WO2016029218A1 - Substituted 1-arylethyl-4-acylaminopiperidine derivatives as opioid/alpha-adrenoreceptor modulators and method of their preparation - Google Patents

Abstract

The invention provides compounds that bind with high affinities to the μ-, δ- and κ- opioid receptors and α₂ - adrenoreceptor. In addition to providing these compounds with novel pharmacological binding properties, the invention also describes detailed novel methods for the preparation of representative compounds and a scheme for the synthesis of related compounds that bind to the opioid receptors and/or α₂ - adrenoreceptor.

Description

Substituted l-arylethyl-4-acylaminopiperidine derivatives as opioid/alpha- adrenoreceptor modulators and method of their preparation. The invention relates to novel pharmacological compounds, and more specifically to the creation of a new class of small molecules which simultaneously exhibit high binding affinities to the μ-, δ-, and κ-opioid receptors and the oc₂- adrenoreceptor. The binding activity is believed to be antagonistic at least with respect to the μ-opioid receptors. In addition to providing these compounds with novel pharmacological binding properties, the invention also describes detailed novel methods for the preparation of representative compounds and a scheme for the synthesis of related compounds that bind to the opioid receptors and/or a₂-adrenoreceptor.

Opioid antagonists are drugs which bind to the opioid receptors with higher affinity than opioid agonists but do not activate the opioid receptors. Commonly known opioid antagonists include drugs such as, for example, naltrexone, naloxone, nelmefene, nalorphine, and nalbuphine. Opioid antagonists effectively block the receptor from the action of both naturally occurring agonists (e.g., morphine, codeine, thebaine) and synthetic agonists (e.g., fentanyl, pethidine, levorphanol, methadone, tramadol, dextropropoxyphene) and uses include counteracting life-threatening depression of the central nervous and respiratory systems and thus are used for emergency overdose and dependence treatment (e.g., naloxone). There are many excellent reviews dedicated to different aspects of opioid antagonists [28-46].

Opioid receptor antagonists are known to modulate numerous central and peripheral effects including those associated with opioid abuse, the development of opioid tolerance and dependence, opioid-induced constipation, alcohol and cocaine abuse, depression, and immune responses [1]. The diverse therapeutic applications of μ- opioid antagonists include opioid-overdose-induced respiratory depression, opioid and cocaine abuse, alcohol dependence, smoking cessation, obesity, psychosis[l ~19] and for the treatment of dyskinesia associated with Parkinson's disease [20-27].

The few opioid antagonists currently on the market are represented by very few drugs (e.g., naloxone, naltrexone, and nalorphine (a partial agonist)) that have been shown to have therapeutic utility in a variety of indications. During last two decades only Alvimopan [13, 14]— a peripherally acting μ-opioid antagonist for the treatment of postoperative ileus— has received approval as new drug. In addition, some azabicyclohexane derivatives and series of bi(hetero)aryl ethers as biological tools have been proposed as new chemical entities in this class of compounds [15].

Every chemical class of compounds with opioid-agonist activity has a structurally similar opioid-antagonist pair. Agonist-antagonist transformation in any of these cases takes place as a result of a small change in the structure of the agonist. The only exceptions, where the corresponding change for agonist-antagonist transformations has not been found, are the compounds of the fentanyl series.

Since the discovery of the "army" of opioid agonists of the fentanyl series (sufentanyl, alfentanyl, carfentanyl, remifentanyl, etc.) beginning in the 1960s, a structurally corresponding antagonist has not been found for any of these compounds. Thus, for decades there has been an evident gap in the art with respect to a possible specific structural change that could make possible the transformation of powerful opioid agonist properties of compounds of fentanyl series into powerful antagonists.

Similar to the general action of the opioid antagonists, antagonists of the adrenoreceptors (adrenergic receptors) bind to the adrenoreceptors and act to inhibit the action of those receptors. Alpha antagonists, or alpha-blockers, may selectively act at the a i -adrenoreceptors or at the a₂-adrenoreceptors, or they may non-selectively act at both receptors. Commonly known oc-blockers include, for example, phenoxybenzamine and phentolamine (non-selective); alfuzosin and prazosin (ai -blockers); and atipamezole, idazoxan, mirtazapine and yohimbine (a₂-blockers). Generally, -blockers have shown to be effective in the treatment of various medical conditions, including Raynaud's disease, hypertension, scleroderma, anxiety and panic disorders, and in the treatment of dyskinesia associated with Parkinson's disease.

The present invention is based on the discovery of certain compounds exhibiting high binding affinity for the μ-, δ-, and κ- opioid receptors and the ₂-adrenoreceptor. The compounds are believed to exhibit antagonistic activity at least with respect to μ-opioid receptors. The compounds are structurally related to the fentanyl series of opioid receptor agonists. Processes for preparing these compounds are also included in this disclosure.

In one embodiment of the disclosure, a compound having the formula

etAr), or COOR₆, or COON(R₆)₂ (III)

is disclosed, wherein R, is H, substituted or unsubstituted Q-C10 alkyl, alkenyl, or alkynyl, or substituted or unsubstituted aryl or hetaryl; R₂ is H, -CH2O-C 1. alkyl; COO- C1 -4 alkyl; -CONR4; R₃ is H, substituted or unsubstituted C1 -C10 alkyl, alkylene, alkynyl, or substituted or unsubstituted aryl or hetaryl; A is substituted or unsubstituted Q - C10 alkyl, alkylene, alkynyl; Ar or HetAr is substituted or unsubstituted monocyclic or polycyclic aromatic or heteroaromatic moiety; COOR₅, or CON(R₆)2, where R₅ and R₆ are H, substituted or unsubstituted C1 -C10 alkyl, alkylene, alkynyl, or substituted or unsubstituted aryl or hetaryl; the central nitrogen-containing ring is a substituted or unsubstituted 5- to 7-membered heterocyclic ring; and pharmaceutically acceptable salts of said compound.

In a preferred embodiment, the prepared compound may belong to the series of N-(l -arylethyIpiperidin-4-yl)acylamides. In another embodiment, the compound may be N-(l -phenethylpiperidin-4-yl)propionamide (Compound I, below). In yet another embodiment the compound may be the oxalate salt, or other pharmaceutically acceptable salt, of N-(l -phenethylpiperidin-4-yl)propionamide.

In another embodiment, a process for preparing a compound of formula III is provided. The process comprising the following steps: (a) reacting a cyclic ketone having a protecting group in a Grignard or Reformatsky reaction to obtain a first product; (b) reacting the product of step (a) in a Ritter reaction to obtain a second product; and (c) deprotecting the product of step (b) with acylation or alkylation to obtain a compound of formula III.

In yet another embodiment, a process for preparing a compound of formula III is provided, comprising the following steps: (a) reacting a cyclic ketone having a protecting group in a Strccker reaction to obtain a first product; (b) reacting the product of step (a) in a selective carbalkoxy group transformation to obtain a second product; and (c) deprotecting the product of step (b) with acylation or alkylation to obtain a compound of formula III.

In another embodiment, a process for preparing N-( 1 -phenethylpiperidin-4-yl) propionamide is provided, comprising the following steps: (a) reacting phenethylpiperidin-4-one with hydroxylamine hydrochloride in ethanol in the presence of a base, to produce l-phenethylpiperidin-4-one oxime; (b) reducing the oxime obtained in step (a) with iso-amyl alcohol and sodium metal to produce l -phenethylpipcridin-4- amine; and (c) acylating the product of step (b) with propionic acid chloride in chloroform in the presence of triethylamine to produce N-(l -phenethylpiperidin-4- y propionamide. The N-(l -phenethylpiperidin-4-yl)propionamide may optionally be further treated with oxalic acid to obtain N-(l -phenethylpiperidin-4-yl)propionamide oxalate.

These and other embodiments, features and advantages of the present invention will become more fully apparent when read in conjunction with the following detailed description taken in conjunction with the accompanying drawings, wherein

Fig. 1 is a (LC/MS) plot of a preferred compound designated HVC-3.

For the purposes of this disclosure, a "salt" is any acid addition salt, preferably a pharmaceutically acceptable acid addition salt, including but not limited to, halogenic acid salts such as hydrobromic, hydrochloric, hydrofluoric and hydroiodic acid salt; an inorganic acid salt such as, for example, nitric, perchloric, sulfuric and phosphoric acid salt; an organic acid salt such as, for example, sulfonic acid salts (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic, benzenesulfonic or -toluenesulfonic), acetic, malic, fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic, oxalic and maleic acid salts; and an amino acid salt such as aspartic or glutamic acid salt. The acid addition salt may be a mono- or di-acid addition salt, such as a di-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid salt. In all cases, the acid addition salt is used as an achiral reagent which is not selected on the basis of any expected or known preference for interaction with or precipitation of a specific optical isomer of the products of this disclosure.

"Pharmaceutically acceptable salt" is meant to indicate those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a patient without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. (1977) J. Pharm. Sciences, vol. 6. 1 -19, which is hereby incorporated by reference in its entirety, describes pharmaceutically acceptable salts in detail.

"Modulation" is meant to refer to the binding activity of a compound with respect to a particular receptor. The binding activity of the compound, or "modulator," may be that of an agonist, inverse agonist, antagonist, allosteric regulator, positive allosteric modulator, negative allosteric modulator, or any other type of ligand-receptor interaction that is known in the art.

In this invention we disclose a new class of molecules that simultaneously bind with high affinity to opioid μ-, δ-, κ- receptors and also to -adrenoreceptors, thereby exhibiting modulation-type interactions with those receptors. The interaction of the molecules with μ-receptors is believed to have the character of antagonist action, based at least in part on the observed high affinity binding of the molecules with respect to the μ-receptors.

Although not wishing to be bound by theory, it appears that the principal structural change for agonist-antagonist transformation is the removal of a phenyl group from an N-phenylpropionamide fragment of fentanyl. This transformation is depicted below, wherein N-(l -phenethylpiperidin-4-yl)-N-phenylpropionamide (II) is transformed to N-(l -phenethylpiperidin-4-yl)-N-propionamide (I), causing a transformation of μ-agonist properties to μ-antagonist with simultaneous modulation of delta-, kappa- and alpha-receptors:

Fentanil (II) μ-Opioid Antagonist/a-agonist (I)

The invention also relates to processes for preparing compounds of general formula III, which are pharmacologically active compounds:

Ar , or (HetAr), or COOR₅, or COON(R₆)₂ (III)

Representative compounds of the present invention also include the following compounds, wherein the central nitrogen containing ring is a substituted or unsubstituted 5- to 7-membered heterocyclic ring:

These compounds may include the following structural and functional groups:

• R] is H, substituted or unsubstituted Ci-Cio alkyl, alkenyl, alkynyl, or substituted or unsubstituted aryl or hetaryl;

• R₂ is H, -CH2O-C₁.4 alkyl, etc.; COO-C alkyl, etc.; -CONR4 etc.;

• R ( is H, substituted or unsubstituted C| -Cio alkyl, alkylene, alkynyl, or

substituted or unsubstituted aryl or hetaryl;

• A is substituted or un-substituted Ci-C₁₀ alkyl, alkylene, alkynyl;

» Ar or HetAr is substituted or un-substituted monocyclic or polycyclic aromatic or heteroaromatic moiety; and

• COOR5, or CON(R₆)₂, where R₅ and R₆ are H, substituted or unsubstituted C_r C 10 alkyl, alkylene, alkynyl, or substituted or un-substituted aryl or hetaryl.

