19g compounds (t1/2 ≤ 20 min across all species). In this instance, the tPSA of the compounds was increased (76–87 Å2), and specifically for the urea 19e, 2 HBD were introduced. Revisiting the amide series, introduction of a more polar side chain with compounds 19h–j resulted in an increase in HLM stability (t1/2 ≥ 79 min) and tPSA (87–107 Å2) while maintaining an appropriate clogDpH 7.4 (0.5–0.9) as well as displaying a moderate to good Caco-2 permeability (Kp 5–40 × 10–6 cm/s). Low DLM stability (t1/2 ≤ 28 min) appeared to be a species outlier for these otherwise attractive compounds. The heterocyclic tetrazole amide 19k showed high metabolic stability across all species with LM t1/2 > 90 min but was of very low permeability (Kp < 1 × 10–6 cm/s). The furyl amide 19l was a low LM stability compound (t1/2 ≤ 17 min across all species).

Due to the attractive features of the R2 = HOCH2C(O)- compound 19h, analogues with varying lipophilic R1 groups were next examined. Following an analogous synthetic sequence to Scheme 2, reductive amination of aldehydes 12b–f with core 17 and Boc-deprotection afforded compounds which were reacted with acetoxyacetyl chloride to provide intermediates 20b–f. The R1 = benzyl and 4,4-diF-c-hexylmethyl analogues 20a and 20g were prepared directly from aldehydes 16a and 16b, respectively. Hydrolysis of the acetyl group of compounds 20a–g yielded ethyl (n = 1) linked compounds 24a–g, Scheme 3.

Scheme 3. Preparation of Compounds 24a−g, 25−27.

Scheme 3

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Reagents and conditions: (i) (a) 12b–f, 13, 14, 15, 16a and b, NaBH(OAc)3, DCM, (b) for intermediates from 12b–f, 13, 14, 15 only: TFA, DCM, 37–78% over 2 steps, then AcOCH2COCl, DIPEA, DCM; (ii) LiOH·H2O, EtOH, H2O; from 20a, g, 49% over 2 steps; from 20b–f, 21, 22, 23, 47–100% over 2 steps.

An alternate synthesis in which the key linker, R1 and R2 groups were introduced in an alternate sequential order was utilized to prepare the ethyl linked, R1 = methyl-tetrahydropyran, R2 = HOCH2C(O)-, compound 29, Scheme 4.

Scheme 4. 'Preparation of Compound 29.

Scheme 4

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Reagents and conditions: (i) (a) (MeO)2CHCHO, NaBH(OAc)3, DCM, (b) 6 N HCl (aq.), 33% over 2 steps; (ii) (a) 4-aminomethyl-tetrahydropyran, NaBH(OAc)3, DCM, 19%, (b) AcOCH2COCl, DIPEA, DCM; (c) LiOH·H2O, EtOH, H2O, 15% over 2 steps.

Additionally, the R1 = 2-ethyl-nbutyl compound 32 was prepared according to Scheme 5.

Scheme 5. Preparation of Compounds 32−33.

Scheme 5

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Reagents and conditions: (i) CbzNHCH2CHO or CbzNHCH2CH2CHO, NaBH(OAc)3, DCM; (ii) (a) H2, Pd(OH)2, EtOH, (b) 2-ethylbutanal, NaBH(OAc)3, DCM, 42–57% over 3 steps, (c) AcOCH2COCl, DIPEA, DCM; (d) 6N NaOH (aq.), MeOH, 60–100% over 2 steps.

