阅读说明：本技术 基于脯氨酸的神经肽ff受体调节剂 (proline-based neuropeptide FF receptor modulators ) 是由 张亚楠 阮水 于 2018-02-13 设计创作，主要内容包括：提供了基于脯氨酸支架的神经肽FF受体调节剂,其提供了纳摩尔范围内的NPFF受体效力和针对NPFF1受体的拮抗选择性。提供了用于调节神经肽FF受体的功能的方法、化合物和组合物,以用于能够影响受神经肽FF受体影响的状况或紊乱的药物疗法。(Proline scaffold-based neuropeptide FF receptor modulators are provided that provide NPFF receptor potency and antagonistic selectivity against the NPFF1 receptor in the nanomolar range. Methods, compounds, and compositions for modulating the function of neuropeptide FF receptors are provided for use in drug therapy capable of affecting conditions or disorders affected by neuropeptide FF receptors.)

1. A neuropeptide FF receptor modulator comprising a compound according to formula (I):

Wherein R2 is selected from-N- (C2-C5 alkyl) 2 and NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; r3 is selected from the group consisting of C3-C9 alkyl, aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocycloalkyl, and arylalkyl; r4 is selected from H and C1-C2 alkyl; and R5 is selected from C3-C9 alkyl, heteroarylalkyl, heteroaryl, heterocycloalkyl, heterocycle, cycloalkylalkyl, and arylalkyl; or a pharmaceutically acceptable salt thereof.

2. The neuropeptide FF receptor modulator of claim 1, wherein R2 is NH-R1 wherein R1 is selected from C3-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl.

3. The neuropeptide FF receptor modulator of claim 2, wherein R1 is a C3-C6 alkyl.

4. The neuropeptide FF receptor modulator of claim 2, wherein R1 is phenethyl substituted with lower alkoxy, nitro, lower alkyl, halogen or halogenated lower alkyl.

5. The neuropeptide FF receptor modulator of claim 1, wherein said compound is according to formula II:

wherein R2 is selected from-N- (C2-C5 alkyl) 2; and X is S, SO2, O, NH, or CH 2.

6. the neuropeptide FF receptor modulator of claim 1, wherein said compound is according to formula IIA:

Wherein R1 is selected from the group consisting of C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; and X is S, SO2, O, NH, or CH 2.

7. The neuropeptide FF receptor modulator according to claim 6, wherein R1 is substituted or unsubstituted C3-C6 alkyl, benzyl, or phenethyl; and X is oxygen.

8. The neuropeptide FF receptor modulator of claim 7, wherein R1 is a C3-C6 alkyl.

9. The neuropeptide FF receptor modulator according to claim 7, wherein R1 is phenethyl substituted with lower alkoxy, nitro, lower alkyl, halogen or halogenated lower alkyl.

10. The neuropeptide FF receptor modulator of claim 1, wherein said compound is according to formula III:

Wherein R3 is selected from the group consisting of C3-C9 alkyl, aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocycloalkyl, and arylalkyl; and R4 is selected from H and C1-C2 alkyl.

11. The neuropeptide FF receptor modulator of claim 10, wherein R3 is benzyl or substituted benzyl, phenethyl or substituted phenethyl, and R4 is H.

12. The neuropeptide FF receptor modulator of claim 10, wherein R3 is benzyl monosubstituted with methoxy; and R4 is H.

13. The neuropeptide FF receptor modulator of claim 10, wherein R3 is benzyl or substituted benzyl, and R4 is methyl.

14. The neuropeptide FF receptor modulator of claim 10, wherein R3 is a C3-C6 alkyl.

15. The neuropeptide FF receptor modulator according to claim 1, wherein said compound is according to formula IV:

Wherein R5 is selected from the group consisting of C3-C9 alkyl, heteroarylalkyl, heteroaryl, heterocycloalkyl, heterocycle, cycloalkylalkyl, and arylalkyl; and X is S, SO2, O, NH, or CH 2.

16. The neuropeptide FF receptor modulator of claim 15, wherein R5 is benzyl or substituted benzyl.

17. The neuropeptide FF receptor modulator of claim 16, wherein R5 is a mono-substituted benzyl group and the substituent is halogen or methoxy at position 2 or 3.

18. The neuropeptide FF receptor modulator according to claim 1, wherein said compound has the structure

Wherein R2 is selected from the group consisting of:

(1)NHMe；

(2)NHEt；

(3)NH(n-Pr)；

(4)NH(n-Bu)；

(5)NH(s-Bu)；

(6)NH(t-Bu)；

(7) NH (n-pentyl);

(8) NH (isopentyl);

(9) NH (n-hexyl);

(10) NH (n-decyl)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)NEt；

(32) N (N-Pr) 2; and

(33)

19. The neuropeptide FF receptor modulator according to claim 1, wherein said compound has the structure

Wherein R1 is selected from the group consisting of:

(i)Et；

(ii) N-pentyl;

(iii)

(iv)

(v) And

(vi)

20. The neuropeptide FF receptor modulator according to claim 1, wherein said compound has the structure

Wherein R4 is H and R3 is selected from the group consisting of:

(a)n-Pr；

(b)n-Bu；

(c) N-pentyl;

(d) N-hexyl;

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l) And

(m)

21. The neuropeptide FF receptor modulator according to claim 1, wherein said compound has the structure

Wherein R4 is Me, and R3 is

22. The neuropeptide FF receptor modulator according to claim 1, wherein said compound has the structure

Wherein R5 is selected from the group consisting of:

(I) N-hexyl;

(II)

(III)

(IV)

(V)

(VI)

(VII)

(VIII)

(IX)

(X)

(XI)

(XII)

(XIII)

(XIV)

(XV) and

(XVI)

23. The neuropeptide FF receptor modulator according to claim 1, wherein said compound is

24. the neuropeptide FF receptor modulator according to claim 1, wherein said compound is

25. A pharmaceutical composition comprising the neuropeptide FF receptor modulator according to any one of claims 1-24, and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition of claim 25, further comprising an opioid.

27. The pharmaceutical composition of claim 26, wherein the opioid comprises at least one selected from the group consisting of: fentanyl, morphine, oxycodone, hydrocodone, and buprenorphine.

28. The pharmaceutical composition of claim 25, further comprising an antipsychotic drug.

29. The pharmaceutical composition of claim 28, wherein the antipsychotic drug comprises at least one selected from the group consisting of haloperidol and aripiprazole.

30. The pharmaceutical composition of claim 25, further comprising a monoamine reuptake inhibitor.

31. The pharmaceutical composition of claim 30, wherein the antipsychotic medication comprises at least one selected from the group consisting of fluoxetine and sertraline.

32. A method for treating a subject suffering from or susceptible to a condition or disorder in which modulation of neuropeptide FF receptor activity is of therapeutic benefit, the method comprising administering to the subject suffering from or susceptible to the condition or disorder a therapeutically effective amount of a neuropeptide FF receptor modulator of any one of claims 1-24.

33. The method of claim 32, wherein the condition or disorder comprises use or abuse of one or more opioids.

34. The method of claim 33, wherein the one or more opioids includes at least one selected from the group consisting of: fentanyl, morphine, oxycodone, hydrocodone, buprenorphine, heroin, and opioid derivatives of the foregoing.

35. The method of claim 32, wherein the therapeutic benefit comprises at least a partial reduction in opioid-induced hyperalgesia.

36. The method of claim 35, wherein the opioid-induced hyperalgesia is induced by an opioid comprising at least one selected from the group consisting of: fentanyl, morphine, oxycodone, hydrocodone, and buprenorphine.

37. The method of claim 32, wherein administration of the neuropeptide FF receptor modulator according to any one of claims 1-24 is performed in a therapeutic intervention comprising co-administering a drug that reduces a side effect of the neuropeptide FF receptor modulator.

38. The method of claim 37, wherein said agent that said neuropeptide FF receptor modulator reduces its side effects is an agent that produces tolerance or hyperalgesia as a side effect.

39. The method of claim 32, further comprising administering an effective amount of a second therapeutically effective agent.

40. The method of claim 32, wherein the condition or disorder in which modulation of neuropeptide FF receptor activity is of therapeutic benefit is selected from the group consisting of: reduction of opioid tolerance and reduction of hyperalgesia.

Description of the Related Art

Neuropeptides FF (NPFF) belong to the family of neuropeptides known as RF amide (RFamide) peptides, the members of which all contain an Arg-Phe-NH2 (RF-amide) motif at their C-terminus. Neuropeptide FF is an endogenous peptide that binds to and activates two G protein-coupled receptors (GPCRs), NPFF1(GPR147) and NPFF2(GPR 74). These receptors are members of the rhodopsin family and are primarily coupled to G α i/o proteins. NPFF and its receptors originally isolated from bovine brain have been identified in the Central Nervous System (CNS) of various animal species. Ligand binding studies in rodents demonstrate that both receptor subtypes are widely expressed in the brain, while only the NPFF2 receptor is expressed at detectable levels in the spine. The NPFF system has been implicated in the regulation of a variety of physiological processes, such as insulin release, food intake, memory, blood pressure, electrolyte balance and nerve regeneration. The NPFF system has also been shown to play an important role in regulating the effects of opioids and several other classes of drug abuse.

It is well documented that NPFF, which has no affinity for opioid receptors, is a modulator of opioid receptor function and reduces tolerance and dependence on opioids. Several studies have shown that the effect of NPFF on opioid modulation is dependent on the route of administration. For example, intracerebroventricular (i.c.v.) administration of NPFF in rats alleviates morphine-induced analgesia and exercise (locomotion) and contributes (preconditioned) to opioid withdrawal syndrome in morphine-dependent rats, while intrathecal (i.t.) administration produces opioid-induced analgesia and also prolongs morphine-induced analgesia (Malin et al, Peptides,11, 277-. Injection (i.c.v.) of 1Dme (peptide NPFF analogue) inhibited morphine-induced analgesia and acquisition of site conditioning (place conditioning) by morphine (Marchand et al, Peptides,27,964-72, 2006). Ventricular injection (ventricular injection) of NPFF antiserum restored analgesic response to morphine in morphine-tolerant rats, but did not affect non-opioid-tolerant rats (Lake et al, neurosci. lett.,132(1):29-32,1991). RF9, a dipeptide NPFF1/2 receptor antagonist, dose-dependently blocked long-term hyperalgesia resulting from acute fentanyl administration or chronic morphine administration (Elhabazi et al, br.j. pharmacol.,165, 424-. This effect appears to be mediated primarily by the NPFF1 receptor, since the selective NPFF1 antagonist AC-262620 also reduces opioid tolerance (Lameh et al, j.pharmacol. exp. ther.,334,244-254, 2010).

Opioids remain the most effective analgesics for many pain conditions, particularly for chronic pain; however, adverse effects associated with opioid use, such as physical dependence, hyperalgesia, and tolerance, prevent proper administration and effective pain control in a large number of pain patients. Combination therapy combining opioids with other drugs that can increase opioid efficacy and/or reduce adverse effects provides a promising alternative strategy for pain management.

Given that biological activity is thought to be modulated by NPFF, the art is looking for compounds and compositions that provide inhibition or activation of the functional effects of NPFF.

SUMMARY

The present disclosure relates to the discovery of neuropeptide FF and proline-based neuropeptide FF receptor modulators. In one aspect, the disclosure relates to such proline-based neuropeptide FF receptor modulators according to formula (I):

Wherein R2 is selected from-N- (C2-C8 alkyl) 2 and NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; r3 is selected from the group consisting of C3-C9 alkyl, aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocycloalkyl, and arylalkyl; r4 is selected from H and C1-C2 alkyl; and R5 is selected from C3-C9 alkyl, heteroarylalkyl, heteroaryl, heterocycloalkyl, heterocycle, cycloalkylalkyl, and arylalkyl. Also provided are proline-based neuropeptide FF receptor modulators according to formula II, formula IIA, formula III, and formula IV.

In another aspect, the present disclosure relates to a pharmaceutical composition comprising a proline-based neuropeptide FF receptor modulator represented by formula I, formula II, formula IIA, formula III, or formula IV, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In a further aspect, the disclosure relates to a method for treating a subject suffering from or susceptible to a condition or disorder in which modulation of neuropeptide FF receptor activity is therapeutically beneficial, comprising administering to the subject suffering from or susceptible to the condition or disorder a therapeutically effective amount of a compound according to formula I, formula II, formula IIA, formula III, or formula IV, or a pharmaceutically acceptable salt thereof.

Other aspects, features and embodiments of the disclosure will be more fully apparent from the ensuing description and appended claims.

Brief Description of Drawings

Figure 1 depicts the chemical structures of various NPFF ligands described in the art.

Figure 2 depicts the chemical structure of proline-based compound 1.

Fig. 3 is a graphical representation of NPFF EC50 in a calcium mobilization assay, showing data obtained when stable NPFF1 and NPFF2 cell lines were treated with NPFF.