In certain embodiments of the invention, compounds of formula (III) can be prepared according to the following general schemes (wherein PG is a protecting group):

etAr), or COOR₅, or COON(R₆)₂ (III)

As shown above, these schemes generally include transformations of different starting cyclic ketones (e.g., a variety of piperidin-4-ones, pyrrolidin-3-ones and azepan-4-one) to desired compounds of formula (III) via, for example:

a) Grignard or Reformatsky type reactions;

b) a Strecker type reaction;

c) a Ritter type reaction;

d) selective carbalkoxy group transformations, deprotection, and further appropriate acylation or alkylation; e) selective carbalkoxy group transformations; or

f) deprotection with further appropriate acylation or alkylation.

The process for the preparation of the first example of μ-opioid modulator/ot- modulator N-(l-phenethylpiperidin-4-yl)propionamide and its salt is described in the present invention in detail below (Scheme 1 ):

(IV) (V) (VI) (I) ( VII )

The synthetic procedure starts with the commercially available 1 - phenethylpiperidin-4-one (IV) of formula:

which may be reacted with hydroxylamine hydrochloride in ethanol in the presence of base, to give 1 -phenethylpiperidin-4-one oxime (V) of formula:

Reducing the C=N double bond of obtained oxime (V) by the Bouveault-Blanc protocol using iso-amyl alcohol and sodium metal as a hydrogen source, 1 - phenethylpiperidin-4-amine (VI) may be obtained:

By treating of compound (VI) with propionic acid chloride in chloroform in the presence of triethylamine the desired N-(l -phenethylpiperidin-4-yl)propionamide (I) may be prepared:

The obtained N-(l -phenethylpiperidin-4-yl)propionamide (I) may be transformed to oxalate by treating with a molar equivalent of oxalic acid in ethanol:

The tables (Tables 1 -3) in the attached Appendix 1 incorporated herein by reference include data from binding assays performed with the N-( 1 -phenethylpiperidin- 4-yl)propionamide compound (I). This data demonstrates the high binding affinities of the compounds of the invention for μ-, δ-, and κ- opioid receptors and for cc2-adreno- receptors. Table 1 illustrates the results of binding assays performed with the N-(l - phenethylpiperidin-4-yl)propionamide (compounds Rl and R2) and various receptors. As can be seen in the table, at a test concentration of 1 .0E-05 M, N-( 1 - phenethylpiperidin-4-yl)propionamide demonstrated the highest percentage binding inhibition of control specific binding with respect to the a2B adrenoreceptor (74% and 55%); the δ-opioid receptor (44% and 69%); the κ-opioid receptor ( 107% and 104%); the μ-opioid receptor (98% and 99%). Table 2 contains reference compound data for the various receptors used in the binding assays. Finally, Table 3 provides summary results of the binding assays, showing the receptors from Table 1 for which the test compound demonstrated the highest percentage binding inhibition of control specific binding.

Appendix 2 incorporated herein by reference includes x-ray crystallography data for a representative compound of the invention, N-( l -phenethylpiperidin-4- yl)propionamide oxalate.

Appendix 3 incorporated herein by reference includes data showing that a representative compound of the invention, N-(l -phenethylpiperidin-4-yl)propionamide, conforms to "Lipinski's rule of five," which provides a general rule of thumb for evaluating the activity of an orally administered drug.

The compounds of the present invention may be utilized in various

pharmaceutical and medical applications in which the use of a compound exhibiting high binding affinities for μ-, δ- or κ- opioid receptors and/or a2-adreno-receptors is indicated. The compounds may be of particular use in applications in which the use of a μ-, δ- or κ- opioid receptor modulator, including particularly a μ· opioid receptor antagonist, and/or an x2 -ad rcn orece pt or modulator is indicated. Accordingly, in certain embodiments, the present invention also includes salts, and particularly pharmaceutically acceptable salts, of the disclosed compounds, as well as processes for preparing the salt forms of the disclosed compounds.

Examples

The following examples illustrate the preparation of N-(l -phenethylpiperidin-4- yl)propionamide and its oxalate salt form, N-(l -phenethylpiperidin-4-yl)propionamide oxalate, in accordance with the present disclosure.

Example 1.

l -Phenethylpiperidin-4-one oxime (Compound V)

1 -Phenethylpiperidin-4-one (10.15 g (0.05 mol) dissolved in 60 mL of ethanol) was added drop-wise at 0°C to a solution of hydroxylamine in water. The water solution of hydroxylamine was preliminarily prepared by adding at 0°C in portions 13.8 g (0.1 mol) of 2CO3 to the solution of 6.95 g (0.1 mol) hydroxylamine hydrochloride in 50 mL of water.

The mixture was set aside for a night. Ethanol was evaporated under slight vacuum. Water (-100 mL) was added, and the mixture was stirred on ice bath for an hour. The separated solid product was filtered, washed with water and allowed to air-dry. The crude oxime (10.71 g (98.25%), m.p. 132-134°C) was reserved for use in the next reaction without further purification. Analysis with electrospray ionization mass spectrometry (MS (ESI)) resulted in a peak at 219.1 (MH+).

Example 2

l -Phenethylpiperidin-4-amine (Compound VI)

l -Phenethylpiperidin-4-one oxime (6.54 g (0.03 mol)) was dissolved in 100 mL of dry i-AmOH on heating. A ten-fold excess of sodium (6.9 g (0.3 mol)) was slowly (1 hour) added to the stirred solution in small pieces, while the temperature was maintained around 1 10°. The solution was stirred on heating at 1 10° for two hours and left to cool to room temperature. 1 50 mL of ether, followed by 75 mL of water, was then added to the solution. The organic layer was separated and dried on gSCv After evaporation of solvents under slight vacuum, the product was distilled to give 4.3 g (70%) of 1- phenethylpiperidin-4-amine (VI) with a boiling point of 138-142°/! .5mm. MS (ESI): 205.0 (MH+).

Example 3

N-(l-Phenethylpiperidin-4-yl)propionamide (Compound I)

Propionyl chloride (2.775 g (0.03 mol)) in 5.55 mL of CHC1₃ was added drop- wise on stirring to the cooled (0°C) solution of 4.08 g (0.02 mol) l -phenethylpiperidin-4- amine and 3.03 g (0.03 mol) of Et₃N in 30 mL of CTICI3 The mixture was left to come to room temperature and stirred overnight. After working up with 5% solution of NaHCO.i (2.52 g (0.03 mol)) in 47.88 H₂0, the organic layer was separated, washed with water and dried on MgSCv After evaporation of solvents under slight vacuum, the residue was crystallized from hexane to give 4.9 g (94%) of

N-(l-phenethylpiperidin-4-yl)propionamide (1) with m.p.134-135⁰. MS (ESI): 261.2 (MH+).

• The results of proton NMR spectroscopy were as follows:

Ή NMR (600 MHz, CDC1₃): δ 7.27 (t, J = 7.4 Hz, 21 1), 7. 19 (m, 3H), 5.32 (d, J =

7.4 Hz, 1H), 3.82 (qt, J = 7.8, 4.2 Hz, 1H), 2.92 (dt, J = 1 1.8, 3.4 Hz, 2H), 2.79 (m*, 2H), 2.59 (m*, 2H), 2.1 9 (q, J = 7.5 Hz, 2H), 2.18 (m, 2H), 1.95 (dtd, J = 12.4, 4.4, 1.7 Hz, 2H), 1 .46 (qd, J = 1 1 .7, 3.8 Hz, 2H), 1.15 (t, J = 7.5 Hz, 3H). * these two multiplets arise from two methylene groups that have magnetically inequivalent protons AA'BB' with ²J_AA- - ¾Β' ⁼ 12 Hz, ³J_AB = 3J_A'B' ⁼ 1 1 Hz,

³JAB' ^{= 3}JA'B ⁼ 4 Hz, consistent with a preferred anti conformation for the phenyl and piperidine rings

• The results of carbon- 1 NMR spectroscopy were as follows:

¹³C NMR (1 50 MHz, CDC1₃): δ 173.0, 140.2, 128.6, 128.4, 126.0, 60.4, 52.3, 46.3, 33.7, 32.3, 29.8, 9.9.

Example 4

N-(l-Phenethylpiperidin-4-yl)propionamide oxalate (Compound VII)

Oxalic acid (1 g (0.01 1 mol)) in 10 mL of ethanol was added drop-wise to the solution of 2.93 g (0.01 1 mol) of N-(l-phenethylpiperidin-4-yl)propionamide (I) in 29.3 mL of ethanol. The mixture was set aside overnight. The obtained crystals were then separated and dried in a desiccator over P2O5 to give 3.5 g of N-(l -phenethylpiperidin-4- yDpropionamide oxalate (VII) with m.p. 216-218 (MS (ESI): 261 .2 ([M + 1 1]) - see Fig. 1

The compound designated as HCV-3 was then subject to cellular functional assay and results reported below:

Cellular functional assays

ExperimenlAssay Catalog Re1 Client Com Batch Compound Test Conce % of Contri % of Control Agonist Response Reference 1 EC50 Ref (M)

1st 2nd Mean

27/07/201! alpha 2B (It 1813 HCV-3 1 1000233221.0E-05 7.1 9.2 5.1 7.1 dexmedetc 1.3E-08 27/07/201! kappa (K02071 HCV-3 1 1000233221.0E-05 -4.2 -10.3 1.8 -4.2 U 50488 1.6E-09 27/07/201! mu (MOP) 1392 HCV-3 1 1000233221.0E-05 33.5 28.1 38.9 33.5 DAMGO 4.2F-09

Cellular functional assays

Experimeni Assay Catalog ReiClient Com Batch Compound Test Conce % Inhibitioi Agonist Response (% of Control) Reference ' IC50 Ref [h Kb Ref |M)

1st 2nd Mean

27/07/201! alpha 28 (h 1814 HCV-3 1 1000233221.0E-05 -28 116.8 138.7 127.8 yohimbine 3.7E-07 4.8E-08

27/07/201! kappa (KO 2072 HCV-3 1 1000233221.0E-05 6 112.4 76.5 94.4 nor-BNI 4.3E-10 7.2E-11

27/07/201! mu (MOP) 1393 HCV-3 1 1000233221.0E-05 -1 100.1 101.8 101.0 CTOP 2.1E-07 2.3E-08

Compound HCV-3 also was tested for hERG inhibition. Over the concentration range tested (up to 25 micromolar) no dose-response was obtained. Therefore the inhibition IC50 was considered as >25 micromolar. There was a hint of some inhibition at the top concentration of 25 micromolar, with 32.5% inhibition observed (insufficient to generate an IC50 value). As such, this compound is categorised as having weak or no hERG inhibition. The control compounds behaved as expected in the assay.