While all of the compounds 24a–g, 29, and 32 maintained μ-opioid receptor antagonist profiles, there was a more pronounced effect on the degree of μ-opioid receptor binding affinity. Relative to the cyclohexylmethyl compound 19h (pKi = 10, DLM t1/2 = 18 min), the benzyl 24a, 2-ethyl-nbutyl 32 and 4,4-diF-cyclohexylmethyl 24g analogues exhibited comparable μ-opioid receptor binding affinity (pKi = 10, 9.8, and 9.9, respectively) while showing an increase in DLM stability (t1/2 = 90, 75, and 90 min, respectively) and maintenance of the favorable target range of properties and Caco-2 permeability values. Extension of the lipophilic phenyl ring of benzyl compound 24a further from the linking N′-amine with the phenethyl 24b and phenpropyl 24c analogues resulted in the observation of a significant reduction in μ-opioid receptor binding affinity (pKi = 9.2 and 9.1, respectively), and this was replicated with the straight chain n-hexyl compound 24f (pKi = 9.1). Contraction of the cyclohexyl ring of compound 19h (pKi = 10, tPSA = 87 Å2) to the cyclopentyl 24d and cyclobutyl 24e compounds resulted in a progressive reduction of μ-opioid receptor binding affinity (pKi = 9.6 and 8.8, respectively), while increasing polarity with the tetrahydropyran analogue 29 (tPSA = 96 Å2) reduced both binding affinity (pKi = 7.9) and Caco-2 permeability (Kp < 1 × 10–6 cm/s). A further element of the SAR that was probed was the length of the alkyl linker, which was done using the constant of R1 = 2-ethyl-nbutyl and R2 = HOCH2C(O)-. The synthesis detailed in Scheme 3 was employed for n = 3–5 using aldehydes 13–15 to afford n-butyl 25, n-pentyl 26, and n-hexyl 27 linked compounds, respectively. An alternate synthetic sequence was used to prepare the ethyl 32 and n-propyl compounds 33, Scheme 5. Similar μ-opioid receptor binding affinity was observed for the ethyl 32 and n-propyl 33 linked compounds (pKi = 9.8 and 10, respectively; clogDpH 7.4 = 0.6 and 0.7 respectively) and this was reduced as lipophilicity increased with the n-butyl 25, n-pentyl 26 and n-hexyl 27 compounds (pKi = 9.2, 9.2 and 9, respectively; clogDpH 7.4 = 0.8, 1.1, 1.4 respectively).

It should be noted that for all the analogues 18, 19a–l, 24a–g, 29, 32, 25, 26, 27, and 33, the binding profile at both the human δ- and guinea pig κ-opioid receptors was also determined, and the selectivity for the three opioid receptor subtypes was in general consistent. The binding affinity for the δ-opioid receptor was reduced by 0.9–2 log, and the binding affinity for the κ-opioid receptor spanned a range of between 0.6 log lower to 0.4 log higher, relative to the affinity at the μ-opioid receptor.

A representative set of compounds 19h, 19i, 19j, and 24g with favorable in vitro properties were advanced to subsequent in vivo pharmacokinetic and pharmacodynamic studies, Table 2. All of the compounds exhibited moderate to good oral bioavailability in rats (%F: 27–61) with significantly improved dose normalized Cmax and AUC relative to both alvimopan 1 and MNTX 2.

Table 2. In Vivo Pharmacokinetic and Pharmacodynamic Screening Data.

Compound Cmaxa (μg/mL) AUC(0–t) (μg h/mL) t1/2 (h) Fb (%) mouse ITc ID50 (mg/kg) mouse GEd ID50 (mg/kg) mouse HPe ID50 (mg/kg) peripheral selectivity index (IT)f peripheral selectivity index (GE)g
naltrexone         0.6 0.5 0.82 1.4 1.6
MNTX 2 0.57 0.49 2.8 2.6 67 25      
alvimopan 1 0.01 0.002   0.1 2.5 0.6 >20 >8 >33
ADL 08-0011 5 0.71 1.20 3.7 53 0.064 0.027 1.1 17 41
19h 0.29 0.43 1.3 61 0.10 0.046 4.2 42 91
19i 0.21 0.47 1.3 27 0.35 0.087 14 40 161
19j 0.88 1.30 1.0 60 0.49 0.33 24 49 73
24g 0.39 1.20 2.0 30 0.23 0.03 4.4 19 147
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a

Pharmacokinetic properties were evaluated in male Sprague–Dawley rats dosed with test compounds via oral gavage (PO) at a dose of 5 mg/kg, n = 3, except for MNTX, which was dosed at 100 mg/kg, and alvimopan, which was dosed at 10 mg/kg.

b

F = oral bioavailability.

c

IT = inhibition of morphine-induced delays in intestinal transit.

d

GE = inhibition of morphine-induced delays in gastric emptying.

e

HP = inhibition of morphine-induced hot plate antinociception.

f

Peripheral selectivity index = ratio of hot plate and intestinal transit ID50 values.

g

Peripheral selectivity index = ratio of gastric emptying and intestinal transit ID50 values.

Pharmacodynamically, following oral administration, 19h, 19i, 19j, and 24g produced a dose-dependent reversal of the morphine-induced inhibition of intestinal transit (ID50 values of 0.1, 0.35, 0.49, and 0.23 mg/kg, respectively) and gastric emptying (ID50 values of 0.046, 0.087, 0.33, and 0.03 mg/kg, respectively) in mice. Both of these assays were viewed as a measure of the peripheral GI activity (non-CNS) of the test compounds. The opioid receptor antagonists naltrexone, alvimopan, and ADL 08-0011 had a similar range of potencies in the intestinal transit (ID50 values of 0.6, 2.5, and 0.064 mg/kg, respectively) and gastric emptying assays (ID50 values of 0.5, 0.6, and 0.027 mg/kg, respectively), while MNTX was significantly less potent in the intestinal transit (ID50 values 67 mg/kg) and gastric emptying assay (ID50 values of 25 mg/kg), Table 2.