Figure 4 shows the concentration-response curves of compound 16 in NPFF1 and NPFF2 calcium mobilization assays, where a plots antagonist activity of compound 16 in NPFF1 calcium mobilization functional Ke assay: NPFF1-RD-HGA16 cells alone (O) and NPFF + 5. mu.M final 16(□) in stable NPFF1-RD-HGA16 cells; and B shows antagonist activity of compound 16 in NPFF2 calcium mobilization functional Ke assay: NPFF2-RD-HGA16 cells stabilized NPFF alone (. smallcircle.) and NPFF + 10. mu.M final 16(□).

figure 5 shows concentration-response curves for compound 16 and compound 33 in NPFF1 and NPFF2 cAMP assays, where a plots antagonist activity of compound 16 and compound 33 in NPFF1 cAMP functional Ke assay: NPFF alone (. smallcircle.), NPFF + 4. mu.M final 16(□) and NPFF + 2. mu.M final 33 (. diamond.) in stable NPFF1-CHO cells; and B shows antagonist activity of compound 16 and compound 33 in NPFF2 cAMP functional Ke assay: stable NPFF 2-individual NPFF (. smallcircle.), NPFF + 10. mu.M final 16(□) and NPFF + 10. mu.M final 33 (. diamond.) in CHO cells.

Figure 6 shows the results of the tests in the fentanyl-induced hyperalgesia model in rats, where a plots the results of fentanyl-induced mechanical hyperalgesia, and where B plots the anti-hyperalgesic effects of compound 16 and compound 33 (N ═ 6/group).

Detailed Description

The present disclosure relates to proline-based neuropeptide FF receptor modulators. Modulators exhibit affinity and activity for the neuropeptide FF receptor. Thus, the molecules may be useful in the treatment of disorders, syndromes and conditions mediated by modulation of the neuropeptide FF receptor.

Studies have found that NPFF contributes to nicotine withdrawal syndrome, which also suggests that NPFF is involved in the process of dependence and drug addiction. (Malin et al, Pharmacol. biochem. Behav.,54,581-585, 1996). Chronic administration of NPFF into the lateral ventricle has also been shown to enhance behavioral sensitization to amphetamines. (Chen et al, Brain Res.,816,220-224, 1999). Recently, it was demonstrated that stimulation of NPFF receptors reduces amphetamine-induced conditional locus-preferred expression, while inhibition of NPFF receptors reduces amphetamine withdrawal anxiety. (Kotlinska et al, Peptides,33, 156-. Furthermore, NPFF has been shown to be involved in the expression mechanism for sensitization to ***e excitatory locomotion (hypercomotion), although this effect may be non-specific. (Kotlinska et al, Peptides,29,933-939, 2008). Consistent with these observations, there appears to be evidence that the NPFF binding site is abundant in the Ventral Tegmental Area (VTA), whereas NPFF-like immunoreactivity is detected in the nucleus accumbens (NAc), two brain areas belonging to the midbrain limbic Dopamine (DA) protrusion known to be involved in drug addiction. (Wu et al, Peptides,31,1374-1382, 2010). Together, these findings render the NPFF system a viable target for the treatment of drug addiction.

In addition to NPFF, several other neuropeptides from the RF amide family have also been found to activate one or two NPFF receptors, including NPSF (neuropeptide SF), NPAF (neuropeptide AF), and NPVF (neuropeptide VF). A number of peptidomimetic NPFF ligands have been reported, which contain a guanidine functional group (figure 1). Among these ligands, acylation of the last two amino acid Residues (RF) has been reported to be critical for NPFF activity (BIBP3226(Bonini et al, J.biol. chem.275(50): 39324-. Modification of RF9 resulted in NPFF1 selective dipeptides biphenyl-RF and peptidomimetic RF313(Bihel et al, ACS Chem Neurosci, 6(3): 438-. These peptides or peptidomimetics were found to act as antagonists, effectively preventing fentanyl-induced hyperalgesia in rats by subcutaneous or oral administration.

Several classes of non-peptide NPFF ligands have also been disclosed. Quinazolinoguanidines, pyrimidinioguanes, thiazologuanes and quinolinoguanes are reported as NPFF ligands (WO 03/026667, chemical structure 5, fig. 1; U.S. patent No. 7,544,691, chemical structure 4, fig. 1). A series of N-benzylpiperidines were found to have mixed activity as agonists/antagonists against NPFF1 and antagonists against NPFF2 (jounnigan et al, j.med.chem.,57,8903-27,2014, chemical structure 6, figure 1). These series have a guanidine functional group. Although guanidine functional groups have been associated in some cases with high plasma protein binding and limited BBB penetration, some of these guanidine-containing ligands have been reported to enter the CNS, albeit to a relatively minor extent. To date, only two series of small molecule NPFF ligands without guanidine functional groups have been reported, but the in vivo effects of these ligands remain to be studied (WO 2009/038012, chemical structure 7, fig. 1; WO 2004/080965, chemical structure 8, fig. 1).

Small non-peptide compounds do not undergo depsipeptide degradation and are therefore more advantageous tools for exploring the biological effects of NPFF receptors. In an effort to develop new small molecule NPFF ligands, high throughput screening of GPCR-oriented compound libraries was performed. Compound 1 with proline scaffold shown in figure 2 emerged as a promising lead compound with moderate activity on two NPFF subtypes, with reasonable physiochemical properties. A synthetic route to this scaffold was developed and a focused library of proline analogues was prepared in order to explore the structure-activity relationship (SAR) in three regions of the scaffold (fig. 2).

Initial SAR studies focused on three regions, the carboxamide, the amino center and the 4-position of proline, and revealed that substitutions at these positions affected NPFF antagonism and subtype selectivity. A number of compounds have been identified that have sub-micromolar NPFF1 potency. For example, compound 16 with n-pentylamino functionality has a Ke value of 720nM against NPFF1, and has a > 4-fold preference over NPFF 2. Compound 33 with a 4-nitrophenylethyl substituent emerged as the most potent analog for NPFF1 (Ke 245nM) and had a-3-fold preference over NPFF 2. In general, these compounds are more potent against the NPFF1 receptor, while selectivity against NPFF2 is only modest. The results of the secondary cAMP assay further confirm the NPFF antagonistic activity of these compounds, and the radioligand binding assay demonstrates that the ligand binds to the NPFF receptor with moderate affinity.

Representative compound 16 and compound 33 have moderate solubility and blood brain barrier permeability, demonstrating that the proline scaffold is a potential drug-like and potent NPFF template.

The obtained compounds were characterized in a calcium mobilization assay to assess their activity against both NPFF receptors. Several compounds were also evaluated to confirm their role in the cellular level of the regulatory cyclic adenosine monophosphate (cAMP) and their binding affinity to the two NPFF receptors. Drug-like properties such as solubility and blood brain barrier permeability were then examined. Finally, the role of these compounds in reversing fentanyl-induced hyperalgesia was investigated.

Traditionally, NPFF activity has been examined using assays such as radiolabel (radioligand) binding, GTP- γ -S or cAMP assays. To establish a platform that allows for low cost high throughput screening, calcium mobilization assays were developed using Chinese Hamster Ovary (CHO) cells that simultaneously overexpress the ga16 protein and the human NPFF1 receptor or the human NPFF2 receptor. NPFF1 and NPFF2 stable cell lines were generated by: expression plasmids were transfected into RD-HGA16 CHO cells (Molecular Devices), positive clones were selected using antibiotic resistance, and functional expression of the NPFF1 receptor and NPFF2 receptor were tested according to the previously disclosed procedures (Zhang, Y. et al, J Med Chem,53, 7048-. Clones were first screened against 10 μ M NPFF to identify clones with functional NPFF receptor. The 8 clones with the highest maximal NPFF response were further evaluated with NPFF concentration-response curves; the clone with the most potent and effective NPFF response was selected as the working clone.

Figure 3 shows data obtained when stable NPFF1 and NPFF2 cell lines were treated with NPFF. NPFF1-RD-HGA16 cells, NPFF had EC50 values of-62 nM and a signal window of 15,000 Relative Fluorescence Units (RFU). NPFF2-RD-HGA16 cells, NPFF had EC50 values of 22nM and a signal window of 8,200 RFU. These NPFF EC50 values are consistent with results from other cAMP assays and GTP- γ -S assays (Gouarders, C. et al, Neuropharmacology,52,376-86, 2007; Lameh, J. et al, J Pharmacol. exp. ther.,334,244-54, 2010; Vyas, N. et al, Peptides,27,990-60, 2006).

in parental RD-HGA-16CHO cells, there was no response to NPFF, confirming its signaling through NPFF receptors. NPFF1 stable cell lines were successfully miniaturized from 96 wells to 384 wells (Z' factor 0.75) for library screening.

NPFF1 FLIPR (fluorescent imaging plate reader) -based calcium mobilization assay was used for high-throughput screening of ligands from internal GPCR-rich libraries, and for characterization of the synthetic compounds obtained. The library screened is highly diverse, comprising 22,000 compounds, and its content and properties are rigorously evaluated for screening against GPCRs based on a variety of factors, including maximal diversity, optimal ADME parameters, structural novelty (minimal overlap with scaffolds found in known GPCR ligands and drugs), and drug compliance with prototypical properties of GPCR ligands.

Library compounds were screened at 10 μ M final concentration for both agonist and antagonist activity as part of the developed 3-addition protocol, enabling evaluation of both activity patterns with a single assay plate, thereby reducing the overall time and cost of screening. Concentration-response curves were run with 37 selected antagonists (27 with > 65% inhibition, and 10 with > 85% inhibition) yielding confirmed activity of these compounds.

Thus, all synthetic compounds were first characterized in NPFF1 and NPFF2 calcium mobilization assays to determine their ability to antagonize NPFF-stimulated calcium influx. Since the NPFF receptor is naturally coupled to G.alpha.i/o proteins and inhibits adenylate cyclase, active compounds with apparent dissociation constant Ke values ≦ 1 μ M in the NPFF1 or NPFF2 calcium assay were selected and further evaluated in the Lance cAMP assay of Perkinelmer.

In both functional assays, EC50 curves for agonist NPFF alone and EC50 curves for agonist NPFF together with the test compound were obtained and the right shift of the agonist curve was measured. As previously described, the apparent dissociation constant, Ke, was calculated from the compound-mediated inhibition of NPFF activity (Perrey et al, J.Med.chem.,56,6901-16, 2013; Perrey et al, ACS chem.Neurosci.6,599-614,2015). As indicated above, all compounds were tested for agonist activity using a calcium mobilization assay; none of the compounds showed any significant agonist activity towards the NPFF1 receptor or the NPFF2 receptor (< 20% NPFF Emax (final 10 μ M)). These compounds were also characterized in radioligand binding assays to measure affinity and confirm that the proline scaffold is the true template for NPFF ligands. Kinetic solubility and two-way MDCK-MDR1 permeability determinations of compounds were performed according to their standard protocol by Paraza Pharma Inc.

As a result of the studies described above, proline-based NPFF receptor modulators according to formula I were found:

Wherein R2 is selected from-N- (C2-C8 alkyl) 2 and NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; r3 is selected from the group consisting of C3-C9 alkyl, aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocycloalkyl, and arylalkyl; r4 is selected from H and C1-C2 alkyl; and R5 is selected from C3-C9 alkyl, heteroarylalkyl, heteroaryl, heterocycloalkyl, heterocycle, cycloalkylalkyl, and arylalkyl; or a pharmaceutically acceptable salt, amide, ester or prodrug thereof.

Formula I represents a proline-based NPFF modulator in which each of the three regions of the proline scaffold of compound 1 can be optimized.

In embodiments of formula I, wherein R1 is selected from heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; r3 is selected from heteroarylalkyl, heterocycloalkyl, and arylalkyl; and/or R5 is selected from heteroarylalkyl, heterocycloalkyl, cycloalkylalkyl, and arylalkyl, the alkyl group being C1, C2, or C3. Suitable examples of such groups are shown in the table below.

In certain embodiments of formula I, R2 is-N- (C5-C6 alkyl) 2.

In certain embodiments of formula I, R2 is NH-R1. In further embodiments of formula I, when R2 is NH-R1, R1 is C3-C6 alkyl.

In some other embodiments of formula I, when R2 is NH-R1, R1 is benzyl or phenethyl, which is substituted or unsubstituted. In certain embodiments wherein R1 is substituted phenethyl, phenethyl is substituted with lower alkoxy such as methoxy, nitro, lower alkyl, halogen or halogenated lower alkyl such as CF 3. The phenethyl group may be mono-or di-substituted.

Embodiments of R2 as described herein may be combined with any combination of embodiments of R3, R4, and R5 described herein.

In certain embodiments of formula I, R3 is a substituted benzyl or substituted phenethyl group having multiple substituents. For example, substituents may be halogen, methoxy, methyl, cyano, N-CH2, and others.

In certain embodiments of formula I, R3 is benzyl or substituted benzyl, and R4 is H. In such embodiments where R3 is substituted benzyl, the benzyl is mono-or di-substituted with methoxy.

In certain other embodiments of formula I, R3 is benzyl or substituted benzyl, and R4 is methyl.

In embodiments of formula I wherein R3 is a mono-substituted benzyl or a mono-substituted phenethyl, the substituent is in the 4 position.