Compound HCV-3 also was tested for CYP inhibition, and was found to inhibit CYP2D6, and to weakly inhibit CYP2C19. However, with CYP2C19 the inhibition was too weak to generate an IC50 value, and we observed just 36.4% inhibition at the top concentration o 25 micromolar. With CYP2D6, an IC50 of 4.2 micromolar was observed. Thus, this compound was considered to be a moderate CYP2D6 inhibitor, and a weak CYP2C 19 inhibitor. No inhibition was observed at CYP2B6, CYP2C9, CYP3A4 (with either substrate), CYP2C8 or CYP1A2. The significance of this CYP2D6 inhibition will depend on the levels of the compound in vivo.

Compound HCV-3 also was tested in cellular and nuclear receptor functional assays, and the results reported in Appendix 4, incorporated herein by reference. Compound HCV-3 was also subjected to AMES testing, and the results reported in Appendix 5, incorporated herein by reference.

In summary, the compound HCV-3 was negative for genotoxicity against both strains used in this assay (TA98 and TA 100) up to a maximum tested concentration of l mg/mL, in both the absence and presence of S9 metabolic activation. The assay controls behaved as expected.

Compound HCV-3 also was subjected to in vitro metabolic disposition in mouse, rat, monkey and human microsomes. The test compound was incubated with pooled liver microsomes, since drip stability in liver microsomes can be predictive of drug stability in vivo. Aliquots were taken at 0, 5, 1 5, 30 and 45 minutes and quenched immediately. The samples were extracted and analyzed by LC-MS/MS. Compound HCV-3 was observed to have low clearance in human, monkey and mouse microsomes, and moderate clearance in rat.

Compound HCV-3 also was subjected to MDCK permeability assay. The compound was observed to be highly permeable in the MDCK assay. There was a slight difference between the plus and minus inhibitor data in terms of the efflux ratio obtained (1.48 minus inhibitor, versus 0.929 plus inhibitor). A ratio of greater than 2 generally indicates that efflux, i.e., blood brain barrier permeability, is occurring. The control compounds behaved as expected, with prazosin (a P-gp substrate) showing efflux in the absence of Cyclosporin A, which was inhibited in its presence.

Various derivatives of the above compounds with potential opiod and alpha antagonist activity were prepared as follows, and characterized by NMR and mass-spec as reported below.

Protocols for the synthesis of substituted l-arylethyl-4-acylammopiperidine derivatives

Synthesis of l-benzylpiperidin-4-one oxime

28.35 g ( 1 equiv., 0.15 mol) of 1 -benzylpiperidin-4-one was dissolved in 60 mL of EtOH and then cooled to 0 °C using an ice bath. A solution containing 20.85 g (2 equiv., 0.30 mol) of hydroxylamine hydrochloride dissolved in 75 mL of H₂0 was prepared and then added dropwise to the reaction mixture followed by dropwise addition of a solution containing 20.7 g (1 equiv., 0.15 mol) of K2CO3 dissolved in 75 mL of I LO. The reaction mixture was then brought to room temperature and stirred overnight. The EtOH was then removed via rotary evaporation and the reaction mixture was then cooled in an ice bath to allow the product to crystallize out of solution. The product was filtered and washed several times with H₂0 and recrystallized in EtOH. Yield: 27.78 g (70.17%). Synthesis of l-benzylpiperidin-4-amine

A solution containing 6.12 g (1 equiv., 0.03 mol) of l -ben/ylpiperidin-4-one oxime dissolved in 90 mL of iso-amyl alcohol was prepared and heated to approximately 1 10 °C. 6.9 g (10 equiv., 0.3 mol) of Na metal was then added slowly to the reaction mixture. After addition of Na, the reaction mixture was allowed to cool to room temperature and stirred until the reaction mixture turned into a thick slurry. The slurry was dissolved in 50 mL of ethyl acetate and 25 mL of H₂0. The organic layer was separated and washed with H₂0 (2 x 20 mL) followed by drying over anhydrous magnesium sulfate. The solvent was removed via rotary evaporation, resulting in a yellow oil. The crude product was purified via column chromatography utilizing silica gel and a DCM:MeOH solvent system in a ratio of 4: 1 with an additional 1% of Et₃N. Yield: 3.7 g (64%).

Synthesis of Aⁱ-(l -ben

A solution of 3.7 g of l -benzylpiperidin-4-amine (1 equiv., 0.019 mol) dissolved in 45 mL of dry dichloromethane was prepared followed by the addition of 5 mL of Et₃N (2.6 equiv., 0.05 mol). The reaction mixture was then cooled to 0 °C using an ice bath, and then 2.17 mL (1 .3 equiv., 0.025 mol) of propionyl chloride dissolved in 10 mL of dry dichloromethane was added dropwise to the reaction mixture. The reaction mixture was then warmed to room temperature and stirred overnight. Once the reaction was complete, 4 mL of NH4OH and 45 mL of H₂0 were added to the reaction mixture. The organic layer was separated, and the aqueous layer was washed with dichloromethane (3 ^χ 20 mL) followed by NaHC0₃ solution and brine. The organic extracts were dried over anhydrous magnesium sulfate and the solvent was removed via rotary evaporation, resulting in a white solid. The product was washed with hexanes to obtain an analytically pure sample. Yield: 2.8 g (72%)

Synthesis of N-(pipcridin-4-yl)propionamide

0.7 g of N-(l -benzylpiperidin-4-yl)propionamide (1 equivalent, 0.003 moles) were added to a parr hydrogenation flask and dissolved in 30 mL of EtOH. The solution was then degassed with argon for 30 min followed by the addition of 0.07 g of 10% Pd/C (0.2 equiv., 6.58 ^χ 10^"4 mol) and 0.07 g of 20% Pd(OH)₂ (0.17 equiv., 4.98 ^χ 10^"4 mol). The black solution was then degassed with argon for an additional 1 5 min. The reaction mixture was then charged with 50 psi of H₂ gas and shaken for 24 h. The product was filtered through celite and the solvent was removed via rotary evaporation. No further purification was required. Yield: 0.467 g (99%)

Synthesis of methyl 3-(4-propionamidopiperidin-l-yl)propanoate (CRAS)

0.1 g of N-(piperidin-4-yl)propionamide (1 equiv., 5.26 ^χ lO^"4 moles) was dissolved in 2 mL of dry acetonitrile followed by the addition of 0.071 niL of methyl acrylate (1.5 equiv., 7.89 ^χ 10^"4 mol). The reaction mixture was re fluxed overnight. The solvent was removed via rotary evaporation. The crude product was purified by washing with hexanes followed by drying under high vacuum. Yield: 0.90 g (71 %)

Synthesis of N-(l-(2-(thiophen-2-yl)ethyl)piperidin-4-yl)propionamide (CRAS1)

0.1 g of N-(piperidin-4-yl)propionamide (1 equiv., 6.40 ^χ 10^"4 mol), 0.145 g of 2- (thiophen-2-yl)ethyl methanesulfonate (1 .1 equiv., 7.04 10^"4 moles), 0.097 g of ₂C0₃ (1.1 equiv., 7.04 ^χ 10^"4 mol), 0.032 g of l (1.92 ^χ 10^"4 mol), and 0.178 mL of Et₃N (2 equiv., 1.28 ^χ 10^"3 mol) were added to a round bottom flask and dissolved in 5 mL of dry acetonitrile. The reaction mixture was stirred and refluxed overnight. The solvent was then removed via rotary evaporation followed by the addition of H₂0. The mixture was extracted with ethyl acetate (3 x mL), and the organic extracts were combined and dried over anhydrous magnesium sulfate. The solvent was removed via rotary evaporation. The crude product was washed with hexanes to obtain an analytically pure sample. Yield: 0.101 g (60%).

Synthesis of l-phenethylpiperidin-4-one oxime

28.35 g (1 equivalent, 0.14 mol) of 1 -benzylpiperidin-4-one were dissolved in 60 mL of EtOH and then cooled to 0° C using an ice bath. A solution containing 19.46 g (2 equivalents, 0.28 mol) of hydroxylamine hydrochloride dissolved in 75 mL of H₂0 was prepared and then added dropwise to the reaction mixture followed by dropwise addition of a solution containing 19.35 g (1 equivalent, 0.14 mol) of K2CO₃ dissolved in 75 mL of H₂0. The reaction mixture was then brought to room temperature and stirred overnight. The EtOH was then removed via rotary evaporation and the reaction mixture was then cooled in an ice bath to allow the product to crystallize out of solution. The product was filtered and washed several times with H₂0 and recrystallized in EtOH. Yield: 25.60 g (83.77%).

Synthesis of l-phenethylpiperidin-4-amine

A solution containing 6.00 g (1 equiv., 0.027 mol) of i -phenethylpiperidin-4-one oxime dissolved in 90 mL of iso-amyl alcohol was prepared and heated to approximately 1 10 °C. 6.21 g (10 equiv., 0.27 mol) of Na metal was then added slowly to the reaction mixture. After addition of Na, the reaction mixture was allowed to cool to room temperature and stirred until the reaction mixture turned into a thick slurry. The slurry was dissolved in 50 mL of ethyl acetate and 25 mL of I LO. The organic layer was separated and washed with H₂0 (2 ^χ 20 mL) followed by drying over anhydrous magnesium sulfate. The solvent was removed via rotary evaporation, resulting in a yellow oil. The crude product was purified via column chromatography utilizing silica gel and a DCM:MeOH solvent system in a ratio of 4: 1 containing an additional 1% of Et₃N.

Synthesis of N-(l-phenethylpiperidin~4~yl)furan-2-carboxamide (CRA8)

A solution of 0.1 g of 1 -phenethylpiperidin-4-amine (1 equiv., 4.89 ^χ 10^"4 mol) dissolved in 2 ml, of dry dichloromethane was prepared followed by the addition of 0.178 niL (2.6 equiv., 1.27 x 10^"3 moles) of Et₃N. The reaction mixture was then cooled to 0 °C using an ice bath, and then 0.063 mL (1.3 equiv., 6.36 ^χ 10^"4 mol) of 2-furoyl chloride dissolved in 0.25 mL of dry dichloromethane was added dropwise to the reaction mixture. The reaction mixture was then warmed to room temperature and stirred overnight. Once the reaction was complete, 4 mL of NH₄OH and 45 mL of H₂0 were added to the reaction mixture. The organic layer was separated, and the aqueous layer was washed with dichloromethane (3 x 5 mL) followed by NaHC0₃ solution and brine. The organic extracts were dried over anhydrous magnesium sulfate and the solvent was removed via rotary evaporation, resulting in a white solid. The product was washed with hexanes to obtain an analytically pure sample. Yield: 0.0845 g (58.3 %).