With respect to the CNS activity of the test compounds following oral administration, 19h, 19i, 19j, and 24g produced a dose-dependent reversal of morphine-induced antinociception in mice (ID50 values of 4.2, 14, 24, and 4.4 mg/kg, respectively; Table 2). By comparison, the opioid receptor antagonists naltrexone, alvimopan, and ADL 08-0011 had ID50 values of 0.82, >20, and 1.1 mg/kg, respectively).

The peripheral selectivity of compounds was defined as the ratio of the CNS-mediated antinociception ID50 value and the peripheral intestinal transit or gastric emptying ID50 values. Compounds 19h, 19i, 19j, and 24g had peripheral selectivities of 42, 40, 49, and 19, respectively, for potencies in the antinociception and intestinal transit assays, and 91, 161, 73, and 147, respectively, upon comparison of potencies in the antinociception and gastric emptying assays, Table 2. The corresponding peripheral selectivities of naltrexone, alvimopan, and ADL 08-0011 were 1.4, >8, and 17, respectively (intestinal transit), and 1.6, >33, and 41, respectively (gastric emptying). The data indicated that compounds 19h, 19i, 19j, and 24g all had a high peripheral selectivity in mice following oral dosing, clearly exceeding that of the centrally penetrant opioid receptor antagonist naltrexone as well as being improved relative to the values for ADL 08-0011, the active metabolite of alvimopan, which at potentially efficacious concentrations in humans was shown to have minimal CNS effects.26 Compounds 19i and 24g appeared to have the most favorable peripheral selectivity indices and each satisfied all the parameters of our target range of properties; in particular, 19i had the highest tPSA (107 Å2) and number of HBD (3).

Compound 19i was selected for further characterization.27,28 Consistent with its high opioid receptor binding affinity, 19i had potent opioid receptor antagonist activity of agonist-induced GTPγS binding in recombinant expression systems (pKb values = 9.6, 8.8, and 9.5, at μ, δ, and κ, respectively) and inhibited agonist-induced inhibition of electric field stimulated contractions of rodent tissue preparations [μ and κ pA2 values = 10.1 and 8.8, respectively (guinea pig ileum), and δ pKb value = 8.4 (hamster vas deferens)]. The selectivity of 19i for opioid receptors was examined; at 1 μM (i.e., a concentration 6300-fold higher than its μ Ki value), 19i had no significant effect on radioligand binding or functional activity at all 80 receptors, ion channels, and enzymes tested.

Oral pharmacokinetics of 19i in fasted female and male beagle dogs (%F: 29 and 45, respectively) were consistent with rat. Additionally, 19i exhibited low plasma protein binding (23–30%) across species, including human. Glucuronidation was the major metabolic pathway in rodents while CYP3A4-mediated hydroxylation was predominant in dog and human based on in vitro studies. Efflux ratios for 19i of 10 and 24 in a Caco-2 and MDR1-MDCK cell line, respectively, indicated the compound is a P-gp substrate which was supported by studies in MDR1a/1b knockout mice which upon oral dosing of 19i had increased CNS drug concentrations relative to wild-type mice. In rats, csf, brain, and plasma concentrations after oral administration (20 mg/kg) of 19i revealed CNS penetration to be <2%.

Following oral dosing to rats, 19i reversed loperamide-induced delays in gastric emptying (ID50 = 0.24 mg/kg) and castor oil-induced diarrhea (ID50 = 0.01 mg/kg). In dogs, 19i inhibited nonproductive GI circular smooth muscle contractility (effective at 3 mg/kg). Orally dosed 19i failed to evoke a CNS-mediated withdrawal response in morphine-dependent mice (30 mg/kg), inhibit morphine-induced antinociception in rat and dog hot plate tests (>60 mg/kg and 3 mg/kg, respectively), or reverse morphine-induced sedation in dogs (at 3 mg/kg). It was concluded that 19i has potent in vivo gastrointestinal activity in mice, rats, and dogs, consistent with opioid receptor antagonism, and possesses a high degree of peripheral selectivity in these species.

Subsequently, compound 19i was nominated as a development candidate, axelopran (TD-1211), and advanced to human clinical trials. Accordingly, an alternate, optimized synthesis of axelopran (TD-1211) 19i was devised to support IND-enabling studies and early clinical manufacture. The previously described13endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenyl carboxamide series core 17 was prepared efficiently from N-benzyl-nortropinone 34. Suzuki reaction of the vinyl triflate 35 yielded styrene 36. Subsequent styrene reduction and concomitant benzyl deprotection under acidic conditions afforded the key intermediate core as a mixture of endo:exo isomers in a ratio of 93:7, which upon crystallization afforded the single endo isomer 17 in >99% purity as its HCl salt, Scheme 6.