In some embodiments of formula I, R5 is benzyl or substituted benzyl. In such embodiments where R5 is substituted benzyl, the substituent may be halogen or methoxy. In further embodiments where R5 is substituted benzyl, the benzyl is mono-substituted, and the substituent may be ortho-substituted halo or methoxy.

In one embodiment, the proline-based NPFF receptor modulator may be represented by formula II:

Wherein R2 is selected from-N- (C2-C8 alkyl) 2 and NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; and X is S, SO2, O, NH, or CH 2. The proline-based NPFF modulators represented by formula II have modifications in region 1, the carboxamide region, of compound 1.

In certain embodiments of formula II, R2 is — N- (C5-C6 alkyl) 2.

In certain embodiments of formula II, X is S or O.

According to some embodiments of formula II, R2 is NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; and X is S, SO2, O, NH, or CH 2. This embodiment is represented by formula IIA:

In certain embodiments of formula IIA, R1 is C3-C6 alkyl; and X is oxygen. In other embodiments of formula IIA, R1 is benzyl or phenethyl, which is substituted or unsubstituted, and X is oxygen. In certain embodiments wherein R1 is substituted phenethyl, phenethyl is substituted with lower alkoxy such as methoxy, nitro, lower alkyl, halogen or halogenated lower alkyl such as CF 3.

Table 1 lists SAR determined as discussed above corresponding to embodiments of formula II, wherein X is oxygen.

TABLE 1

values for a are mean ± SEM of at least three independent experiments in duplicate.

b values are mean ± SEM of two independent experiments in duplicate.

Pre-incubation of antagonist and test compound was 45min or 1 hour.

Compound d was inactive in antagonist screening at final 10 μ M (N ═ 2).

The e compound appeared cytotoxic in the assay and potency was undetermined.

Analogues with the 4-methoxybenzyl group at the 4-position of the proline core, instead of the 4- (methylthio) benzyl group in compound 1, were used for SAR studies in the carboxamide region, since the methoxy moiety is known to have better metabolic stability. Furthermore, the corresponding starting materials for the methoxy analogues are more readily available than 4- (methylthio) benzaldehyde.

As can be seen from table 1, the series of NPFF1 antagonist activities is sensitive to the length of the R1 substituent of the amide functionality. Methyl and ethyl analogs (10 and 11) were inactive at 10 μ M against both NPFF receptors. Antagonist activity against the NPFF1 receptor increased from n-propyl to n-pentyl (12, 13, 16), then decreased slightly in the case of n-hexyl (18) and completely eliminated in the case of n-decyl chain (19). Among the three butyl isomers (13-15), NPFF1 antagonist activity decreased slightly in the order of n-butyl, sec-butyl and tert-butyl. In contrast, NPFF2 antagonist activity increased significantly in the same order, indicating that linear is preferred for NPFF1 selectivity. Similar trends were also observed in the case of the n-pentyl and isopentyl isomers (16 and 17). Between these two isomers, n-pentyl is a more potent and selective NPFF1 antagonist (NPFF 1Ke 720nM, NPFF2 Ke 3,090 nM).

When the cyclohexyl group (21) is replaced by the phenyl group (24), NPFF1 activity is slightly increased while maintaining selectivity over the NPFF2 receptor. Shortening (23) or lengthening (25) the distance between the phenyl ring and the proline core results in weaker potency. Compound 24(NPFF 1Ke ═ 850nM) emerged as a potent and selective NPFF1 antagonist.

among the electron-donating substituents at the para-position of the phenyl ring (26-31), 4-methoxy (26, NPFF1Ke 670nM) is the most potent antagonist of NPFF 1.3, 4-dimethoxy (27) and 4-dimethylamino (28) were slightly less potent. Bulky groups such as 4-acetamido (29), 4- (methylamino) carbonyl (30) and tert-butyl (31) are not well tolerated, consistent with the limited space at the binding pocket previously observed. Turning to the electron withdrawing substituent, the 4-nitro group (33, NPFF1Ke ═ 250nM) demonstrated the best NPFF1 antagonist efficacy among all proline analogues. The results indicate that a strong electron withdrawing group is advantageous for good NPFF1 antagonist activity, as 3, 4-difluoro (34, NPFF1Ke 610nM) is more potent than 4-chloro (32, NPFF1Ke 990nM), 4-fluoro (35, NPFF1Ke 1,130nM) and 4-trifluoromethyl (36, NPFF1Ke 1,670 nM). 4-pyridyl (37), which has been used as an isosteric replacement for 4-nitrophenyl, showed only moderate NPFF1 activity (Ke ═ 1,820 nM).

Finally, the NPFF1 antagonist activity of the two bulky electron withdrawing groups acetyl (38) and 4-methylsulfonyl (39) was significantly suppressed compared to the 4-nitro analog. These data suggest that small, strong electron withdrawing substituents are preferred, while bulky groups have proven detrimental to NPFF1 antagonist activity. Similar to the aliphatic series, these phenethyl analogs are not effective against the NPFF2 receptor, except for 4-methoxy (26), diethylamino (28), 4-chloro (32), 4-nitro (33), and 3, 4-difluoro (34).

Next, the effect of the disubstituted amide in this region was also investigated. Diethylamino and dipropylamino analogs (40, NPFF1Ke ═ 2,900nM and 41, NPFF1Ke ═ 880nM) were more potent at the NPFF1 receptor than their monosubstituted amide counterparts (11, NPFF1Ke >10,000nM and 12, NPFF1Ke ═ 9,080 nM). Compound 42(NPFF 1Ke ═ 1,330nM) with a rigid spacer between the phenyl ring and the amide was less active against NPFF1 receptors than 24 with a flexible ethylene linker (NPFF 1Ke ═ 850 nM).

TABLE 2 SAR of the carboxamide region of the 4- (4-methylthio) benzylamino series.

Values for a are mean ± SEM of at least three independent experiments in duplicate.

b values are mean ± SEM of two independent experiments in duplicate.

The pre-incubation of the antagonist and test compound was 1 hour.

Since the starting compound 1 has 4-methylthio at the 4-benzyl group on the proline scaffold, after substituting the various substituents exploring the carboxamide region with 4- (4-methoxybenzyl) of the proline scaffold, several more potent analogs were selected from table 1 and the effect of the 4-methylthio substitution was examined. As shown in table 2, the re-synthesized 43(NPFF 1Ke ═ 1,080nM, NPFF2 Ke 5,600nM) had potency against both receptors comparable to 1(NPFF 1Ke ═ 1,620nM, NPFF2 Ke ═ 7,250nM) from the screening library. The two (methylthio) benzyl analogs with an alkyl group at the carboxamide are more potent NPFF1 antagonists than their methoxy counterparts (NPFF 1: 43Ke ═ 1,080nM versus 11Ke >10,000 nM; 44Ke ═ 360nM versus 16Ke ═ 720 nM). Similarly, phenethyl (45) and 3, 4-difluorophenethyl (48) analogs demonstrated slightly better NPFF1 activity in the thioether series than their methoxy counterparts (45 Ke-530 nM versus 24 Ke-850 nM; 48 Ke-470 nM versus 34 Ke-610 nM). In another aspect, the 4-methoxy (46) and 4-nitro (47) analogs are slightly less potent than their methoxy equivalents (46 Ke-870 nM versus 26 Ke-670 nM, 47 Ke-370 nM versus 33 Ke-250 nM). Against the NPFF2 receptor, except 46 and 47, this series appeared to be more active and therefore less selective for the NPFF1 receptor than the 4- (4-methoxybenzyl) amino analogue.

Overall, these results highlight the importance of substituent size and preference for lipophilicity as well as some flexibility at the binding pocket. The most potent NPFF1 ligand is 4-nitro (33), with NPFF1Ke being 250 nM. Several ligands with moderate activity against NPFF1, no NPFF2 activity/weak NPFF2 activity were also identified. Throughout the course of these studies, most of the compounds we tested showed competitive antagonism in the curve-shift assay; however, some compounds (23, 32, 33, 41, 42, 46) shifted right through the curve and also suppressed the maximum NPFF signal to show evidence of insurmountable antagonism. Although allosteric modulators usually produce such a response, antagonism of this type can also be observed in the case of competitive orthosteric antagonists with slow off-rates. Such antagonists have been used previously and show that by performing a curve-shift assay with a longer antagonist-receptor incubation period, the system reaches equilibrium and thus the compound produces a typical competitive antagonist profile. Indeed, when longer incubation periods were applied to NPFF assays, the compounds showed a typical competitive antagonist activity profile.

In another embodiment, NPFF modulators may be represented by formula III:

Wherein R3 is selected from the group consisting of C3-C9 alkyl, aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocycloalkyl, and arylalkyl; and R4 is selected from H and C1-C2 alkyl.

In certain embodiments of formula III, R3 is a substituted benzyl or substituted phenethyl group having multiple substituents. For example, substituents may be halogen, methoxy, methyl, cyano, N-CH2, and others.

In certain embodiments of formula III, R3 is benzyl or substituted benzyl, phenethyl or substituted phenethyl, and R4 is H. In some embodiments wherein R3 is substituted benzyl, the benzyl is substituted with methoxy.

In certain other embodiments of formula III, R3 is benzyl or substituted benzyl, and R4 is methyl.

In other embodiments of formula III, R3 is C3-C6 alkyl.

table 3 lists the SAR determined as discussed above for analogs of compound 1 substituted in region 2, including embodiments corresponding to formula III.

TABLE 3

The a values are the mean ± SEM of at least three independent experiments performed in duplicate.

b pre-incubation of antagonist and test compound was 1 hour.

To explore the SAR of region 2, 4-nitrophenylethylamino was chosen as the best substituent at R1, and various substituents were introduced at the amine center at position 4 of the proline scaffold (table 3). Similar trends in substituent size in the aliphatic series were observed as discussed above (52-55). NPFF1 activity against both receptors became more potent as the side chain length increased from n-propyl to n-hexyl. With side chains reaching 5 to 6 carbons, activity appears to reach a plateau. Benzyl (56) and phenethyl (58) have similar activity against both receptors. N-methylation of the amino group leading to the tertiary amine analogue (57) slightly suppressed activity against both NPFF subtypes. This trend is believed to mean that the bonded bag also has limited space similar to zone 1.

The effect of substituents at different positions on the benzyl ring was also probed (33, 59-64). Conversion of 4-OMe to position 3 or addition of another OMe at position 3 on the phenyl group reduced activity on both receptor subtypes. Electronic effects do not appear to be a determinant of ring substitution. Replacement of the 4-OMe with another electron donating group, 4-OH, results in loss of activity. The electron-withdrawing trifluoromethyl group is not active, while the other two halogen analogs have good activity against NPFF 1. Notably, compound 64 has over 10-fold selectivity for the NPFF1 receptor.

In another embodiment, the NPFF modulator may be represented by formula IV:

wherein R5 is selected from the group consisting of C3-C9 alkyl, heteroarylalkyl, heteroaryl, heterocycloalkyl, heterocycle, cycloalkylalkyl, and arylalkyl; and X is S, SO2, O, NH, or CH 2.

In certain embodiments of formula IV, R5 is selected from C3-C9 alkyl, heteroarylalkyl, heterocycloalkyl, cycloalkylalkyl, and arylalkyl, and X is O.

In certain embodiments of formula IV, R5 is selected from C3-C9 alkyl, heteroarylalkyl, heterocycloalkyl, cycloalkylalkyl, and arylalkyl, and X is S.

in certain embodiments of formula IV, R5 is selected from C3-C9 alkyl, heteroarylalkyl, heterocycloalkyl, cycloalkylalkyl, and arylalkyl, and X is NH.

In some embodiments of formula I, R5 is benzyl or substituted benzyl, and X is O. In such embodiments where R5 is substituted benzyl, the substituent may be halogen or methoxy. In further embodiments where R5 is substituted benzyl, the benzyl is mono-substituted, and the substituent may be halogen or methoxy at the 2 or 3 position.

Table 4 lists the SAR determined as discussed above for analogs of compound 1 substituted in region 3 of compound 1, including embodiments corresponding to formula IV.

TABLE 4

Values for a are mean ± SEM of at least three independent experiments in duplicate.

During synthesis, the nitro group is readily reduced under conditions used to remove the proline nitrogen and the protecting group for the amino group at the 4-position. To simplify the synthetic effort to prepare a series of analogs in region 3, analogs having a phenethyl group in region 1 were explored instead of 4-nitrophenylethyl, and a 4-methoxybenzyl group was used in region 2.

As shown in table 4, R5 can be an acyclic substituent (i.e., n-hexyl, 74), a cyclic alkyl substituent (i.e., cyclohexylmethyl, 75), or a benzyl substituent (24, 76-88). The position of the substituents has a strong influence on the activity. In substituted benzyl analogs, substitution at the 2 and 3 positions is preferred over the 4 position. Of the three chloro-substituted isomers, the substitution at position 3 is the most potent analog (77, Ke 580nM), with moderate subtype selectivity (>10 fold) compared to the NPFF2 receptor. In another aspect, the methoxy (79) group and the methyl (82) group appear to be preferred at the 2-position. Similarly, other substituents such as 4-hydroxy (84) group, 4-dimethylamino (85) group, and 4-cyano (86) group attenuate NPFF activity. Bulky substituents such as 2-naphthylmethyl (87) or 3, 4-methylenedioxybenzyl (88) are also not advantageous.