Synthesis of N-(l-phenethylpiperidin~4~yl)furan-3-carboxamide (CRA9)

A solution of 0.1 g of 1 -phenethylpiperidin-4-amine (1 equiv., 4.89 10^"4 mol) dissolved in 2 mL of dry dichloromethane was prepared followed by the addition of 0.178 mL (2.6 equiv., 1.27 ^χ 10^"3 mol) of Et₃N. The reaction mixture was then cooled to 0 "C using an ice bath, and then 0.063 mL (1.3 equiv., 6.36 x 10^"4 mol) of 3-furoyl chloride dissolved in 0.25 mL of dry dichloromethane was added dropwise to the reaction mixture. The reaction mixture was then warmed to room temperature and stirred overnight. Once the reaction was complete, 4 niL of NH₄OH and 45 mL of 1 LO were added to the reaction mixture. The organic layer was separated, and the aqueous layer was washed with dichloromethane (3 < 5 mL) followed by NaHCC solution and brine. The organic extracts were dried over anhydrous magnesium sulfate and the solvent was removed via rotary evaporation, resulting in a white solid. The product was washed with hexanes to obtain an analytically pure sample. Yield: 0.104 g (72%).

Synthesis of 8-bromo-N-(l-phenethylpiperidin-4-yl)octanamide (CRAIO)

A solution of 0.101 g of l -phenethylpiperidin-4-amine (1.1 equiv., 4.93 ^χ 10^"4 mol), 0.1 g of 8-bromooctanoic acid (1 .0 equiv., 4.48 x 10^"4 mol), 0.170 g of HATU (1.0 equiv., 4.48 x 10^"4 mol), 0.061 g of HO At (1.0 equiv., 4.48 10^"4 mol), and 0.314 mL of DIEPA (4.0 equiv., 0.0018 mol) in dry DMF was prepared. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then quenched with 0.5 M KHSO₄ solution followed by the addition of dichloromethane. The organic and aqueous layers were separated, and the aqueous layer was extracted with dichloromethane (3 x 5 mL) followed by washing with NaHCO;, solution and Brine. The organic extracts were then dried over anhydrous magnesium sulfate.

Synthesis of N-(l-phenethylpiperidin-4-yl)henzamide (CRA11)

A solution of 0.1 g of l-phenethylpiperidin-4-amine (1 equiv., 4.89 ^χ 10 mol) dissolved in 2 ml., of dry dichloromethane was prepared followed by the addition of 0.178 mL (2.6 equiv., 1.27 ^χ 10^"3 mol) of Et₃N. The reaction mixture was then cooled to 0 °C using an ice bath, and then 0.074 ml , (1 .3 equiv., 6.36 x 10⁴ mol) of 3-furoyl chloride dissolved 5 in 0.25 mL of dry dichloromethane was added dropwise to the reaction mixture. The reaction mixture was then warmed to room temperature and stirred overnight. Once the reaction was complete, 4 niL of NH₄OH and 45 niL of I LO were added to the reaction mixture. The organic layer was separated, and the aqueous layer was washed with dichloromethane (3 5 ml .) followed by NaHC0₃ solution and brine. The organic 10 extracts were dried over anhydrous magnesium sulfate and the solvent was removed via rotary evaporation, resulting in a white solid. The product was washed with hexanes to obtain an analytically pure sample.

Synthesis of 2-(thiophen-2-yl)ethyl methanes ulfonate

15 2.6 mL of 2-(thiophen-2-yl)ethanol (1 equiv., 0.023 mol) was dissolved in 45 mL of dry dichloromethane followed by the addition of 3.63 mL of Et₃N (1 .13 equiv., 0.026 mol). The reaction mixture was stirred at room temperature for 1 h. It was then cooled to -5 °C using an ice bath and solid NaCl. Once cooled, 1 .92 mL of methane sulfonyl chloride was added dropwise over the course of 10 min. The reaction mixture was then warmed to

20 room temperature and stirred for 1 h. Once the reaction was complete, 30 mL of Nal ICO3 solution was added followed by separation of the organic and aqueous layers. The aqueous layer was extracted with dichloromethane (3 x 30 mL). The combined organic extracts were dried over anhydrous magnesium sulfate and the solvent was removed via rotary evaporation, resulting in a brown oil. No further purification was

25 required.

NMR (Ή & ^UC) & mass-spec data of novel substituted l -arylethyl-4- acylaminopiperidine derivatives (total six compounds)

(dtd, J=3.70, 11.10, 11.13, 12.61 Hz, 2H), 1.91 (m, 211), 2.17 (m, 4 H), 2.49 (m, 211). 2.68 (m, 2H), 2.81 (m, 2H), 3.67 (s, 3H), 3.78 (dddd, J=4.29, 4.36, 11.92, 15.26, IH), 5.25 (d, J= 7.96 Hz, IH).¹³C NMR (100 MHz, CDC ) δ 10.02, 29.99, 32.41, 46.40,

5 51.76, 52.25.55.63, 173.14, 174.05.

CRA8 NMR data: Ή NMR (400 MHz, CDC1₃) δ 1.63 (d, J =11.93 Hz, 211), 2.05 (d, J- 12.33 Hz, 2H), 2.26 (t, .1=11.35, 11.35 Hz, 2H), 2.64 (dd, .1=6.16.10.21 Hz, 2H), 2.83 (dd, J=6.12, 10.24 Hz, 2H), 2.99 (d, J= 12.01 Hz, 2H), 3.98 (m, IH), 6.21 (d, J=8.20 Hz, IH), 6.49 (dd, J^~ 1.79.3.47 Hz, IH), 7.10 (dd, J-0.84, 3.47 Hz, IH), 7.24 (m, 5H), 7.43 0 (dd.0.84, 1.77 Hz. III). ^,3C NMR (100 MHz, CDCh) δ 32.67, 34.17, 46.27.46.61, 52.76, 60.88, 112.60, 114.57, 126.56, 128.87, 129.13, 140.58, 144.16, 148.48, 158.13. CRA9 NMR data: Ή NMR (400 MHz, CDC1₃) δ 1.63 (m, 2H), 2.06 (d, J=l 1.35 Hz, 211), 2.26 (m, .1=11.35, 2H), 2.65 (m, 2H), 2.84 (m, 2H), 3.02 (d, J= 11.86 Hz, 2H), 3.99 (m, IH), 5.65 (d, J=7.99 Hz, IH), 6.59 (dd, .1=0.91.1.93 Hz, IH), 7.24 (m, 5H), 7.42 (dd, 5 1.58, 1.91 Hz, IH), 7.91 (dd, J=0.90, 1.59 Hz, IH).¹³C NMR (100 MHz, CDC1₃) δ 32.04, 33.49, 46.43, 52.35, 60.26, 108.25, 122.66, 126.19, 128.46, 128.68, 139.88, 143.72, 144.69, 161.95

CRAIS NMR data: Ή NMR (400 MHz, CDC1₃) 61.15 (t. J=7.58, 7.58 Hz, 311).1.47 (m, 2H), 1.95 (m, 2H), 2.18 (m, 4H), 2.64 (dd, J =6.87.8.62 Hz, 2H), 2.90 (m, 2H), 3.00 0 (m, 2H), 3.82 (dddd, J=4.20, 8.31, 10.86, 15.17 Hz, IH), 5.32 (d, J=7.95 Hz, IH), 6.81 (dq, J- 1.02.1.02, 1.02, 3.20 Hz, IH), 6.91 (dd, .1= 3.39.5.14 Hz, IH), 7.11 (dd, .1=1.21, 5.13 Hz, 1 H).¹³C NMR (100 MHz, CDC1₃) δ 10.04, 28.06, 30.03, 32.15.46.52, 52.42, 59.98, 123.62, 124.71, 126.69, 142.87, 173.15.

CRA10 NMR data: Ή NMR (400 MHz, CDC1₃) δ 1.32-2.34 (m, 14H), 2.82-3.01 (m, 5 1111), 3.16 (dd, .1=6.53, 10.91 Hz, IH), 3.49 (d, .1=11.91 Hz, IH), 4.63 (dt, J=5.61, 5.61, 8.76 Hz, IH), 7.24 (m, 4H), 7.42 (dd, .1=4.46, 8.37, 1 I I), 8.02 (m, HI).¹³C NMR (100 MHz, CDCI₃) δ 25.17, 25.45, 27.85, 28.70, 28.75, 31.46, 36.55, 38.61, 81.47, 120.73. 128.66, 128.86, 129.19, 151.29, 162.71, 173.62.

CRA11 NMR data: \ll NMR (400 MHz, CDC1₃) δ 1.35 (t, J- 7.29, 7.29 Hz, 2H), 1.64 (qd, J =3.80, 11.29, 11.29, 11.35 Hz, 211).2.08 (m, 211), 2.27 (td, J=2.57, 11.61. 11.65 Hz, 2H), 2.64 (m, 2H), 2.83 (m, 2H), 3.00 (m, 3H), 4.04 (dddd, J -4.28, 8.29, 10.85, 15.24 Hz, III), 6.04 (d, J=7.94 Hz, III).7.25 (m, 511), 7.44 (m, 311), 7.75 (m, 211). C NMR (100 Mi l/.. CDCI3) δ 32.22, 33.71, 45.83.46.97.52.38, 60.42, 114.25, 126.12, 126.86, 128.42, 128.56, 128.68, 131.42, 134.75, 140.11, 166.88

Many other variations and modifications may be made to the above-described embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of the present disclosure and protected by the following claims.

Appendix 1

Docket No. UA 15-023

Sliidy Number 3235

Table 1

Binding Assays

Snmmnry Results

8255-1 RSAlOIc I.OE-05 -21 8255-2 Rl I. E-05 74 8255-3 R2 l.OE-05 55

BZD (central) ;

8255-1 RSAlOIc I.OE-05 6 8255-2 Rl l.OE-05 17 8255-3 R2 l.OE-05 12

CORP (h)

8255-1 RSAlOIc l.OE-05 -7 8255-2 Rl ^•l.OE-05 -8 8255-3 R2 l.OE-05 3 ^"

CO, ()

8255-1 RSAlOIc l.OE-05 -3 8255-2 Rl 1, OE-05 -3 8255-3 R2 l.OE-05 12

CBi (li)

8255-1 RSAlOIc Ί.0Ε-05 5 8255-2 l l.OE-05 15 8255-3 R2 l.OE-05 25

OABAB

8255-1 RSAlOIc i .OE-05 6 8255-2 Rl l.OE-05 -1 8255-3 R2 l.OE-05 -11

Galaiiiii (lion-selective)

' 8255-1 RSAlOIc 1 ,0E-O5 -191 8255-2 R! !. OE-05 -30 8255-3 R2 J, OE-05 -30

MG,

8255-1 RSAlOIc 1.OE-05 6 8255-2 Rl l.OE-05 7 82S5-3 R2 l.OE-05 13

MO,

8255-1 RSAlOIc ! .OE-05 46 S255-2 Rl l.OE-05 I 8255-3 R2 I.OE-05 2

NK| (Ii)

8255-1 RSAlOIc l.OE-05 -7 8255-2 l 1.OE-05 11 8255-3 R2 l.OE-05 6 δ (DOP) Study Number S255

Assny Tost % Inhibition of

Olien! Compound l.D.