Scheme 6. Preparation of Compound 17.

Scheme 6

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Reagents and conditions: (i) PhNTf2, NaHMDS, THF and (ii) 3-carbamoylphenyl boronic acid, Pd(OAc)2, dppf, KF, THF, reflux, 78% over 2 steps; (iii) H2, 10% Pd/C, EtOH, 6M HCl, H2O, 50°C, endo:exo 93:7, then crystallization from EtOH, 75% and >99% single endo isomer.

The aldehyde 40, generated in situ from the bench-stable sodium bisulfite aldehyde adduct 39 (Scheme 7) was coupled with the intermediate core 17 via reductive amination, Scheme 8. Deprotection of the Cbz group to the secondary amine 18 and subsequent amide coupling with the lithium salt of the protected diol chiral acid afforded the acetal protected compound 41. Removal of the acetal group and crystallization afforded the monosulfate salt of axelopran (TD-1211) 19i.

Scheme 7. 'Preparation of Aldehyde Intermediate 40.

Scheme 7

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Reagents and conditions: (i) (a) H2NCH2CH(OEt)2, H2, Raney-Ni, Me-THF, 0°C, (b) CbzCl, DIPEA, Me-THF, and (ii) (a) HCl, AcCN, 30°C, (b) NaHSO3, EtOAc, 75% over 4 steps; (iii) 1M NaOH (aq.), Me-THF.

Scheme 8. Preparation of Axelopran (TD-1211) 19i.

Scheme 8

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Reagents and conditions: (i) (a) Me-THF solution of 40, NaBH(OAc)3, DMF, (b) H2, 10% Pd/C, MeOH, 6M HCl, 65% over 2 steps; (ii) lithium-(4S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate, PyBOP, DMF, and (iii) 2M H2SO4 (aq.), then crystallization from 10% H2O in MeOH, 73% over 2 steps.

A crystal structure of the monosulfate salt of axelopran (TD-1211) 19i was obtained to unambiguously define its chemical structure, highlighting the phenyl-carboxamide endocyclic and hydrophilic group 2S stereochemistry as well as the phenyl axial conformation, Figure 4.29

Figure 4.

Figure 4

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X-ray crystal structure of the monosulfate salt of axelopran (TD-1211) 19i.

Axelopran (TD-1211) 19i has successfully completed Phase 1a and 1b and three Phase 2 studies which were carefully designed to identify the best dose and regimen for Phase 3 studies.30−32 The key efficacy highlight from the most recent 200 OIC patient Phase 2b study revealed a statistically significant (p = 0.0001) increase in complete spontaneous bowel movements (CSBMs) relative to placebo during week 5 of the 5 week treatment with patients transitioning from a baseline of 0.2 CSBMs to almost 3 at the top dose of 15 mg qd. Axelopran (TD-1211) 19i has been well-tolerated across all studies with observation of similar treatment emergent adverse events relative to placebo. Importantly, in the relevant studies there has been no impact on the use of opioid analgesics at efficacious doses, confirming the peripheral selectivity of the drug. Axelopran (TD-1211) 19i is positioned as Phase 3 ready for the treatment of OIC.

In summary, Theravance’s multivalent approach to drug discovery in combination with a physicochemical property driven design strategy was applied to optimize the N-substituted-endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenyl carboxamide series A of μ-opioid receptor antagonists to afford an orally absorbed, non-CNS penetrant profile that resulted in the clinical compound axelopran (TD-1211) 19i, targeted for the treatment of OIC. Extensive preclinical characterization of the pharmacological and DMPK properties of axelopran (TD-1211) 19i are highlighted along with an optimized synthesis applied to its clinical manufacturing. Axelopran (TD-1211) 19i has successfully completed Phase 2 studies in patients with OIC and is Phase 3 ready.

Acknowledgments

The authors would like to thank Dr. David L. Bourdet, Dr. R. Murray McKinnell, and Dr. Kris Josephson from Theravance Biopharma US, Inc. for their assistance in reviewing this article.

Glossary

Abbreviations

Cmax

maximal concentration

AUC

area under the curve

ID50

50% inhibitory dose

GTPγS

guanosine 5′-O-[gamma-thio]triphosphate

MDCK

Madin–Darby canine kidney.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00406.

  • Select assay protocols and representative synthetic procedures (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml9b00406_si_001.pdf (302.2KB, pdf)

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Supplementary Materials

ml9b00406_si_001.pdf (302.2KB, pdf)

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