Several compounds were further characterized in radioligand binding assays and cAMP assays (table 5). In general, the data obtained from all three assays were compared to each other adequately. Compounds that are effective antagonists in calcium mobilization assays are also effective antagonists in assays measuring native G protein-coupled secondary cAMP. Furthermore, the binding assay showed that all compounds bound to NPFF receptors, and that the effective compounds in the functional assay of NPFF1 (e.g., compound 33 and compound 34) had effective binding affinities.

Table 5 results of cAMP assay, radioligand binding assay and calcium mobilization assay for representative compounds.

The a values are mean ± SEM of at least two independent experiments in duplicate.

b values are mean ± SEM of at least three independent experiments in duplicate.

Pre-incubation of antagonist and receptor was 1 hour.

d is not determined

In Drug-Like Properties: Concepts,Structure Design and Methods from ADME to Toxicity Optimization, Concepts,Structure Design and Methods from ADME to Toxicity Optimization,One of the major challenges of CNS drugs is their ability to cross the Blood Brain Barrier (BBB) and reach the CNS. For most drugs, blood-brain barrier permeability is affected by two factors: the ability to passively penetrate through the BBB and avoid efflux via transport proteins such as P-glycoprotein. Thus, representative compound 33 was evaluated, which had good antagonist activity in a bidirectional transport assay using MDCK-MDR1 cells stably transfected with human MDR1 cDNA, such that they expressed higher levels of P-glycoprotein (Pgp) than the wild-type. Compound 33 crossed the cellular barrier from apical (A) to basolateral (B) at a rate of 2.7X 10-6cm/s and in the reverse direction B to A at a rate of 3.6X 10-6cm/s, demonstrating moderate BBB permeability (in the range of 3X 10-6 cm/s-6X 10-6 cm/s). The 33 efflux ratio (PB → a/PA → B) is 1.3, indicating that the compound is not a Pgp substrate. (Di et al, In Drug-Like Properties: Concepts, Structure Design and Methods from ADME to sensitivity Optimization, Academic Press, pp.141-159 (2016)). In addition, the compounds also have a solubility of 45.9. + -. 7.7. mu.M (mean. + -.% CV) which falls within the range of 10. mu.g/ml to 60. mu.g/ml for compounds with moderate to good solubility, In Drug-Like Properties: Concepts, Structure Design and Methods from ADME to sensitivity Optimization, Academic Press, pp.56-85 (2016), according to Di et al.

The activities shown indicate that the proline-based neuropeptide FF receptor modulators are useful as NPFF antagonists.

the terms are used within their accepted meaning. The following definitions are intended to illustrate, but not to limit, the defined terms.

as used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, identification of carbon number ranges, for example, in C1-C12 alkyl, is intended to include each of the component carbon number moieties within such ranges, such that each intervening carbon number within the recited range and any other recited carbon number value or intervening carbon number value is encompassed, it is also understood that sub-ranges of carbon numbers within the specified carbon number ranges may independently be included within the smaller carbon number ranges within the scope of the present invention, and carbon number ranges specifically excluding one or more carbon numbers are included in the invention, and sub-ranges excluding either or both of the carbon number limits of the specified ranges are also included in the present disclosure.

thus, for example, C3-C9 alkyl is intended to include propyl, butyl, pentyl, hexyl, heptyl, octyl and nonyl, including such types of straight and branched chain groups as isopropyl and tert-butyl. Thus, it is understood that the identification of carbon number ranges as broadly applicable to substituent moieties, such as C1-C12 or C1-C6, enables the carbon number range to be further limited, in particular embodiments of the present disclosure, as a subset of moieties having carbon number ranges within the broader specification of substituent moieties. For example, in particular embodiments of the present disclosure, a carbon number range, e.g., C1-C12 alkyl, may be more restrictively designated to encompass sub-ranges, such as C1-C4 alkyl, C2-C8 alkyl, C2-C4 alkyl, C3-C5 alkyl, or any other sub-range within a broad carbon number range. Thus, for example, the range C1-C6 would include sub-ranges within a broader range such as C1-C3, C1-C4, C2-C6, C4-C6, and the like and may be further limited by the provision of sub-ranges within a broader range such as C1-C3, C1-C4, C2-C6, C4-C6, and the like.

The term "lower alkyl" includes any of C1 alkyl, C2 alkyl, or C3 alkyl.

When the term "alkyl" is used in conjunction with a second group as a suffix, as in "arylalkyl", "aminoalkyl", "cycloalkylalkyl", or "heterocycloalkyl", then the second group is attached to the remainder of the molecule via the alkyl group. For example, when "arylalkyl", "aminoalkyl", "cycloalkylalkyl", or "heterocycloalkyl" or the like is used, the alkyl group may be any of C1 alkyl, C2 alkyl, C3 alkyl, or C4 alkyl.

"cycloalkyl" refers to an optionally substituted non-aromatic cyclic hydrocarbon ring. Unless otherwise indicated, cycloalkyl groups include three to eight carbon atoms. Exemplary "cycloalkyl" groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Cycloalkyl groups may be substituted or unsubstituted, for example by halogen or C1-C3 alkyl.

"heterocycle" refers to a saturated cyclic group containing one or more heteroatoms (e.g., O, N, S) as part of a ring structure and having two to seven carbon atoms in the ring. In embodiments, the heterocyclic ring may be fused with an aryl group such as phenyl. The heterocyclic ring may be substituted or unsubstituted, for example, by halogen, amino, cyano, nitro, carbonyl, amido, acetyl, carboxymethyl, C1-C3 alkyl or C1-C3 alkoxy.

"heteroaryl" refers to an unsaturated aromatic cyclic group containing one or more heteroatoms (e.g., O, N, S) as part of the ring structure and having two to seven carbon atoms in the ring. Heteroaryl groups may include furyl, thienyl, pyridyl, pyrrolyl (pyrrolyl), pyrrolo (pyrrolo), pyrimidinyl, pyrazinyl, imidazolyl, and the like. Heteroaryl groups may be substituted or unsubstituted, for example, by halogen, amino, cyano, nitro, carbonyl, amido, acetyl, carboxymethyl, C1-C3 alkyl or C1-C3 alkoxy.

Furthermore, the term "heteroaryl" is used to include fused bicyclic ring systems containing one or more heteroatoms. Examples of such heteroaryl groups include benzodioxole, benzisoxazolyl, benzofuranyl.

As used herein, "aryl" includes hydrocarbons derived from benzene or benzene derivatives which are unsaturated aromatic carbocyclic groups of from 6 to 10 carbon atoms. The aryl group may have a single ring or multiple rings. Examples include phenyl, benzyl or naphthyl. The aryl group may be substituted or unsubstituted, for example, by halogen, amino, cyano, nitro, carbonyl, amido, acetyl, carboxymethyl, C1-C3 alkyl or C1-C3 alkoxy.

"arylalkyl" includes, for example, benzyl and phenethyl.

"alkylcycloalkyl" includes, for example, methylcyclohexyl groups.

The compounds of the present disclosure may be further specified in specific embodiments by conditions or limitations that exclude specific substituents, groups, moieties, or structures, with reference to the various descriptions and examples thereof set forth herein. Thus, the present disclosure contemplates restrictively defined compositions, such as compositions wherein R is C1-C12 alkyl, with the proviso that when R1 is a specified molecular component and i is a specified carbon number, R ≠ Ci alkyl. The substituents may be selected in any manner and in combination with each other to give a compound according to formula I, formula II, formula IIA, formula III or formula IV.

Certain of the compounds disclosed herein may exist as stereoisomers, including optical isomers. The scope of the present disclosure includes all stereoisomers as racemic mixtures of such stereoisomers as well as individual enantiomers which may be separated according to methods well known to those of ordinary skill in the art. When a chiral center is present, the stereochemistry of the structure includes both the R and S configurations, unless otherwise indicated.

The present disclosure as variously set forth herein with respect to various described features, aspects, and embodiments, can be configured in particular embodiments to include, consist of, or consist essentially of: some or all of such features, aspects, and embodiments, as well as elements and components thereof, which are aggregated, constitute various additional embodiments of the present disclosure. The present disclosure contemplates such features, aspects, and embodiments in various permutations (permutations) and combinations within the scope of the disclosure. Accordingly, the present disclosure may be specified as comprising, consisting of, or consisting essentially of: any such combinations and permutations of these specific features, aspects and embodiments, or selected ones or more thereof.

The term "effective amount" means an amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. The term "therapeutically effective amount" means any amount that results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or disorder, as compared to a corresponding subject not receiving such an amount. The term also includes within its scope an amount effective to enhance normal physiological function.

For example, NPFF receptor modulators may be useful in pain management to reduce opioid tolerance and hyperalgesia, addictive disorders, anti-inflammation, feeding, blood pressure, insulin release, antipyretics, reduce anxiety, reduce borderline seizure activity, reduce opioid-induced hypothermia, and cardiovascular regulation.

in one aspect, NPFF receptor modulators may be used in combination with other drugs or agents, or in combination with various psychotherapies that may be used to treat conditions and types of disorders modulated by NPFF receptors. Drugs or agents that may be used with NPFF receptor modulators and compositions containing NPFF receptor modulators may include typical and/or atypical antipsychotics, such as haloperidol (haloperidol) and aripiprazole (aripiprazole) or monoamine reuptake inhibitors such as fluoxetine and sertraline.

In view of the opioid modulating properties of the NPFF system, other combination therapy applications of the NPFF receptor modulators and compositions of the present disclosure include their concurrent administration with opioids, such as fentanyl, morphine, oxycodone, hydrocodone, buprenorphine, and the like, to combat the hyperalgesic and tolerogenic effects of opioids.

In another aspect of the disclosure, there is provided a method for treating a subject suffering from or susceptible to a condition or disorder in which modulation of neuropeptide FF receptor activity is of therapeutic benefit, comprising administering to the subject suffering from or susceptible to the condition or disorder an effective amount of a neuropeptide FF modulator according to formula I.

Wherein R2 is selected from-N- (C2-C5 alkyl) 2 and NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; r3 is selected from the group consisting of C3-C9 alkyl, aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocycloalkyl, and arylalkyl; r4 is selected from H and C1-C2 alkyl; and R5 is selected from C3-C9 alkyl, heteroarylalkyl, heteroaryl, heterocycloalkyl, heterocycle, cycloalkylalkyl, and arylalkyl; or a pharmaceutically acceptable salt, amide, ester or prodrug thereof.

in embodiments of formula I, wherein R1 is selected from heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; r3 is selected from heteroarylalkyl, heterocycloalkyl, and arylalkyl; and/or R5 is selected from heteroarylalkyl, heterocycloalkyl, cycloalkylalkyl, and arylalkyl, the alkyl group being C1, C2, or C3. Suitable examples of such groups are shown in the tables above.

In certain embodiments of formula I, R2 is NH-R1. In further embodiments of formula I, when R2 is NH-R1, R1 is C3-C6 alkyl.

in some other embodiments of formula I, when R2 is NH-R1, R1 is benzyl or phenethyl, which is substituted or unsubstituted. In certain embodiments wherein R1 is substituted phenethyl, phenethyl is substituted with lower alkoxy such as methoxy, nitro, lower alkyl, halogen or halogenated lower alkyl such as CF 3. In certain embodiments, the phenethyl group is mono-substituted or di-substituted.

Embodiments of R2 as described herein may be combined with any combination of embodiments of R3, R4, and R5 described herein.

In certain embodiments of formula I, R3 is a substituted benzyl or substituted phenethyl group having multiple substituents. For example, substituents may be halogen, methoxy, methyl, cyano, N-CH2, and others.

In certain embodiments of formula I, R3 is benzyl or substituted benzyl, and R4 is H. In such embodiments where R3 is substituted benzyl, the benzyl is mono-or di-substituted with methoxy.

In certain other embodiments of formula I, R3 is benzyl or substituted benzyl, and R4 is methyl.

In embodiments of formula I wherein R3 is a mono-substituted benzyl or a mono-substituted phenethyl, the substituent is in the 4 position.

In some embodiments of formula I, R5 is benzyl or substituted benzyl. In such embodiments where R5 is substituted benzyl, the substituent may be halogen or methoxy. In further embodiments where R5 is substituted benzyl, the benzyl is mono-substituted, and the substituent may be ortho-substituted halo or methoxy.

In one embodiment, the proline-based NPFF modulator may be represented by formula II:

Wherein R2 is selected from-N- (C2-C5 alkyl) 2 and NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; and X is S, SO2, O, NH, or CH 2.

According to some embodiments of formula II, R2 is NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; and X is S, SO2, O, NH, or CH 2. This embodiment is represented by formula IIA:

In certain embodiments of formula IIA, R1 is C3-C6 alkyl; and X is oxygen. In other embodiments of formula IIA, R1 is benzyl or phenethyl, which is substituted or unsubstituted, and X is oxygen. In certain embodiments wherein R1 is substituted phenethyl, phenethyl is substituted with lower alkoxy such as methoxy, nitro, lower alkyl, halogen or halogenated lower alkyl such as CF 3.