Cerep Compound l.D. Concentration Control Specific Bi ding

8255-1 RSA!Olc 1.0E-05 98 8255-2 J I.OE-05 Ι 8255-3 R2 l.OE-OS 60 ic (KOP)

8255-1 RSAlOIc I.OE-05 79 S255-2 Rl I.OE-05 107 8255-3 R2 I.OE-05 104 μ 00 (MQP)

825S-3 R2 I.OE-05 18

P2Y

8255-1 RSAlOIc l.OE-05 -3

8255-2 Rl I.OE-05 -1 8255-3 R2 I.OE-05 -1 σ (iion-selectivc)

8255-1 RSAlOIc I.OE-05 33

8255-2 Rl I.OE-05 104 8255-3 R2 I.OE-05 101

K v channel

S255-I RSAlOIc l. E-05 -1

S255-2 Rl I.OE-05 -1

8255-3 R2 I .OE-05 0

NE\ transporter (V '

8255-1 RSAlOIc 1.OE-05 -7

8255-2 l l.OE-05 -16

8255-3 R2 1, OE-05 29

GABA transporter

8255-1 RSAlOIc I ,ΟΕ-05 5

8255-2 Rl 1.OE-05 -1

8255-3 R2 1. OE-05 17

2015/046585

Study Number 8255 p. 3At Table 2

Binding Assays

Reference Compound Data

dATPaS 2.4E-OS 1 .2E-08 0.8 σ (non-selective)

aiopon'dol 7.9E-OS 6.2E-OS 0.9

K*v channel

o.-dei lrotoxin 7.7E- 10 ό. Ι Ε- 10 2.8

NE transporter (It)

protrlptyline !.OE-08 7.9E-09 1.1

GABA transporter

nipecotic acid 2.SE-06 2.7E-06 0.9 Study Number 8255 p.4/4

Table^'3

Binding Assnys

Summary Results

Tusl % Inhibition of

Assay Client Compound l.D, Concentration Control Specific Binding

Corcp Compound I.D.

μ (7, (MOP)

8255-2 I !.OE-05 98

8255-3 R2 !.OE-05 99

(KOP)

8255-2 l !.OE-05 107

104

8255-3 2 !.OE-05

cf (non-selective)

8255-2 R.I 1.0E-0S 104

8255-3 1.0E-05 101

82 -2 Rl 1.0E-05 7

8255-3 R2 !.OE-05 55

Appendix 2

Docket No. UA 15-023

1

X-ray Diffraction Facility

Department of Chemistry and Biochemistry

The University of Arizona

Your code: OxEtOH Our code: rvl 01

Comment

The crystals submitted were small needles which diffracted poorly. Reflections were streaky, indicating that the crystal was not single but comprised of more than one not-exactly-oriented component. Diffraction was observed only to about 1 A resolution. Because of the poor resolution, A and B level cheokcif alerts are generated. The quality of the structure is such that the identity of the molecule and its conformation is confirmed, but derived parameters (bond distances, angles, thermal motion) are not reliable.

Nevertheless, the structure could be determined and refined, and the molecule is shown in Figure 1. Figure 2 shows the contents of the unit cell, which also includes an oxalate molecule.

Figure 3 shows the unit cell viewed down the crystal lographic a axis. The hydrogen bonding network in the unit cell connects the organic molecule to oxalate along the crystallographic b axis (ydrogen bonds between the Nl and 02 of the oxalate and between N2 and 04 of the oxalate). The oxalate molecule are connected by hydrogen bonds 04 and 05 of the oxalate, along the crystallographic a axis.

C8 has been modeled with disorder in two positions. There is unmodeled (probably rotational) disorder in the aromatic ring (C1 1 -C 1 6). Both distance and planarity restraints were applied to this ring during refinement. Not all hydrogen atoms were visible in the electron density map. Because of the low resolution and streaky diffraction pattern, the structure could not be refined without constraints. Constraints used in the refinement are included at the end of the report.

Figure 1 . The molecule with displacement ellipsoids at the 50% probability level. C8 is disordered, the minor component has been removed for clarity. An oxalate molecule also in the unit cell is not shown in this figure. 2

Figure 2. The contents of the asymmetric unit with displacement ellipsoids at the 50% probability level. B8 is disordered, and the minor compont is not shown. Fog has been added to show depth.

Figure 3. The contents of the unit cell viewed down the short a axis. Hydrogen bonds are shown between the Nl and 02 of the oxalate. Hydrogen bonds also exist between N2 and 04 of the oxalate and between 04 and 05 of the oxalate, connecting the oxalate molecules along the crystallographic a axis (looking down into the page). 46585

Acknowledgement:

The APEXII DUO was purchased with funding from NSF grant CHE-0741837.

References:

APEX2 (data collection)

Bruker (2007). APEX2. Bruker AXS Inc., Madison, Wisconsin, USA.

SAINT (integration and reduction)

Bruker (2007). SAINT. Bruker AXS inc., Madison, Wisconsin, USA.

SADABS (absorption correction)

Sheldrick, G. M. (1 996). SADABS. University of Gottingen, Germany.

SHELXTL (structure solution and refinement)

Sheldrick, G. M. (2008). Acta Cryst. A64, 1 12-122.

MERCURY (molecular graphics - hydrogen bonding and packing)

Macrae, C. F., Bruno, i. J ., Chisholm, J. A., Edgington, P. R., McCabe, P. Pidcock, E„ Rodriguez - Monge, L. Taylor, R., van de Streek, J. & Wood, P. A. (2008). J. Appl. Cryst. 41, 466-470.

PLATON

Spek, A. L. (2003). J, Appl. Cryst. 36, 7-13.

OLEX2

Dolomanov, O.V., Bourhis, L.J ., Gildea, R.J., Howard, J.A. ., Puschmann H.

(2008), J. Appl. Cryst. 42, 339-341.

Table 1 Crystal data and structure refinement

Identification code rvlOl

Empirical formula C₁₈H₂₇N₂0₅

Formula weight 351.41

Temperature/K 150.0

Crystal system triclinic

Space group P-l

a/A 5.701(3)

b/A 12.024(6)

c/A 14.105(7)

/⁰ 100.473(14)

β/° 93.997(14)

γ/° 95.571(15)

Volume/A³ 942.5(8)

Z 2

Pcalcg/Cm³ 1.238

umrn^"' 0.090

F(000) 378.0

Crystal size/mm³ 0.3 0.08 x 0.05

Radiation ΜοΚα (λ = 0.71073)

2Θ range for data collection/⁰ 2.948 to 41.622

Index ranges -5<h<5,-ll<k<11,-14<[< 14 Reflections collected 7408

Independent reflections 1965 [R_im = 0.0625, R_sigma = 0.0684] Data/restraints/parameters 1965/237/222

Goodness-of-fit on F² 1.592

Final R indexes [1>=2σ (I)] R, = 0.0970, w .2 = 0.2383

Final R indexes [all data] R, =0.1319, wR₂ = 0.2545

Largest diff. peak/hole / e A^"3 0.57/-0.46

5

Table 2 Fractional Atomic Coordinates ( ΐθ⁴) and Equivalent Isotropic Displacement

(A² l0³)

U_eq is defined as 1/3 of of the trace of the orthogonalised Uu tensor.

Atom x y z U(eq)

04 2499(7) 898(4) 1556(3) 26.4(12)

05 -3506(7) 78(4) 1467(3) 28.1(12)

03 -1621(7) 1811 (4) 1840(3) 34.3(13)

02 605(7) -782(4) 1662(3) 29.6(13)

Nl -2989(8) -2451(4) 1622(3) 20.6(13)

N2 -2795(9) -3369(4) -1448(4) 25.0(14)

C2 -1073(11) -3368(6) 206(5) 26.8(16)

01 -2673(8) -5260(4) -1872(3) 35.3(13)

CI -1627(11) -3398(5) 1239(5) 27.3(17)

C3 -3356(11) -3435(5) -460(5) 26.4(16)

C9 -3409(12) -2486(6) 2654(4) 28.0(17)

C4 -4744(11) -2458(6) -50(5) 25.9(16)

C18 -1623(11) 803(6) 1645(4) 16.6(14)

C5 -5248(10) -2510(6) 986(4) 22.8(16)

C17 690(11) 242(6) 1604(4) 16.3(14)

C7 -1722(13) -4117(6) -3046(5) 38.1(19)

CIO -4412(13) -1434(6) 3188(5) 36.6(19)

C6 -2419(11) -4299(6) -2080(5) 24.0(16)

Cll -4740(9) -1569(5) 4222(3) 39.1(18)

C12 -2894(8) -1194(5) 4941(4) 62(3)

C13 -3168(9) -1341(5) 5883(3) 76(3)

C14 -5288(10) -1864(5) 6107(3) 59(2)

C15 -7134(8) -2239(6) 5388(4) 120(5)

C16 -6860(8) -2091(6) 4445(4) 1.14(5)

C8 -1320(80) -5210(40) -3670(30) 66(3)

C8A 680(20) -4565(10) -3240(8) 66(3)

Table 3 Anisotropic Displacement Parameters (A²* 10³).

Ui2+...]-

04 7(2) 29(3) 48(3) 18(2) 4(2) 7.9(19)

05 10(2) 29(3) 47(3) 13(2) 4(2) 2.6(19)

03 20(3) 26(3) 57(3) 6(2) 6(2) 4(2)

02 14(2) 23(2) 57(3) 15(2) 9(2) 7.7(19)

Ni 9(3) 21(3) 34(3) 11(2) -1(2) 5(2)

N2 18(3) 24(3) 36(3) 11(2) 7(2) 5(2)

C2 17(3) 24(4) 43(4) 12(3) 5(3) 11(3)

01 37(3) 23(3) 48(3) 9(2) 5(2) 6(2)

CI 12(3) 26(4) 43(4) 6(3) -1(3) 4(3)

C3 16(3) 27(4) 40(3) 13(3) 4(3) 4(3)

C9 17(4) 36(4) 33(3) 15(3) 0(3) 3(3)

C4 17(4) 29(4) 36(3) 11(3) 7(3) 7(3)

C18 9(2) 20(3) 22(3) 7(2) 3(2) 2(2)

C5 9(3) 26(4) 36(3) 13(3) -1(3) 6(3)

C17 7(2) 23(3) 22(3) 9(2) 5(2) 4(2)

C7 47(4) 39(4) 35(4) .14(3) 7(3) 15(4)

CIO 37(4) 41(4) 38(4) 14(3) 6(3) 18(4)

C6 13(4) 26(3) 36(3) 9(3) 0(3) 8(3)

Cll 32(4) 49(5) 43(4) 16(3) 9(3) 20(3)

C12 60(5) 81(7) 43(4) .17(4) -1(3) -12(5)

C13 88(6) 98(7) 39(4) 13(4) 4(4) 2(5)

C14 66(5) 72(6) 50(4) 20(4) 19(3) 42(4)

C15 55(5) 238(14) 77(5) 77(6) 11(4) -20(7)

C16 40(5) 238(14) 72(5) 78(7) -6(4) -29(6)

C8 72(7) 78(7) 59(6) 17(5) 26(5) 35(6)

C8A 72(7) 78(7) 59(6) 17(5) 26(5) 35(6)

Table 4 Bond Lengths

Atom Atom Length/A Atom Atom Length/A

04 CI 7 1.250(7) C9 CIO 1.532(9)

05 C18 1.295(7) C4 C5 1.519(8)

03 C18 1.194(7) CI 8 C17 1.538(9)

02 C17 1.245(7) C7 C6 1.493(9)

Nl CI 1.482(7) C7 C8 1.49(4) l C9 1.499(8) C7 C8A 1.542(13)

Nl C5 1.505(7) CIO CI 1 1.519(7)

N2 C3 1.465(8) C11 C12 1.3900

N2 C6 1.342(8) C11 C16 1.3900

C2 CI 1.519(9) CI 2 C13 1.3900

C2 C3 1.538(9) CI 3 CI 4 1.3900

01 C6 1.242(7) CI 4 C15 1.3900

C3 C4 1.529(8) CI 5 C16 1.3900

U 2015/046585

Table 5 Bond Angles

Atom Atom Atom Angle/⁰ Atom Atom Atom Angle/⁰

C1 Nl C9 109.4(5) 02 C17 04 126.8(6)

CI Nl C5 110.0(5) 02 C17 C18 118.2(6)

C9 Nl C5 112.8(5) C6 C7 C8A 110.5(6)

C6 N2 C3 121.5(6) C8 C7 C6 111.4(17)

CI C2 C3 111.0(5) Cll CIO C9 109.3(5)

Nl CI C2 111.3(5) N2 C6 C7 116.7(6)

N2 C3 C2 110.4(5) 01 C6 N2 121.3(6)

N2 C3 C4 110.9(5) 01 C6 C7 122.0(6)

C4 C3 C2 108.4(5) C12 Cll CIO 119.9(4)

Nl C9 CIO 114.4(5) CI 2 CI 1 C16 120.0

C5 C4 C3 110.5(5) C16 Cll CIO 120.1(4)

05 C18 CI 7 113.5(5) C13 C12 Cll 120.0

03 C18 05 124.7(6) C14 C13 C12 120.0

03 CI 8 C17 .121.7(6) C13 C14 C15 120.0

Nl C5 C4 111.1(5) C1.6 CI 5 C14 120.0

04 C17 C18 114.9(6) C15 C16 Cll 120.0

Table 6 Torsion Angles

A B c D Angle/⁰ A B C D Angle/'

05 C18 C17 04 164.8(5) C9 CIO Cll CI 6 -89.5(6)

05 C18 C17 02 -18.1(8) C5 Nl CI C2 57.9(7)

03 CIS C17 04 -16.5(8) C5 Nl C9 CIO -67.5(7)

03 C18 C17 02 160.6(6) CIO CM C12 C13 =178.4(5)

Nl C9 CIO CI 1 -179.6(5) CIO Cll C16 CI 5 178.4(5)

N2 C3 C4 C5 -178.1(5) C6 N2 C3 C2 85.2(7)

C2 C3 C4 C5 -56.7(7) C6 N2 C3 C4 -154.6(6)

0.0

CI Nl C9 CIO 169.7(5) Cll C12 CI 3 C14

CI Nl C5 C4 -58.3(6) C12 Cll C16 C15 0.0

CI C2 C3 N2 178.2(5) CI2 C13 C14 C15 0.0

CI C2 C3 C4 56.5(7) CI 3 C14 C15 C16 0.0

C3 N2 C6 Ol 4.4(9) CI 4 CI 5 C16 CI 1 0.0

C3 N2 C6 C7 -176.5(6) C16 Cll C12 CI 3 0.0

C3 C2 CI N -58.2(7) C8 C7 C6 N2 179(2)

C3 C4 C5 Nl 58.5(7) C8 C7 C6 01 -2(2)

C9 Nl CI C2 -177.7(5) C8A C7 C6 N2 122.0(8)

C9 Nl C5 C4 179.3(5) C8A C7 C6 01 -58.9(10)

C9 CIO CI 1 C12 88.9(6)

Table 7 Hydrogen Atom Coordinates (A*10⁴) Isotropic Displacement Parameters (A²xl0³)

Atom X y z U(eq)

H4 3698 545 1578 40

115 -3360 -442 1785 42

HI -2014 -1718 1607 25

H2 -2702 -2706 -1630 30

H2A -188 -4016 -34 32

H2B -59 -2656 191 32

HI A -2553 -4133 1259 33

MB -131 -3342 1655 33

H3 -4332 -4175 -469 32

H9A -1893 -2570 3007 34

H9B -4520 -3167 2668 34

H4A -3821 -1722 -67 31

H4B -6255 -2508 -454 31

H5A -6132 -1868 1242 27

H5B -6247 -3228 997 27

H7AA -2983 -3771 -3369 46

H7AB -255 -3582 -2958 46

H7BC -1605 -3295 -3067 46

H7BD -2948 -4520 -3558 46

HI OA -3313 -744 3188 44

H I OB -5951 -1347 2854 44

HI 2 -1446 -837 4788 75

H13 -1907 -1085 6375 91

H14 -5475 -1965 6751 71

H15 -8583 -2596 5540 144

H16 -8121 -2347 3954 137

H8A . -734 -5723 -3269 100

H8B -150 -5058 -4125 100

H8C -2812 -5564 -4039 100

H8AA 1887 -4172 -2730 100

H8AB 1128 -4424 -3869 100

H8AC 543 -5385 -3244 100 Table 8 Atomic Occupancy

Atom Occupancy Atom Occupancy Atom Occupancy H7AA 0.215(12) H7AB 0.215(12) H7BC 0.785(12) M7BD 0.785(12) C8 0.215(12) H8A 0.215(12) 118 B 0.215(12) H8C 0.215(12) C8A 0.785(12) H8AA 0.785(12) H8AB 0.785(12) H8AC 0.785(12)

IP-

Experimental Details

Needle shaped crystals of C18H27N2O5 were submitted for structure determination. The crystals were small and were grown together. Even apparently single crystals were stacks of needles A crystal was selected and attached to a Micromount using paratone oil, then mounted on a Bruker Kappa APEX-11 Duo diffractometer. The crystal was kept at 150.0 K during data collection. Using Olex2 [1 ], the structure was solved with the ShelXS [2] structure solution program using Direct Methods and refined with the ShelXL [3] refinement package using Least Squares minimisation.

1 . Dolomanov, O. V., Bourhis, L.J., Gildea, R.J, Howard, J.A.K. & Puschmann, H. (2009), J. Appl. Cryst. 42, 339-341.

2. Sheldrick, G.M. (2008). Acta Cryst. A64, 1 12-122.

3. Sheldrick, G.M. (2008). Acta Cryst. A64, 1 12-122.

Crystal Data for C18H27 2O5 (M =351 .41 g/mol): triclinic, space group P-l (no. 2), o = 5.701 (3) A, b = 12.024(6) A, c = 14.105(7) A, a = 100.473(14)°, β = 93.997(14)°, γ = 95.573 (15)°, V =

942.5(8) A³, Z= 2, T- 1 50.0 K, μ(Μο α) = 0.090 mm^"1, Dcalc = 1 .238 g/cm³, 7408 reflections measured (2.948° < 2Θ < 41 .622°), 1965 unique (R_mt = 0.0625, R_sjgma = 0.0684) which were used in all calculations. The final R was 0.0970 (I > 2σ(Ι)) and wR_z was 0.2545 (all data).

Refinement model description

Number of restraints - 237, number of constraints - Details:

1. Fixed Uiso

At 1 .2 times of:

All C(H) groups, All C(H,H) groups, All C(H,H,H,H) groups, All N(H) groups

At 1 .5 times of:

All C(H,H,H) groups, Ail O(H) groups

2. Restrained planarity

C1 1, C12, C 13, C14, C15, C 16, C 10

with sigma of 0, 1

3. Rigid bond restraints

02, 05, CI 8, 04, C I 7, 03

with sigma for 1 -2 distances of 0.0.1 and sigma for 1 -3 distances of 0.01.

C1 1, C16, C 15, C 14, C 13, C 12

with sigma for 1 -2 distances of 0.01 and sigma for 1 -3 distances of 0.01

4. Uiso/Uaniso restraints and constraints

Uanis(03) « Ueq, Uanis(C 1 8) « Ueq, Uanis(05) « Ueq, Uanis(02) «

Ueq, Uanis(Cl 7) « Ueq, Uanis(04) « Ueq: with sigma of 0.005 and sigma

for terminal atoms of 0.01

Uanis(C8) = Uanis(C8A)

5. Rigid body (RIGU) restrains

All non-hydrogen atoms

with sigma for 1 -2 distances of 0.004 and sigma for 1 -3 distances of 0.004

6. Others

^' Sof(H7BC)=Sof(H7BD)=Sof(C8A)=Sof(H8A A)=Sof(H8AB)=Sof(H8AC)=l -FVAR(1 )

Sof(H7AA)=Sof(H7AB)=Sof(C8)=Sof(H8A)=Sof(H8B)=Sof(M8C)=FVAR( l)

7. a Ternary CIT refined with riding coordinates:

N1(H.1 ), C3(H3)

7.b Secondary CH2 refined with riding coordinates;

C2(H2A,H2B), C I (H I Α,Η Ι Β), C9(H9A,H9B), C4(H4A,H4B), C5(H5A,H5B), C7(H7AA,

13

H7AB), C7(H7BC,H7BD), C 10(H 10A,H1 OB)

7.c Aromatic/amide H refined with riding coordinates:

N2(H2), C12(HI 2), C 13(H 13), C 14(H 14), C 15(H 15), C16(H 16)

7.d Fitted hexagon refined as free rotating group:

C1 1 (C12,C13,C14,C15,C16)

7.6 Idealised Me refined as rotating group:

C8(H8A,H8B,H8C), C8A(H8AA,H8AB,H8AC)

7. f Idealised tetrahedral OH refined as rotating group:

04(H4), 05(H5)

This report has been created with 01ex2, compiled on 2014.07.22 svn.r2960 for OlexSys. Please let us know if there are any errors or if you would like to have additional features.