In other embodiments of the method for treating a subject, the NPFF receptor modulator may be according to any embodiment disclosed herein for formula I, formula II, or formula IIA.

In certain embodiments of the methods for treating a subject, the NPFF receptor modulator may be according to any of formula III or formula IV as described herein.

In certain embodiments of the methods for treating a subject, the modulation of neuropeptide FF receptor activity is antagonistic activity.

In certain embodiments, methods are provided for treating a subject having a condition or disorder in which modulation of neuropeptide FF receptor activity is therapeutically beneficial, comprising administering to the subject having or susceptible to the condition or disorder an effective amount of a compound according to one of formula I, formula II, formula IIA, formula III, or formula IV that exhibits selective binding to neuropeptide FF receptors and functional antagonist activity against neuropeptide FF receptors.

The condition or disorder to be treated may involve pain management, addictive disorders, anti-inflammatory, eating, blood pressure, insulin release, fever abatement, anxiety reduction, borderline seizure activity reduction, opioid-induced hypothermia, cardiovascular regulation, hyperalgesia and/or opioid tolerance reduction, or other conditions or disorders modulated by NPFF receptors.

In embodiments, the administered NPFF receptor modulator or antagonist is a pharmaceutically acceptable salt, amide, ester, or prodrug of any of the modulators of formula I, formula II, formula IIA, formula III, or formula IV described previously. In this aspect, any compound of formula I, formula II, formula IIA, formula III, or formula IV can be combined with a pharmaceutically acceptable carrier.

Salts of the compounds of the present disclosure may be prepared by methods known to those skilled in the art. The acid may be an inorganic acid or an organic acid. Suitable acids include, for example, hydrochloric acid, hydroiodic acid, hydrobromic acid, sulfuric acid, phosphoric acid, citric acid, acetic acid, and formic acid.

A variety of administration techniques may be used, including oral, transdermal or parenteral techniques, such as subcutaneous, intravenous, intraperitoneal, intracerebral and intraventricular injections, catheterization and the like. Such methods of administration are well known to those skilled in the art. For a general discussion of drug delivery systems and modes of administration, see Kirk-Othmer Encyclopedia of Chemical Technology, fourth edition, volume 8, pages 445-475.

The average amount of compound may vary depending on the binding properties of the compound (i.e. affinity, onset and duration of binding) and in particular should be based on recommendations and prescriptions of qualified physicians.

Therapeutic compositions useful in practicing the methods of treatment of the present disclosure may comprise, in admixture, a pharmaceutically acceptable excipient (carrier) and, as an active ingredient, one or more of the NPFF receptor modulators as described herein.

NPFF receptor ligands can be administered by a variety of methods. Thus, those products of the invention which are active by the oral route may be administered in the form of solutions, suspensions, emulsions, tablets (including sublingual and intraoral tablets), soft gelatin capsules (including solutions used in soft gelatin capsules), aqueous or oily suspensions, emulsions, pills, lozenges (lozenge), troches (troche), tablets, syrups or elixirs and the like. NPFF receptor modulators active upon parenteral administration may be administered by depot injection, implants including silastic and biodegradable implants, skin patches, skin creams or intramuscular injection and intravenous injection.

Compositions comprising NPFF receptor modulators may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets are acceptable. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil (arachis oil), liquid paraffin or olive oil.

Aqueous suspensions of the present disclosure contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl ethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; and dispersing or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame or saccharin. As is known in the art, ophthalmic formulations will be adjusted with respect to osmotic pressure.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil (arachis oil), olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules of the present disclosure suitable for preparation of an aqueous suspension by the addition of water may be formulated from the active ingredient in admixture with a dispersing, suspending and/or wetting agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical composition may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragacanth, naturally-occurring phosphatides such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent (demulcent), a preservative, a flavouring or a colouring agent.

The pharmaceutical compositions may be in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution of 1, 3-butanediol. Acceptable vehicles (vehicles) and solvents that may be employed are water and Ringer's solution, isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Sterilization may be performed by conventional methods known to those of ordinary skill in the art, such as, for example, by sterile filtration or irradiation.

aqueous formulations (i.e., oil-in-water emulsions, syrups, elixirs and injectable formulations) can be formulated at a pH that achieves optimum stability. Determination of the optimum pH can be carried out by conventional methods known to those of ordinary skill in the art. Suitable buffers may also be used to maintain the pH of the formulation.

NPFF receptor modulators may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Non-limiting examples of such materials are cocoa butter and polyethylene glycols.

They may also be administered by intranasal, intraocular, intravaginal and intrarectal routes, including suppository, insufflation, powder and aerosol formulations.

The products disclosed herein, preferably administered by the topical route, may be administered as a stick, solution, suspension, emulsion, gel, cream, ointment, paste, jelly, paint, powder and aerosol.

If desired, the compositions may be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. Pharmaceutical compositions comprising NPFF modulators as described herein and formulated in compatible pharmaceutical carriers can also be prepared, placed in a suitable container, and labeled for treatment of an indicated condition.

Advantages and features of the present disclosure are further illustrated by reference to the following examples, which are not to be construed as limiting the scope of the present disclosure in any way, but rather as illustrations of embodiments of the present disclosure.

Example (b):

And (5) carrying out a chemical process. Compounds 10-48 were synthesized following the procedure depicted in scheme 1. trans-4-hydroxy-L-proline methyl ester (2) undergoes reductive amination by sodium triacetoxyborohydride in 1, 2-dichloroethane to give intermediate 3, which intermediate 3 is then converted to the tosylated derivative 4 in the presence of pyridine in dichloromethane. Substitution with SN2 of sodium azide provided azide 5, which azide 5 subsequently underwent Staudinger reduction with triphenylphosphine to give amine 6. The second reductive amination yields 7a and 7b, respectively. This three-step conversion of tosylate 4 to amine 7a-7b gives higher yields than direct displacement with 4-methoxybenzylamine or 4- (methylthio) benzylamine. The free amino group is then protected with a Boc group and the resulting intermediate 8a-8b is subsequently hydrolyzed under basic conditions to give the acid 9a-9 b. HBTU-assisted amide coupling between 9a-9b and the corresponding amine followed by cleavage of the Boc protecting group provided the final product (10-48). All target compounds were characterized by 1H and 13C NMR, MS and HPLC.

Scheme 1. Synthesis of Compounds 10-48.

The reagents and conditions of scheme 1 are as follows: (a) 2-chlorobenzaldehyde, Na (OAc)3BH, 1,2-DCE (1, 2-dichloroethane), rt, 24H (b) TsCl, pyridine, DCM (dichloromethane), rt, 24H (c) NaN3, DMF, 70 ℃, 16H (d) PPh3, THF, H2O, reflux, 16H (e)4-MeOPhCHO or 4-mesph, Na (OAc)3BH, 1,2-DCE, rt, 24H (f) 2O, aqueous Na2CO3, 1, 4-dioxane, rt, 16H (g) aqueous LiOH, Boc, rt, 16H (H) corresponding amine, HBTU, DIEA, DMF, rt, 24H (i) TFA, DCM, rt, 1H.

Compounds 52-64 were obtained via a slightly different route (scheme 2). Ester 3 is hydrolyzed to give acid 49. Amide coupling of HBTU-assisted 49 with 4-nitrophenylethylamine yielded intermediate 50. Conversion of 50 to tosylated derivative 51 then underwent displacement with SN2 of the corresponding amine to give final product 52-64. The low yield of the SN2 substitution improves upon the addition of more amine equivalents. However, the excess of unreacted amine complicates the purification of the final product.

Scheme 2. Synthesis of Compounds 52-64.

The reagents and conditions of scheme 2 are as follows: (a) aqueous LiOH, MeOH, RT, 16h (b)4-NO2CH2CH2PhNH2, HBTU, DIEA, DMF, RT, 24h (c) TsCl, pyridine, DCM, RT, 24h (d) the corresponding amine, Et3N, THF, reflux, 24 h.

Scheme 3 illustrates the synthesis of compounds 74-88. trans-4-hydroxy-L-proline methyl ester (5) was protected by N-Boc as intermediate 65 and converted to 4-tosyl derivative 66, which 4-tosyl derivative 66 underwent displacement with SN2 of sodium azide (67) and Staudinger reduction to afford intermediate 68. Reductive amination with 4-methoxybenzaldehyde by sodium triacetoxyborohydride gave secondary amine 69, which secondary amine 69 was subsequently protected with an N-Troc protecting group as intermediate 70. Hydrolysis of the methyl ester (71) followed by HBTU-assisted amide coupling with phenethylamine yielded intermediate 72. Removal of the N-Boc group by TFA yielded intermediate 73, which intermediate 73 was used to prepare a series of analogs of region 3. These analogs (74-88) were obtained by reductive amination of 73 with the corresponding aldehyde and removal of the N-Troc group by Zn in the presence of acetic acid in methanol at reflux.

Scheme 3 Synthesis of Compounds 74-88

The reagents and conditions of scheme 3 are as follows: (a) boc2O, aqueous NaOH, 1, 4-dioxane, 0 ℃ to room temperature, 16H (b) TsCl, pyridine, DCM, 0 ℃ to reflux, 16H (c) NaN3, DMF, 70 ℃, 16H (d) PPh3, THF, H2O, reflux, 16H (e)4-OMePHCHO, Na (OAc)3BH, 1,2-DCE, room temperature, 24H (f) TrocCl, Et3N, DCM, room temperature, 16H (g) aqueous LiOH, MeOH, room temperature, 3H (PhCH 2CH2NH2, HBTU, DIEA, DCM, room temperature, 24H (i) TFA, DCM, room temperature, 2H (j) the corresponding aldehyde, Na (OAc)3BH, AcOH, 1,2-DCE, room temperature, 48H (k) Zn, AcOH, MeOH, reflux, 1H.

Experimental part

And (5) carrying out a chemical process. All solvents and chemicals were reagent grade. Unless otherwise mentioned, all reagents and solvents were purchased from commercial suppliers and used as received. Flash column chromatography was performed on a Teledyne ISCO CombiFlash Rf system using pre-filled columns. Solvents used included hexane, ethyl acetate (EtOAc), dichloromethane, methanol, and chloroform/methanol/ammonium hydroxide (80:18:2) (CMA-80). Purity and characterization of the compounds was established by a combination of HPLC, TLC, mass spectrometry and NMR analysis. 1H and 13C NMR spectra were recorded on a Bruker Avance DPX-300(300MHz) spectrometer and determined in CDCl3, DMSO-d6 or CD3OD, with Tetramethylsilane (TMS) (0.00ppm) or solvent peak as internal reference. Chemical shifts are reported in ppm relative to a reference signal and coupling constant (J) values are reported in hertz (Hz). Thin Layer Chromatography (TLC) was performed on EMD pre-coated silica gel 60F254 plates and spots were visualized by UV light or iodine staining. Low resolution mass spectra were obtained using a Waters Alliance HT/Micromass ZQ system (ESI). All tested compounds were greater than 95% pure as determined by HPLC using an Agilent Zorbax SB-Phenyl, 2.1mm x 150mm, 5 μm column on an Agilent 1100 system, eluting with a mobile phase (a) H2O containing 0.1% CF3COOH and (B) MeCN at a gradient of flow rate of 1.0 mL/min.

(2S,4R) -1- [ (2-chlorophenyl) methyl ] -4-hydroxypyrrolidine-2-carboxylic acid methyl ester (3). To a solution of methyl trans-4-hydroxy-L-proline (16.5mmol, 3.00g) and 2-chlorobenzaldehyde (21.5mmol, 2.1ml) in 1, 2-dichloroethane (55ml) was added acetic acid (0.9ml) and sodium triacetoxyborohydride (24.8mmol, 5.26 g). The reaction was stirred at rt for 24 h. After quenching with saturated sodium bicarbonate solution, the reaction mixture was extracted three times with dichloromethane. The combined organic layers were washed sequentially with water and brine, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography to give the desired product as a yellow liquid (4.00g, 90%). 1H NMR (300MHz, CDCl3) δ 7.44(dd, J ═ 1.98,7.44Hz,1H),7.34(m,1H),7.20(m,2H),4.43(m,1H),3.92(m,2H),3.63-3.74(m,4H),3.35(dd, J ═ 5.56,10.08Hz,1H),2.52(dd, J ═ 3.58,10.17Hz,1H),2.24(m,1H),2.12(m, 1H). MS (ESI) [ M ] + 270.2.

(2S,4R) -1- [ (2-chlorophenyl) methyl ] -4- [ (4-methylbenzenesulfonyl) oxy ] pyrrolidine-2-carboxylic acid methyl ester (4). To a solution of 13821-125(14.8mmol, 4.00g) in pyridine (11.4ml) and anhydrous dichloromethane (11.4ml) was added dropwise tosyl chloride (17.8mmol, 3.39g) at 0 ℃. The reaction was refluxed for 24 h. After removal of the solvent in vacuo, the residue was dissolved in dichloromethane and washed with saturated copper sulfate, water and brine. The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, ethyl acetate/hexane) to provide the desired product as a colorless liquid (3.77g, 60%). 1H NMR (300MHz, CDCl3) δ 7.74-7.79(m,2H),7.37-7.46(m,1H),7.28-7.35(m,3H),7.17-7.23(m,2H),5.01(d, J ═ 5.46Hz,1H),3.74-4.04(m,2H),3.69(s,1H),3.66(s,3H),3.29(dd, J ═ 6.03,11.11Hz,1H),2.67-2.73(m,1H),2.44(s,3H),2.28(dd, J ═ 5.46,7.54Hz, 2H). MS (ESI) [ M ] + 424.2.

(2S,4S) -4-azido-1- [ (2-chlorophenyl) methyl ] pyrrolidine-2-carboxylic acid methyl ester (5). To a solution of 4(6.87mmol, 2.91g) in DMF (40ml) was added sodium azide (13.74mmol, 0.89 g). After stirring at 65 ℃ for 16h, the reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, hexane/ethyl acetate) to give the desired product as a yellow liquid (1.47g, 73%). 1H NMR (300MHz, CDCl3) δ 7.54(dd, J ═ 1.70,7.54Hz,1H),7.34(dd, J ═ 1.51,7.72Hz,1H),7.16-7.29(M,2H),4.02-4.09(M,1H),3.90-3.98(M,1H),3.81-3.87(M,1H),3.72(s,3H),3.45(dd, J ═ 6.03,9.23, 1H),3.13(dd, J ═ 1.51,10.36Hz,1H),2.71(dd, J ═ 5.75,10.27Hz,1H),2.54(dd, J ═ 7.72,9.23,14.13, 1H),2.12 (dd, J ═ 5.75,10.27Hz,1H),2.54 (esi ═ 7.72,9.23,14.13, 1H),2.12 ═ 2.21.84 (J ═ 3.84, 10.84 Hz, 10.14M ++ 1H.

(2S,4S) -4-amino-1- [ (2-chlorophenyl) methyl ] pyrrolidine-2-carboxylic acid methyl ester (6). To a solution of azide 5(4.3mmol, 1.27g) in THF (19ml) under nitrogen was added PPh3(8.6mmol, 2.26g) and water (0.2 ml). The reaction mixture was refluxed for 6h with stirring. After removal of the solvent, the residue was dissolved in ether, treated with 0.1N HCl for 5min, and then extracted twice with ether. The aqueous layer was then treated with 1N NaOH until pH 10 and then extracted with dichloromethane. The combined dichloromethane fractions were dried over anhydrous MgSO4 and concentrated in vacuo to provide the desired product as a yellow liquid (1g, 86%). 1H NMR (300MHz, CDCl3) δ 7.50(dd, J ═ 1.9,7.54Hz,1H),7.33(m,1H),7.16-7.20(m,2H),3.95(d, J ═ 14.7Hz,1H),3.81(d, J ═ 13.9Hz,1H),3.66(s,3H),3.45(m,1H),3.39(dd, J ═ 5.5,9.6Hz,1H),2.85(m,1H),2.68(dd, J ═ 5.5,9.4Hz,1H),2.48(m,1H),1.79(m,1H),1.74(br, 2H). MS (ESI) [ M + H ] +: 269.3.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxylic acid methyl ester (7 a). To a solution of amine 6(3.72mmol, 1g) in 1, 2-dichloroethane (12.4ml) was added 4-methoxybenzaldehyde (3.72mmol, 0.45ml), sodium triacetoxyborohydride (5.58mmol, 1.18g) and glacial acetic acid (3.72mmol, 0.21 ml). After stirring at room temperature for 16h, the reaction was quenched with saturated sodium bicarbonate and extracted three times with dichloromethane. The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, dichloromethane/methanol) to give the desired product as a colorless liquid (0.68g, 47%). 1H NMR (300MHz, CDCl3) δ 7.49(dd, J ═ 1.79,7.44Hz,1H),7.33(m,1H),7.16-7.25(m,4H),6.84(m,2H),3.95(d, J ═ 14.30Hz,1H),3.78-3.84(m,4H),3.67(d, J ═ 3.20Hz,5H),3.39(dd, J ═ 6.03,9.04Hz,1H),3.28(m,1H),3.03(dd, J ═ 2.54,9.51Hz,1H),2.61(dd, J ═ 6.12,9.51Hz,1H),2.40(m,1H),1.93(m, 1H). MS (ESI) [ M + H ] + 389.4.

Methyl (2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4- (methylhydrothio) phenyl) methyl ] amino } pyrrolidine-2-carboxylate (7b) was synthesized from 6 and 4- (methylthio) benzaldehyde according to the procedure used for the synthesis of 7a as a yellow liquid (45% yield). 1H NMR (300MHz, CDCl3) δ 7.42(br.s.,0H),7.32(d, J ═ 6.78Hz,0H),7.13-7.24(m,6H),3.90(d, J ═ 7.91Hz,1H),3.66(s,3H),3.64(br.s.,1H),3.22-3.30(m,1H),2.87-2.97(m,1H),2.47-2.50(m,2H),2.46(s,3H),2.02-2.12(m, 1H). MS (ESI) [ M + H ] + 405.3.

Methyl (2S,4S) -4- { [ (tert-butoxy) carbonyl ] [ (4-methoxyphenyl) methyl ] amino } -1- [ (2-chlorophenyl) methyl ] pyrrolidine-2-carboxylate (8 a). To a solution of amine 7(1.75mmol, 0.68g) in 1, 4-dioxane (10ml) was added a 10% w/v Na2CO3 solution (2ml) and Boc2O (1.92mmol, 0.42 g). After stirring at room temperature for 16h, 1, 4-dioxane was removed under reduced pressure. The remaining aqueous solution was extracted three times with dichloromethane. The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo, and the crude product was used in the next step without further purification. 1H NMR (300MHz, CDCl3) δ 7.30-7.38(m,2H),7.16-7.22(m,2H),7.06(d, J ═ 8.67Hz,2H),6.80(d, J ═ 8.67Hz,2H),4.47-4.63(m,1H),4.26-4.44(m,2H),3.83(d, J ═ 6.97Hz,2H),3.78(s,3H),3.65(s,3H),3.61(d, J ═ 6.97Hz,1H),3.06(t, J ═ 8.29Hz,1H),2.67(dd, J ═ 7.44,8.95Hz,1H),2.13-2.25(m,2H),1.53(s, 9H). MS (ESI) [ M ] + 489.6.

Methyl (2S,4S) -4- { [ (tert-butoxy) carbonyl ] [ (4- (methylhydrogen-thio) phenyl) methyl ] amino } -1- [ (2-chlorophenyl) methyl ] pyrrolidine-2-carboxylate (8b) was synthesized from 7b as a yellow liquid (quantitative yield) according to the procedure used for the synthesis of 8 a. 1H NMR (300MHz, CDCl3) d 7.34(d, J ═ 6.22Hz,1H),7.26-7.32(m,1H),7.08-7.19(m,4H),7.01-7.06(m,2H),4.79-5.03(m,1H),4.58(s,2H),3.91(d, J ═ 13.75Hz,1H),3.64(s,3H),3.58(d, J ═ 13.94Hz,1H),3.27(t, J ═ 8.29Hz,1H),2.87-2.96(m,1H),2.49-2.60(m,2H),2.45(s,3H),1.87-1.99(m,1H),1.34(s, 9H). MS (ESI) [ M ] + 505.6.

(2S,4S) -4- { [ (tert-butoxy) carbonyl ] [ (4-methoxyphenyl) methyl ] amino } -1- [ (2-chlorophenyl) methyl ] pyrrolidine-2-carboxylic acid (9 a). To a solution of ester 8(1.75mmol, 0.86g) in methanol (16ml) and water (16ml) was added LiOH (8.75mmol, 0.21 g). After stirring at room temperature for 3h, methanol was removed under reduced pressure. The remaining solution was diluted in water and acidified to pH 5 with 1N HCl. The mixture was then extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to provide the desired product as a white solid (0.83g, quantitative yield). 1H NMR (300MHz, CDCl3) δ 7.35(d, J ═ 8.10Hz,2H),7.17-7.24(m,2H),7.02(d, J ═ 8.48Hz,2H),6.77(d, J ═ 8.48Hz,2H),4.26-4.55(m,3H),4.06-4.17(m,1H),3.71-3.82(m,4H),3.40-3.52(m,1H),3.08(d, J ═ 6.40Hz,1H),2.70-2.82(m,1H),2.44-2.58(m,1H),2.07-2.17(m,1H),1.40(s, 9H). MS (ESI) [ M ] +475.7, [ M-H ] -473.8.

(2S,4S) -4- { [ (tert-butoxy) carbonyl ] [ (4- (methylhydrogen thio) phenyl) methyl ] amino } -1- [ (2-chlorophenyl) methyl ] pyrrolidine-2-carboxylic acid (9b) was synthesized from 8b as a white solid (quantitative yield) according to the procedure used for the synthesis of 9 a. 1H NMR (300MHz, CDCl3) d 7.42(br.s.,1H),7.34(d, J ═ 7.72Hz,1H),7.16-7.26(m,2H),7.13(d, J ═ 8.10Hz,2H),7.02(d, J ═ 8.10Hz,2H),4.47-4.61(m,1H),4.31-4.46(m,2H),4.23(d, J ═ 10.93Hz,1H),3.77-3.88(m,1H),3.58(d, J ═ 10.74Hz,1H),3.02-3.13(m,1H),2.81(d, J ═ 10.36Hz,1H),2.52-2.66(m,1H),2.44(s, 3.44, 2H), 2.06(m,1H), 1H, 8H, 1H). MS (ESI) [ M-H ] -489.5.

General procedure a: to a solution of acid 9(0.2mmol, 1 equiv) in DMF (0.6ml, 0.3M) was added HBTU (0.22mmol, 1.1 equiv), the corresponding amine (0.22mmol, 1.1 equiv), DIEA (0.66mmol, 3 equiv). After stirring at room temperature for 16h, the reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic fractions were dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was dissolved in 20% v/v trifluoroacetic acid (1ml) in dichloromethane and stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (SiO2, methanol/dichloromethane) to afford the desired product.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N-methylpyrrolidine-2-carboxamide (10) was prepared as a yellow oil (9%) according to general procedure A. 1H NMR (300MHz, CDCl3) δ 7.43(dd, J ═ 1.79,7.44Hz,1H),7.28-7.34(m,2H),7.12-7.25(m,3H),6.87(s,2H),3.90-3.96(m,2H),3.75(s,3H),3.58-3.69(m,2H),3.28(s,2H),3.09(dd, J ═ 4.14,7.54Hz,1H),2.68(d, J ═ 4.90Hz,3H),2.43-2.59(m,1H),2.03-2.14(m, 2H). 13C NMR (75MHz, CDCl3)

173.3,160.5,134.4,133.9,131.4,131.0,129.6,129.0,127.1,114.6,66.2, 55.6,55.2,54.9,54.8,53.6,42.0,33.5,11.8。MS(ESI)[M]388.3。

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N-ethyl-4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (11) was prepared according to general procedure A as a yellow oil (32%). 1H NMR (300MHz, CDCl3) δ 7.41(br.s.,1H),7.30-7.38(m,2H),7.14-7.25(m,4H),6.84(d, J ═ 8.67Hz,1H),3.83-3.91(m,1H),3.79(s,3H),3.58-3.73(m,3H),3.33(td, J ═ 2.80,5.89Hz,1H),3.19-3.28(m,1H),3.10-3.19(m,2H),3.00(d, J ═ 9.98Hz,1H),2.62(dd, J ═ 5.56,10.08Hz,1H),2.49(d, J ═ 6.40,9.84, 13.70H), 1.70H (d, 1.87, 1H, 1.02, 1H, 3.87 (dd, 1H, 3.40, 9.84, 13.70H), 1H. 13C NMR (75MHz, CDCl3) delta 173.9,158.9,135.7,134.3,131.1,129.7,129.5,128.9,126.9,113.9,67.1,59.5,57.4,56.2,55.3,51.1,37.0,33.7, 14.6. MS (ESI) [ M ] + 402.2.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N-propylpyrrolidine-2-carboxamide (12) was prepared according to general procedure A as a yellow oil (48%). 1H NMR (300MHz, CDCl3) δ 7.82(t, J ═ 5.27Hz,1H),7.29-7.50(m,4H),7.24(d, J ═ 8.67Hz,2H),6.85(d, J ═ 8.67Hz,2H),4.26-4.46(m,3H),4.14(d, J ═ 13.00Hz,2H),3.94-4.03(m,1H),3.78(s,3H),3.46(d, J ═ 5.09Hz,2H),3.07-3.22(m,2H),2.88-3.02(m,1H),2.27-2.40(m,1H),1.42-1.54(m,2H),0.87(t, J ═ 7.44, 3H). 13C NMR (75MHz, CDCl3) delta 174.0,158.9,135.7,134.1,131.0,129.7,129.6,128.8,126.9,113.9,67.1,59.2,57.2,56.1,55.3,51.0,40.7,36.8,22.7, 11.4. MS (ESI) [ M ] + 416.5.