Appendix 3 Docket No. UA 15-023

μ-Opioid Antagonist/ -agonist (I)

Lipinski and Related Properties Value Condition Note

Freely Rotatable Bonds 5(1 )

H Acceptors 3(1 )

H Donors 1 (1 )

H Donor/Acceptor Sum 4(1 )

logP2.216 0.355Temp:25 C(1 )

Molecular Weight260.37(1 )

Lipinski's rule of five

Lipinski's rule of five also known as the Pfizer's rule of five or simply the Rule of five (R05) is a rule of thumb to evaluate druglikeness or determine if a chemical compoundwith a

certain pharmacological or biological activity has properties that would make it a likely orally active drug in humans. The rule was formulated by Christopher A. Lipinski in 1997, based on the observation that most orally administered drugs are relatively small and

moderately lipophilic molecules.^11I[21

The rule describes molecular properties important for a drug's pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion ("AD E"). However, the rule does not predict if a compound is pharmacologically active.

The rule is important to keep in mind during drug discovery when a pharmacologically active lead structure is optimized step-wise to increase the activity and selectivity of the compound as well as to ensure drug-like physicochemical properties are maintained as described by Lipinski's

rule.¹³' Candidate drugs that conform to the R05 tend to have lower attrition rates during clinical trials and hence have an increased chance of reaching the market.'²"''¹

Components of the rule Lipinski's rule states that, in general, an orally active drug has no more than one violation of the following criteria:

• No more than 5 hydrogen bond donors (the total number of nitrogen-hydrogen and oxygen hydrogen bonds)

• Not more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms)

• A molecular mass less than 500 daltons

• An octanol-water partition coefficient'⁵' log P not greater than 5

Note that all numbers are multiples of five, which is the origin of the rule's name. As with many other rules of thumb, (such as Baldwin's rules for ring closure), there are many exceptions to Lipinski's Rule.

Appendix 4

Docket No. UA 15-023

. STUDY REFERENCES

Study title In Vitro Pharmacology

Study of One Compound

Study number 100023322 FINAL REPORT

Experimental period

. PERSONS INVOLVED IN THE STUDY

Investigator Eurofins Cerep Thierry JOLAS.Ph.D.

Le Bois I'Eveque Principal Scientist, Pharmacology B.P. 30001

86 600 Celle I'Evescault Tel: +33 (0)5 49 89 39 89

France Fax: +33 (0)5 49 43 21 70

Tel: +33 (0)5 49 89 30 00

Fax: +33 (0)5 49 43 21 70

Study sponsor TECH LAUNCH ARIZONA Dr. Paul EYNOTT

University of Arizona Licensing Manager, The College of Science

220 W 6th Street

4th Floor

TUCSON, AZ 85701

U.S.A.

. APPROVAL

Investigator Eurofins Cerep Thierry JOLAS.Ph.D.

Le Bois I'Eveque Principal Scientist, Pharmacology B.P. 30001

86 600 Celle I'Evescault Tel: +33 (0)5 49 89 39 89

France Fax: +33 (0)549 43 21 70

Tel: +33 (0)5 49 89 30 00

Fax: +33 (0)5 49 43 21 70

I certify that this report accurately reflects all relevant data collected in this study.

Date

Quality assurance statement Eurofins Cerep's Quality Unit certifies that results presented in this report were generated using the materials and methods mentioned and that these results accurately reflect the raw data.

Date

Signature

14. TABLE OF CONTENTS

1. STUDY REFERENCES 2

2. PERSONS INVOLVED IN THE STUDY 2

3. APPROVAL · 3

4. TABLE OF CONTENTS · - ·· - 4

5. SUMMARY ··■■ 5

5.1. Study Design ■·■■■ 5

5.2. Measurements ■■ ■ 5

5.3. Results ■ 5

6. COMPOUNDS ■ 6

6.1. Test Compounds ■ ■ 6

6.2. Reference Compounds 6

7. RESULTS 7

7.1. In Vitro Pharmacology: Cellular and Nuclear Receptor Functional Assays 7

7.1.1. Agonist Effect: Test Compound Results 7

7.1.2. Reference Compound Results 7

7.1.3. Antagonist Effect: Test Compound Results 8

7.1.4. Reference Compound Results 8

8. RESULTS INTERPRETATION GUIDE 9

9. MATERIALS AND METHODS 10

9.1. Experimental Conditions ■ 10

9.1.1. In Vitro Pharmacology: Cellular and Nuclear Receptor Functional Assays 10

9.2. Analysis and expression of results ■ 11

9.2.1. In Vitro Pharmacology: Cellular and Nuclear Receptor Functional Assays 11

10. BIBLIOGRAPHY - ■ 12

I 5. SUMMARY

The purpose of this study was to test HCV-3 in cellular and nuclear receptor functional assays.

5.1. Study Design

HCV-3 was tested at 1.0E-05 M.

5.2. Measurements

Cellular agonist effect was calculated as a % of control response to a known reference agonist for each target and cellular antagonist effect was calculated as a % inhibition of control reference agonist response for each target.

5.3. Results

Results showing an inhibition or stimulation higher than 50% are considered to represent significant effects of the test compounds. Such effects were not observed at any of the receptors studied.

6. COMPOUNDS 6.1» Test Compounds

Manufacturer: TECH LAUNCH ARIZONA

Clienf Compound ID Compound ID Reference Number Batch Number FW MW Purity Received Form Stock solution Rag

HCV-3 ^" 100023322-1 - 1 296.84 260.38 - Powder 1.E-02 M H2O

FW: Formula Weight - MW: Molecular Weight

6.2. Reference Compounds

In each experiment and if applicable, the respective reference compound was tested concurrently with HCV-3, and the data were compared with historical values determined at Eurofins. The experiment was accepted in accordance with Eurofins validation Standard Operating Procedure.

7. RESULTS

7.1. In Vitro Pharmacology: Cellular and Nuclear Receptor Functional Assays

7.1.1. Agonist Effect: Test Compound Results of Control Agonist esponse

-10 10 30 40 50 60 100 e;2B(h) (agonist effect)

52 (OOP) (agonist effect)

K (HOP) (agonist effect) μ (MOP) (h) (agonist effect)

I Test Concentration: 1.0E-05 M

Figure 1. Histogram for HCV-3

Compound I.D. Client Compound I.D. Test % of Control Agonist Response

Concentration 1^s' 2^nd Mean a_2!l ^(a priist effect)

100023322-1 HCV-3 1.0E-O5 M 9.2 5.1 7.1

100023322-1 HCV-3 1.0E-05 M 4.0 4.0 4.0 (KOP) (agonist effect)

100023322-1 HCV-3 1.DE-05 -10.3 -4.2 μ (MOP) (h) (agonist effect)

100023322-1 HCV-3 1.0E-05 M 28.1 38.9 33.5

7.1.2. Reference Compound Results

Compound I.D. EC₅₀ (M) nH t (W (agonist effect)

dexmedetomidine 1.3E-08 n/a

(DOP) (ayoiiist effect)

DPDPE 1.5E-09 M n/a

(KOP) (agonist effect)

U 50488 1.6E-09 M

μ (MOP) (h) (agonist effect)

DAM GO 4.2E-09 M n/a 7.1.3. Antagonist Effect: Test Compound Results

<M> Inhibition of Control Agonist Response

-10 0 10 20 30 40 50 60 80 90 100

«2B<h) ⁽antagonist effect)

02 (OOP) (antagonist effect) (KOP) (antagonist effect) μ (MOP) (h) (antagonist effect)

Test Concentration: l.OE-05 M

Figure 2. Histogram for HCV-3

Compound I.D. Client Compound I.D. Test % Inhibition of Control Agonist Response

Concentration 1^s1 2^nd Mean ¾<^an&3oniSt: ffect)

100023322-1 HCV-3 1.0E-05 -16.8 -38.7 -27.8

6₂ (DOP) (antagonist effect)

100023322-1 HCV-3 1.0E-05 M 0.8 5.9 3.3 (KOP) (antagonist effect)

100023322-1 HCV-3 1.0E-05 M -12.4 23.5 5.6

■i fWKJl'i ill !a :rf£,uiiist uifc-ct)

100023322-1 HCV-3 1.0E-05 M -0.1 -1.8 -1.0

7.1.4. Reference Compound Results

Compound I.D. ICso M . ^ K_B ( ) ^ nH t (h) (antagonist effect)

yohimbine 3.7E-07 M 4.8E-08 M n/a o_? iuur|

naltrindole 2.7E-10 M 4.6E-11 M n/a (KOP) (antagonist effect)

nor-BNI 4.3E-10 M 7.2E-11 M n/a μ (MOP) (h) (antagonist effect)

CTOP 2.1 E-07 M 2.3E-08 M n/a

8. RESULTS INTERPRETATION GUIDE

In Vitro Pharmacology

Results showing a stimulation or an inhibition higher than 50% are considered to represent significant effects of the test compounds. 50% is the most common cut-off value for further investigation (determination of EC₅₀ or IC₅₀ values from concentration-response curves).

Results showing a stimulation or an inhibition between 25% and 50% are indicative of weak to moderate effects (in some assays, they may be confirmed by further testing as they are within a range where more inter-experimental variability can occur).

Results showing a stimulation or an inhibition lower than 25% are not considered significant and mostly attributable to variability of the signal around the control level.

J 9. MATERIALS AND METHODS 9.1. Experimental Conditions

Minor variations to the experimental protocol described below may have occurred during the testing, they have no impact on the quality of the results obtained.

9.1.1. In Vitro Pharmacology: Cellular and Nuclear Receptor Functional Assays

Assay Source Stimulus Incubation Measured Detection Method Bibl.

Component

Receptors

a_2B (l>) human recombinant none 30 min cA P HTRF 915

(agonist effect) (HEK-293 cells) (3 μ 37°C

dexrnedeiomidine

for control)

(h) human recombinant dexmedetomidine 30 min cA P HTRF 915

(antagonist effect) (HEK-293 cells) ( 100 nM ) 37°C

δ₂ (OOP) NG- 10815 cells none 37°C impedance Cellular dielectric 934

(agonist effect) (endogenous) (300 nM DPDPE spectroscopy

for control)

5₂ (DOP) NG-10815 cells DPDPE 37°C impedance Cellular dielectric 934

(antagonist effect) (endogenous) ( 10 nM ) spectroscopy

( OP) rat recombinant none 10 min cAMP HTRF 819

(agonist effect) (CHO cells) (0.3 μΜ U 50488 37"C

for control)

(KOP) rat recombinant U 50488 10 min cAMP HTRF 819

(antagonist effect) (CHO cells) ( 3 nM ) 37°C

(MOP) (h) human recombinant none 10 min cAMP HTRF 260

(agonist effect) (CHO cells) (0.3 μΜ DAMGO 37°C

for control)

μ (MOP) (h) human recombinant DAMGO 10 min cAMP HTRF 260

(antagonist effect) (CHO cells) ( 20 nM ) 37°C

9.2. Analysis and expression of results

9.2.1. In Vitro Pharmacology: Cellular and Nuclear Receptor Functional Assays

The results are expressed as a percent of control agonist response

measured response

_. : *100

control response and as a percent inhibition of control agonist response

measured response

100-( MOO)

control response obtained in the presence of HCV-3.