(2S,4S) -N-butyl-1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (13) was prepared according to general procedure A as a yellow oil (12%). 1H NMR (300MHz, CDCl3) δ 7.74(br.s.,1H),7.47(d, J ═ 7.72Hz,1H),7.39-7.45(m,2H),7.34(dd, J ═ 2.07,7.16Hz,1H),7.24(d, J ═ 8.67Hz,2H),6.87(d, J ═ 8.29Hz,2H),4.34-4.56(m,3H),4.19(d, J ═ 13.37Hz,2H),3.99-4.07(m,1H),3.78(s,3H),3.56(d, J ═ 3.20Hz,2H),3.13-3.27(m,2H),2.94-3.08(m,1H),2.33-2.46(m,1H), 1.46 (t, 1H), 1.29H, 1H),7.15 (d, 1H), 1H),7.15 (d, 1H),3.15 (1H, 1H), 3.90H). 13C NMR (75MHz, CDCl3) delta 173.2,159.8,135.1,133.9,130.8,130.6,129.6,128.8,127.0,114.3,66.7,57.2,55.8,55.5,55.2,49.6,39.0,34.8,31.4,20.0, 13.6. MS (ESI) [ M ] + 430.1.

(2S,4S) -N- (but-2-yl) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (14) was prepared according to general procedure A as a colorless oil (74%). 1H NMR (300MHz, CDCl3) δ 7.29-7.39(m,3H),7.16-7.25(m,4H),6.83(d, J ═ 8.67Hz,1H),3.87-3.94(m,1H),3.74-3.84(m,5H),3.61-3.72(m,3H),3.20-3.33(m,2H),2.97(td, J ═ 1.62,10.13Hz,1H),2.60(ddd, J ═ 3.01,5.60,10.03Hz,1H),2.45-2.54(m,1H),1.87-1.96(m,1H),1.76(br.s, 1H),1.29-1.39(m,2H),1.02(d, 6.59, 6H), 0.59H, 0.85H, 0.59H, 74H, 0.85 Hz, 0.5H). 13C NMR (75MHz, CDCl3) delta 173.4,173.4,158.7,158.7,135.9,134.3,134.2,131.9,131.2,130.9,129.8,129.8,129.4,128.9,128.8,126.9,126.9,113.8,113.8,67.3,67.2,59.7,59.6,57.7,57.5,56.2,56.1,55.3,51.3,46.0,45.8,37.4,37.2,29.6,20.1,10.5, 10.4. MS (ESI) [ M ] + 430.4.

(2S,4S) -N-tert-butyl-1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (15) was prepared according to general procedure A as a colorless oil (88%). 1H NMR (300MHz, CDCl3) δ 7.30-7.39(m,2H),7.15-7.25(m,5H),6.82-6.87(m,2H),3.76-3.82(m,5H),3.60-3.75(m,2H),3.28-3.36(m,1H),3.14(dd, J ═ 5.27,9.80Hz,1H),3.06(d, J ═ 10.17Hz,1H),2.64(dd, J ═ 5.65,9.98Hz,1H),2.47(ddd, J ═ 6.40,9.80,13.56Hz,1H),1.89(td, J ═ 3.67,13.56Hz,1H),1.18(s, 9H). 13C NMR (75MHz, CDCl3) delta 173.5,158.8,136.0,134.4,131.0,129.7,129.4,128.9,127.0,113.9,67.8,59.9,57.6,56.2,55.3,51.2,50.2,37.2, 28.5. MS (ESI) [ M ] + 430.0.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N-pentylpyrrolidine-2-carboxamide (16) was prepared according to general procedure A as a yellow oil (25%). 1H NMR (300MHz, CDCl3) δ 7.32-7.43(m,3H),7.17-7.24(m,4H),6.81-6.86(m,2H),3.84-3.91(m,1H),3.78(s,3H),3.64-3.74(m,3H),3.31-3.38(m,1H),3.25(dd, J ═ 5.65,9.80Hz,1H),2.99-3.16(m,3H),2.61(dd, J ═ 5.56,10.27Hz,1H),2.48(dd, J ═ 2.83,6.78Hz,1H),1.92(d, J ═ 18.08Hz,1H),1.31-1.43(m,2H), 1.16-1.84 (t, t ═ 0, 6.84H), 1.97 (t, 3H). 13C NMR (75MHz, CDCl3) delta 173.9,158.9,135.7,134.1,130.9,129.7,129.6,128.8,126.9,113.9,67.1,59.2,57.2,56.1,55.3,51.0,38.9,36.8,29.1,29.1,22.3, 13.9. MS (ESI) [ M ] + 444.4.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (3-methylbutyl) pyrrolidine-2-carboxamide (17) was prepared according to general procedure A as a colorless oil (71%). 1H NMR (300MHz, CDCl3) δ 7.23-7.34(m,3H),7.10-7.18(m,4H),6.76(d, J ═ 8.67Hz,2H),3.77-3.83(m,1H),3.71(s,3H),3.57-3.67(m,3H),3.27(td, J ═ 2.87,5.93Hz,1H),3.17(dd, J ═ 5.46,9.80Hz,1H),3.05(dt, J ═ 7.72,12.90Hz,2H),2.92-2.98(m,1H),2.54(dd, J ═ 5.65,10.17Hz,1H),2.42(d, J ═ 6.59,9.80,13.75, 1H, 1.54 (dd, J ═ 5.65,10.17Hz,1H),2.42(dd, J ═ 6.59,9.80,13.75, 1H),1.7, 1H, 1.7, 13.7, 13.. 13C NMR (75MHz, CDCl3) delta 172.9,157.9,134.7,133.1,130.0,128.7,128.6,127.8,125.9,112.9,66.1,58.2,56.2,55.1,54.2,49.9,37.3,36.2,35.8,24.8, 21.4. MS (ESI) [ M ] + 444.7.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N-hexylpyrrolidine-2-carboxamide (18) was prepared according to general procedure A as a colorless oil (49%). 1H NMR (300MHz, CDCl3) δ 7.45(t, J ═ 5.65Hz,1H),7.31-7.38(m,2H),7.16-7.24(m,4H),6.81-6.85(m,2H),3.84-3.91(m,1H),3.79(s,3H),3.58-3.72(m,3H),3.31(td, J ═ 2.76,5.98Hz,1H),3.25(dd, J ═ 5.56,9.89Hz,1H),3.11(dt, J ═ 7.06,12.86Hz,2H),3.00(d, J ═ 10.17Hz,1H),2.60(dd, J ═ 5.56,10.08, 1H, 2.49(dd, J ═ 6.84, 1.84H), 1.31, 13.85H, 1H, 13.84 (td, 13.6H, 1H), 1H, 1.6, 13.85H, 13.6, 1H, 13.84 (td, 1H). 13C NMR (75MHz, CDCl3) delta 174.0,158.8,135.8,134.2,131.8,131.0,129.7,129.4,128.8,126.9,113.9,67.2,59.6,57.4,56.2,55.2,51.2,38.9,37.2,31.5,29.4,26.6,22.5, 14.0. MS (ESI) [ M ] + 458.0.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N-decyl-4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (19) was prepared according to general procedure A as a colorless oil (58%). 1H NMR (300MHz, CDCl3) δ 7.42(t, J ═ 5.75Hz,1H),7.33-7.37(m,2H),7.16-7.25(m,5H),6.83(d, J ═ 8.67Hz,1H),3.84-3.91(m,1H),3.78(s,3H),3.62-3.75(m,4H),3.33-3.40(m,1H),3.25(dd, J ═ 5.65,9.61Hz,1H),2.90-3.16(m,6H),2.61(dd, J ═ 5.65,10.17Hz,1H),2.49(ddd, J ═ 6.59,9.61,13.75, 1H),1.89-1.97(m, 1.97, 1.42H), 1.42H (m, 3.85H), 3.42H (m, 1H). 13C NMR (75MHz, CDCl3) delta 173.9,159.0,135.7,134.1,130.9,129.7,129.7,128.8,127.0,114.0,67.1,59.1,57.1,56.1,55.3,50.9,39.0,36.7,31.9,29.6,29.5,29.5,29.3,27.0,22.7, 14.1. MS (ESI) [ M ] + 514.8.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- [2- (morpholin-4-yl) ethyl ] pyrrolidine-2-carboxamide (20) was prepared as a colorless oil (18%) according to general procedure A. 1H NMR (300MHz, CDCl3) δ 7.52(t, J ═ 5.27Hz,1H),7.34-7.38(m,2H),7.18-7.25(m,4H),6.84(d, J ═ 8.67Hz,2H),3.84-3.91(m,1H),3.74-3.78(m,4H),3.68-3.73(m,2H),3.59-3.65(m,4H),3.42(t, J ═ 7.72Hz,1H),3.22-3.37(m,4H),2.35-2.45(m,7H),2.13-2.21(m, J ═ 1.00Hz, 2H). 13C NMR (75MHz, CDCl3) delta 173.5,159.2,135.6,134.2,130.9,129.8,129.7,128.9,126.9,114.0,66.8,66.4,57.4,57.3,55.4,55.2,53.4,51.4,36.7,35.3, 30.9. MS (ESI) [ M ] + 487.6.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- (2-cyclohexylethyl) -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (21) was prepared according to general procedure A as a colorless oil (53%). 1H NMR (300MHz, CDCl3) δ 7.32-7.41(m,3H),7.15-7.25(m,4H),6.84(d, J ═ 8.67Hz,2H),3.83-3.90(m,1H),3.79(s,3H),3.59-3.73(m,3H),3.32(td, J ═ 2.80,5.89Hz,1H),3.25(dd, J ═ 5.56,9.70Hz,1H),3.08-3.19(m,2H),3.02(d, J ═ 10.17Hz, ddh), 2.60(d, J ═ 5.65,9.98Hz,1H),2.49(ddd, J ═ 6.50,9.80,13.66, 1H, 1.86(m,1H), 1.70H (m,1H), 1.93-1H, 1.70H, 1H, 6.50 (m,1H), 1H, 0.70H, and 1H). 13C NMR (75MHz, CDCl3) delta 173.9,158.8,135.8,134.1,130.9,129.7,129.5,128.8,126.9,113.9,67.2,59.4,57.3,56.2,55.2,51.1,37.0,36.8,36.7,35.3,33.1,33.1,30.9,26.5, 26.2. MS (ESI) [ M ] + 484.4.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N, N-diethyl-4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (22) was prepared according to general procedure A as a colorless oil (11%). 1H NMR (300MHz, CDCl3) δ 7.78(br.s.,1H),7.46(dd, J ═ 2.26,6.97Hz,1H),7.33-7.38(m,1H),7.16-7.25(m,4H),6.80-6.88(m,2H),3.83-3.91(m,1H),3.79(s,3H),3.64-3.76(m,3H),3.16-3.36(m,4H),3.03(d, J ═ 9.61Hz,1H),2.43-2.61(m,7H),1.79-2.05(m,3H),0.96(t, J ═ 7.06Hz, 6H). 13C NMR (75MHz, CDCl3) delta 174.2,158.8,135.8,134.0,130.8,129.5,129.4,128.6,126.9,113.9,67.2,56.8,56.1,55.3,51.5,51.2,46.6,37.3,36.2, 11.1. MS (ESI) [ M ] + 473.5.