The EC₅o values (concentration producing a half-maximal response) and IC₅₀ values (concentration causing a half-maximal inhibition of the control agonist response) were determined by non-linear regression analysis of the concentration-response curves generated with mean replicate values using Hill equation curve fitting

A-D

Y=D+[ -

1 +(C/C₅₀)"H where Y = response, A = left asymptote of the curve, D = right asymptote of the curve, C = compound concentration, and C₅₀= EC50 or IC50, and nH = slope factor.

This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot ® 4.0 for Windows ® (© 1997 by SPSS Inc.).

, the apparent dissociation constants (K_B ) were calculated using the modified Cheng Prusoff equation

where A = concentration of reference agonist in the assay, and EC₅₀A = EC₅₀ value of the reference agonist.

110. BIBLIOGRAPHY

260. Wang, J.B. et al, (1994), FEBS Lett., 338„: 217-222.

819. Avidor-Reiss, T. et al. (1995), FEBS Lett., 361 ; 70-74.

915. Eason, .G. et al. (1992), J. Biol. Chem., 267 : 15795-15801.

934. Law, P.Y. and Loh, H.H. (1993), Mol. Pharmacol., 43_: 684-693.

Appendix 5

Docket No. UA 15-023

Company Name: Tech Launch Arizona 2 Cyprotex Study Number: CYP 1 199-R l

Table of Contents

AUTHENTICATION STATEMENT . 3

1 PURPOSE 4

2 STUDY CONDITIONS 4

3 EXPERIMENTAL DESIGN 4

3. 1 AMES MFP: Experimental Conditions 4 RESULTS AND CONCLUS IONS 5

4.1 AMES MPF: Data Summary 5

4.2 AMES MPF: Individual Data 6 REFERENCE 8

Cyprotex Study Number: CYP1 199-R 1

AUTHENTICATION STATEMENT

I the undersigned, hereby declare that the work described in this report was performed according to the study protocol and/or standard procedures, and to the best of my knowledge, this report provides a correct record of results obtained.

Study Manager

La :

Rarry Press. Ph.D.

Principal Scientist &

Quality Manager

Company Name: Tech Launch Arizona 4 Cyprotex Study Number: CYP 1 1 99-R1

1 PURPOSE

The purpose of this study was to evaluate the mutagenicity potential of a test article (HCV-3) in the Ames assay using the microplate fluctuation (MPF) method.

STUDY CONDITIONS

This study was performed under non-GLP conditions. All work was performed with appropriate local health regulations and ethical approval.

3 EXPERIMENTAL DESIGN

3.1 AMES MFP: Experimental Conditions

*The top concentration in the dose response reflects the highest soluble concentration of the test article in Ames assay conditions.

Experimental Procedure: Approximately ten mi l lion bacteria are exposed in triplicate to test agent (six concentrations), a negative control (vehicle) and a positive control for 90 minutes i n medium containing a low concentration of histidine (sufficient for about 2 doublings.) The cultures are then diluted into indicator medium lacking histidine, and dispensed into 48 wells of a 384 wel l plate (micro-plate format, MPF). The plate is incubated for 48 hr at 37°C, and cells that have undergone a reversion will grow in a well, resulting in a color change in wells with growth. The number of wel ls showing growth are counted and compared to the vehicle control. An increase in the number of colonies of at least two-fold over baseline (mean + SD of the vehicle control) and a dose response indicates a positive response. An unpaired, one-sided Student's T-test is used to identify conditions that are significantly different from the vehicle control.

Where indicated, S9 fraction from the livers of Aroclor 1254-treated rats is included in the incubation at a final concentration of 4.5%. An NADPH-regenerating system is also included to ensure a steady supply of reducing equivalents.

Strains used in this study:

S. typhimurium TA98: hisD3052, rfa, uvrB / pK l O l ; detects frame-shift mutations.

S. typhimurium TA 100: hisG45, rfa, uvrB / pKM l O l ; detects base-pair substitutions.

Cyprotex Study Number: CYP 1 199-R1

RESULTS AND CONCLUSIONS

4.1 AMES MPF: Data Summary

*Highest soluble concentration of test article in Ames assay conditions.

Conclusion: Test article HCV-3 was assessed for its mutagenic potential in the Ames reverse mutation assay. This test was performed in the absence and presence of S9 metabolic activation. HCV-3 was found to be negative for genotoxicity against both strains used in this study (TA98 and TA 100) up to a maximum tested concentration of 1 mg/ml. The positive controls behaved as expected.

U 2015/046585

Company Name: Tech Launch Arizona

Cyprotex Study Number: CYP 1 199-R1

4.2 AMES MPF: Individual Data

Significant fold increase over baseline values (>2-fold) are indicated in bold red. Significant T-test probabilities (P < 0.05) appear red and (P<0.01 ) are indicated in bold red.

HGV-3

TA 98 +S9 Assay 8/10/2015

~— Forar— " t-test mean # increase p-value

Cone. pos. Corr. Base(over (unpaired, 1 - n Wells mean SD line baseline) sided)

0 3 4.00 1 .00 5.00

31.3 3 5.67 2.52 1 .13 0.1732

62.5 3 6.67 2.31 1.33 0.0702

125 3 6.00 3.61 1 .20 0.2035

250 3 6.00 4.36 1.20 0.2409

500 3 3.67 2.08 0.73 0.4075

1000 3 5.00 4.00 1.00 0.3480

Pos. Control 3 34.33 8.33

Cyprotex Study Number: CYPl 199-RI

HCV-3

Company Name: Tech Launch Arizona

Cyprotex Study Number: CYP1199-R]

REFERENCE

Umbuzeiro, G. et al. (2010). "Comparison of the Salmonella/microsome micro-suspension assay with the new microplate fluctuation protocol for testing the mutagenicity of environmental samples". Environ. Mol. Mutagen.51(l):31-38,

Appendix - References Cited UA 15-023 PCT

Appendix - Literature Cited

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4. Husbands, S.M., Lewis, J.W., Opioid ligands having delayed long-term antagonist activity: Potential pharmacotherapies for opioid abuse, Mini-Revi. Med. Chem., (2003), 3(2), 137- 144.

5. Schmidhammer, H., Opioid receptor antagonists, Prog. Med. Chem., (1998), 35, 83- 1 32,.

6. Boothby, L.A.,Doering, P.L., Buprenorphine for the treatment of opioid dependence, Am. J. Health-System Pharm., (2007), 64(3), 266-272.

7. Lowengrub, K., Iancu, I., Aizer, A., Kotler, M., Dannon, P.N., Pharmacotherapy of pathological gambling: review of new treatment modalities, Exp. Rev. Neurotherapeut., (2006), 6(12), 1845-1 851 .

8. Krishnan-Sarin, S., O'Malley, S.S., Opioid antagonists for the treatment of nicotine dependence, Med. Treat. Nicotine Depend., (2007) 123-135.

9. White, J.M., Lopatko, O.V., Opioid maintenance: a comparative review of pharmacological strategies, Expert Opin. Pharmacotherapy, (2007), 8( 1), 1-1 1 .

10. Cunningham, C.W., Coop, A.,Therapeutic applications of opioid antagonists, Chimica Oggi, 24(3), 54-57 (2006).

1 1. Lauretti, G.R., Highlights in opioid agonists and antagonists, Expert Rev.

Neurotherapeut., 6(4), 613-622 (2006).

12. Capasso, A., D'Ursi, A., Pharmacological activity of new mu, k, delta receptor agonists and antagonists. Studies in Natur. Prod. Chem. (2005), 30, 797-823.

13. Anon, N.Z., Alvimopan: ADL 8-2698, ADL 82698, entrareg, LY 246736, Drugs in R&D, (2006), 7(4), 245-253.

14. Leslie, J. B., Alvimopan: a peripherally acting Mu-. Opioid receptor

antagonists, Drugs of Today (2007), 43(9), 61 1 -625.

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Incorporation by reference

Each document, patent, patent application or patent publication cited by or referred to in this disclosure is incorporated by reference in its entirety. However, no admission is made that any such reference constitutes prior art, and the right to challenge the accuracy and pertinence of the cited documents is reserved.

Claims

Claims:

1. A compound having the formula

Ar , or (HetAr), or COOR₅, or COON(R₆)₂ (III)

wherein

Ri is I I, substituted or unsubstituted Q-Cio alkyl, alkenyl, or alkynyl, or substituted or unsubstituted aryl or hetaryl;

R₂ is H, -CH₂0-C, _4 alkyl; COO-C, ₄ alkyl; -CONR₄;

R₃ is H, substituted or unsubstituted Q-C10 alkyl, alkylene, alkynyl, or substituted or unsubstituted aryl or hetaryl;

A is substituted or unsubstituted Cj-C 10 alkyl, alkylene, alkynyl;

Ar or HetAr is substituted or unsubstituted monocyclic or polycyclic aromatic or heteroaromatic moiety;

COOR₅, or CON(R₆)2, where R₅ and R₆ are H, substituted or unsubstituted C _r C10 alkyl, alkylene, alkynyl, or substituted or unsubstituted aryl or hetaryl;

the central nitrogen-containing ring is a substituted or unsubstituted 5- to 7- membered heterocyclic ring; and pharmaceutically acceptable salts of said compound.

2. The compound of claim 1, wherein the compound belongs to a series of N-(l- arylethylpiperidin-4-yl)acylamides.

3. The compound of claim 1, wherein the compound is N-( 1 -phenethylpiperidin-4- yl)propionamide.

4. The compound of claim 1, wherein the compound is N-( 1 -phenethylpiperidin-4- yl)propionamide oxalate.

5. A process for preparing a compound of claim 1 , comprising the following steps:

(a) reacting a cyclic ketone having a protecting group in a Grignard or

Reformatsky reaction to obtain a first product;

(b) reacting the product of step (a) in a Ritter reaction to obtain a second product; and

(c) deprotecting the product of step (b) with acylation or alkylation to obtain a compound of claim 1 .

6. A process for preparing a compound of claim 1 , comprising the following steps: (a) reacting a cyclic ketone having a protecting group in a Strecker reaction to obtain a first product;

(b) reacting the product of step (a) in a selective carbalkoxy group

transformation to obtain a second product; and

(c) deprotecting the product of step (b) with acylation or alkylation to obtain a compound of claim 1.

A process for the preparation of N-(l -phenethylpiperidin-4-yl)propionamide, comprising the following steps:

(a) reacting phenethylpiperiding-4-one with hydroxy lam ine hydrochloride in ethanol in the presence of a base, to produce 1 -phenethylpiperidin-4-one oxime;

(b) reducing the oxime obtained in step (a) with iso-amyl alcohol and sodium metal to produce l-phenethylpiperidin-4-amine; and

(c) acylating the product of step (b) with propionic acid chloride in

chloroform in the presence of triethylamine to produce N-(l - phenethylpiperidin-4-yl)propionamide.

The process of claim 7, wherein the N-(l -phenethylpiperidin-4-yl)propionamide is further treated with oxalic acid to produce N-( 1 -phenethylpiperidin-4- yl)propionamide oxalate.