(2S,4S) -N-benzyl-1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (23) was prepared according to general procedure A as a yellow oil (44%). 1H NMR (300MHz, CDCl3) δ 7.82(t, J ═ 5.75Hz,1H),7.22-7.27(m,5H),7.10-7.21(m,6H),6.77-6.83(m,2H),4.32(dd, J ═ 5.93,8.95Hz,2H),3.87(d, J ═ 13.19Hz,1H),3.77(s,3H),3.58-3.71(m,3H),3.34-3.41(m,1H),3.31(dd, J ═ 5.56,9.70Hz,1H),2.98(d, J ═ 10.36Hz,1H),2.60(dd, J ═ 5.46,10.36, 1H),2.49(d, J ═ 6.92, 13.78 Hz,1H), 1.02 (dd, J ═ 5.46,10.36, 1H). 13C NMR (75MHz, CDCl3) delta 173.9,159.1,138.3,135.4,134.1,131.0,129.7,129.7,128.8,128.6,127.8,127.3,126.9,114.0,67.0,58.7,56.9,56.0,55.3,50.7,43.1, 36.3. MS (ESI) [ M ] + 464.4.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (24) was prepared according to general procedure A as a yellow oil (59%). 1H NMR (300MHz, CDCl3) δ 7.52(t, J ═ 5.84Hz,1H),7.32-7.37(m,1H),7.09-7.24(m,11H),6.83(d, J ═ 8.67Hz,1H),3.73-3.81(m,4H),3.63-3.67(m,1H),3.56-3.62(m,2H),3.43-3.55(m,1H),3.30-3.42(m,1H),3.19-3.28(m,2H),2.92(d, J ═ 10.17Hz,1H),2.67-2.76(m,2H),2.42-2.55(m,2H),1.96(br.s.,1H),1.81(m, 1H). 13C NMR (75MHz, CDCl3) delta 174.1,158.7,138.9,135.8,134.1,132.0,130.7,129.6,129.4,128.7,128.6,128.5,126.9,126.4,113.9,67.3,59.5,57.2,56.0,55.3,51.2,39.8,37.3, 35.5. MS (ESI) [ M ] + 478.4.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (3-phenylpropyl) pyrrolidine-2-carboxamide (25) was prepared according to general procedure A as a colorless oil (70%). 1H NMR (300MHz, CDCl3) δ 7.44(t, J ═ 5.37Hz,1H),7.13-7.36(m,10H),7.09(d, J ═ 6.78Hz,1H),6.80(d, J ═ 8.67Hz,2H),3.83-3.90(m,1H),3.75(s,3H),3.66-3.73(m,3H),3.34-3.41(m,1H),3.25(dd, J ═ 5.56,9.70Hz,1H),3.11-3.20(m,2H),3.05(d, J ═ 10.55Hz,1H),2.43-2.64(m,4H),1.96(d, J ═ 3.20Hz,1H), 1.77-1.7 (m, 2H). 13C NMR (75MHz, CDCl3) delta 173.9,159.1,141.5,135.6,134.1,130.9,129.8,129.7,128.9,128.4,128.3,127.0,125.9,114.0,67.0,58.9,56.9,56.0,55.2,50.8,38.6,36.5,33.2, 31.1. MS (ESI) [ M ] + 492.7.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-methoxyphenyl) ethyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (26) was prepared according to general procedure A as a colorless oil (20%). 1H NMR (300MHz, CDCl3) δ 7.40(t, J ═ 5.65Hz,1H),7.32-7.36(m,1H),7.15-7.22(m,4H),7.10-7.14(m,1H),7.05(d, J ═ 8.67Hz,2H),6.85(d, J ═ 8.67Hz,2H),6.77(d, J ═ 8.48Hz,2H),3.78(s,3H),3.75(s,3H),3.70-3.73(m,1H),3.65(d, J ═ 9.04Hz,2H),3.42-3.52(m,1H),3.37(dd, J ═ 5.65,9.61, 1H),3.23-3.32(m, 3.32H), 3.42-3.52(m,1H),3.37(dd, J ═ 5.65,9.61, 1H),3.23-3.32(m, 3.8H), 3.01-2H, 2H), 3.68(m,2H), 3.01-2H, 2H), 3.01(m,2H), 2H), 3.68H, 2H). 13C NMR (75MHz, CDCl3) delta 173.8,158.8,158.2,135.7,134.2,132.1,131.0,130.9,129.6,129.6,129.2,128.8,126.9,114.0,113.9,66.7,60.1,57.8,56.1,55.3,55.2,52.1,40.0,37.9,34.8, 30.9. MS (ESI) [ M ] + 508.6.

(2S,4S) -N- [2- (4-chlorophenyl) ethyl ] -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (27) was prepared according to general procedure A as a colorless oil (55%). 1H NMR (300MHz, CDCl3) δ 7.46(t, J ═ 5.93Hz,1H),7.27-7.31(m,1H),7.08-7.25(m,7H),7.00-7.04(m,2H),6.81-6.86(m,2H),3.75-3.80(m,0H),3.73(s,3H),3.68(d, J ═ 13.19Hz,2H),3.53-3.60(m,1H),3.31-3.50(m,3H),3.18(dd, J ═ 6.78,9.04Hz,1H),3.08(d, J ═ 10.93Hz,1H),2.69(q, J ═ 7.03Hz,2H),2.42-2.55(m,2H), 2.92 (m,1H), 1.06H, 3.06H, 1H, J ═ 7.06. 13C NMR (75MHz, CDCl3) delta 173.1,159.8,137.2,134.9,133.7,132.1,130.6,130.2,130.0,129.6,128.7,128.5,126.9,114.3,67.1,57.3,55.8,55.2,55.2,49.7,39.7,34.9, 34.7. MS (ESI) [ M ] + 512.4.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (3, 4-dimethoxyphenyl) ethyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (28) was prepared as a white solid (61%) according to general procedure A. 1H NMR (300MHz, CDCl3) δ 7.29-7.35(m,1H),7.18-7.25(m,3H),7.09-7.15(m,2H),6.82(d, J ═ 8.67Hz,2H),6.68(s,3H),3.63-3.88(m,13H),3.45-3.60(m,3H),3.28-3.41(m,1H),3.12-3.20(m,2H),2.70(qd, J ═ 7.21,14.93Hz,2H),2.43-2.58(m,2H),1.99(ddd, J ═ 3.49,6.26,13.99Hz, 1H). 13C NMR (75MHz, CDCl3) delta 172.6,160.3,148.9,147.6,134.6,133.6,131.2,131.1,130.4,129.5,128.7,126.9,120.6,114.5,111.8,111.1,66.8,56.0,55.8,55.2,55.0,54.9,48.9,40.1,34.8, 33.8. MS (ESI) [ M ] + 538.4.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- {2- [4- (dimethylamino) phenyl ] ethyl } -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (29) was prepared according to general procedure A as a yellow oil (25%). 1H NMR (300MHz, CDCl3) δ 7.30-7.34(m,1H),7.15-7.25(m,5H),6.98(d, J ═ 8.67Hz,2H),6.84(d, J ═ 8.67Hz,2H),6.59(d, J ═ 8.48Hz,2H),3.62-3.81(m,7H),3.33-3.45(m,3H),3.20(dd, J ═ 5.46,9.42Hz,1H),3.05(d, J ═ 10.36Hz,1H),2.86(s,6H),2.53-2.69(m,3H),2.40-2.51(m,1H),1.91(d, J ═ 13.56Hz, 1H). 13C NMR (75MHz, CDCl3) delta 173.7,159.3,149.4,135.4,133.9,130.8,130.1,129.5,129.2,128.6,126.9,126.5,114.1,112.9,66.8,58.2,56.1,55.8,55.3,50.3,40.7,40.3,35.8, 34.4. MS (ESI) [ M ] + 521.8.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-acetamidophenyl) ethyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (30) was prepared as a colorless liquid (50%) according to general procedure A. 1H NMR (300MHz, CDCl3) δ 8.44(br.s.,1H),7.67(t, J ═ 5.75Hz,1H),7.37(dd, J ═ 1.60,7.44Hz,1H),7.23-7.31(m,6H),7.11-7.21(m,2H),7.03(d, J ═ 8.29Hz,2H),6.86(d, J ═ 8.67Hz,2H),3.84(s,2H),3.76(s,3H),3.62-3.74(m,2H),3.39-3.48(m,2H),3.34(td, J ═ 6.31,12.62Hz,1H),3.11-3.23(m,2H),2.66-2.75(m,2H), 56.56 (m, 56H), 14.02 (d, 2H), 14.07 (d, 2H), 3.19H). 13C NMR (75MHz, CDCl3) delta 173.1,168.9,160.1,136.1,134.5,134.5,133.5,130.9,130.5,129.2,129.0,128.5,126.8,120.6,114.3,65.6,55.0,54.8,54.4,53.5,48.7,39.8,34.4,33.7, 23.9. MS (ESI) [ M ] + 535.6.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- {2- [4- (methylcarbamoyl) phenyl ] ethyl } pyrrolidine-2-carboxamide (31) was prepared according to general procedure A as a colorless oil (28%). 1H NMR (300MHz, CDCl3) d 7.75(t, J ═ 5.84Hz,1H),7.53(d, J ═ 8.10Hz,2H),7.37(dd, J ═ 2.17,6.88Hz,1H),7.22(d, J ═ 8.67Hz,2H),7.09-7.18(m,4H),6.82(d, J ═ 8.48Hz,3H),3.86(d, J ═ 2.45Hz,2H),3.71-3.77(m,3H),3.57-3.68(m,2H),3.33-3.54(m,3H),3.12-3.23(m,2H),3.06(q, J ═ 7.41, 1H),2.89(d, 4.4, 3H),3.12-3.23(m,2H),3.06(q, J ═ 7.41, 1H), 1H, 2.89(d, 4, 3.54(m,3H), 3.13, 13H), 1.55H, 1H, 13-3.55H, 1H, 14H, 1H, 14H, 1H, and 14H. 13C NMR (75MHz, CDCl3) d 173.0,168.3,160.4,142.5,134.7,133.6,132.7,131.3,130.5,129.5,128.8,128.7,127.1,114.5,66.5,55.2,54.9,54.7,53.7,48.9,42.0,39.8,35.0,33.8,26.7, 11.8. MS (ESI) [ M ] + 535.5.

(2S,4S) -N- [2- (4-tert-butylphenyl) ethyl ] -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (32) was prepared according to general procedure A as a colorless oil (36%). 1H NMR (300MHz, CDCl3) δ 7.51(br.s.,1H),7.31-7.36(m,1H),7.14-7.25(m,8H),7.05(d, J ═ 8.29Hz,2H),6.83(d, J ═ 8.67Hz,2H),3.75-3.83(m,5H),3.57-3.67(m,3H),3.40-3.50(m,1H),3.30-3.39(m,1H),3.20-3.26(m,2H),2.92(d, J ═ 10.17Hz,1H),2.67(q, J ═ 7.03Hz,2H),2.41-2.56(m,2H),1.80-1.89(m,1H),1.26 (m,1H), 9H). 13C NMR (75MHz, CDCl3) delta 174.1,158.7,149.2,135.8,135.8,134.1,132.1,130.9,129.6,129.3,128.7,128.3,126.9,125.4,113.8,67.2,59.6,57.2,56.1,55.2,51.2,39.9,37.3,35.1,34.3, 31.3. MS (ESI) [ M ] + 534.3.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-methoxyphenyl) ethyl ] -4- { [ (4-nitrophenyl) methyl ] amino } pyrrolidine-2-carboxamide (33) was prepared according to general procedure A as a yellow oil (22%). 1H NMR (300MHz, CDCl3) δ 8.06(d, J ═ 8.67Hz,2H),7.45-7.51(m,1H),7.31-7.36(m,1H),7.27(s,1H),7.13-7.26(m,6H),6.82-6.89(m,2H),3.79(s,3H),3.62-3.74(m,4H),3.34-3.53(m,3H),3.11-3.22(m,2H),2.83(dt, J ═ 2.92,6.92Hz,2H),2.22-2.33(m,1H),2.08(t, J ═ 7.54Hz,2H),1.46-1.72(m, 2H). 13C NMR (75MHz, CDCl3) delta 174.1,158.9,146.7,146.7,135.5,134.1,130.9,129.7,129.5,129.2,129.0,128.8,127.0,123.7,113.9,66.6,60.1,58.1,55.9,55.3,52.0,39.2,37.8, 35.6. MS (ESI) [ M ] + 523.7.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (3, 4-difluorophenyl) ethyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (34) was prepared as a white solid (62%) according to general procedure A. 1H NMR (300MHz, CDCl3) δ 7.55(t, J ═ 6.03Hz,1H),7.29(d, J ═ 2.26Hz,1H),7.11-7.25(m,5H),6.88-6.96(m,2H),6.75-6.86(m,3H),3.72-3.79(m,4H),3.54-3.71(m,3H),3.30-3.47(m,3H),3.19(dd, J ═ 6.69,9.14Hz,1H),3.07(d, J ═ 10.93Hz,1H),2.67(q, J ═ 6.91Hz,2H),2.43-2.56(m,2H),1.94(dd, J ═ 3.11,6.50, 1H). 13C NMR (75MHz, CDCl3) delta 173.2,159.7,135.8,135.0,133.7,130.5,130.2,129.6,128.7,126.9,124.5,124.5,124.4,124.4,117.5,117.2,117.1,116.9,114.2,67.2,57.6,56.0,55.3,55.2,49.8,39.6,38.6,35.1, 34.5. MS (ESI) [ M ] + 514.5.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-fluorophenyl) ethyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (35) was prepared according to general procedure A as a colorless oil (74%). 1H NMR (300MHz, CDCl3) δ 7.38(t, J ═ 5.84Hz,1H),7.16-7.26(m,4H),7.01-7.12(m,4H),6.77-6.86(m,4H),3.73-3.87(m,2H),3.70(s,3H),3.60-3.67(m,1H),3.47-3.58(m,3H),3.24-3.37(m,1H),3.10-3.21(m,2H),2.63-2.81(m,2H),2.44-2.56(m,2H),1.93-2.04(m, 1H). 13C NMR (75MHz, CDCl3) delta 172.3,160.5,134.4,134.3,134.3,133.5,131.4,130.0,129.9,129.5,128.6,126.9,121.8,115.3,115.0,114.5,67.1,55.8,55.2,54.9,54.6,48.7,40.1,34.4, 33.4. MS (ESI) [ M ] + 496.7.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- {2- [4- (trifluoromethyl) phenyl ] ethyl } pyrrolidine-2-carboxamide (36) was prepared according to general procedure A as a yellow oil (69%). 1H NMR (300MHz, CDCl3) δ 7.54(t, J ═ 5.84Hz,1H),7.43(d, J ═ 8.10Hz,2H),7.32(dd, J ═ 1.88,7.35Hz,1H),7.14-7.24(m,7H),6.81-6.86(m,2H),3.73-3.80(m,4H),3.57-3.70(m,3H),3.38-3.48(m,2H),3.29-3.36(m,1H),3.23(dd, J ═ 5.84,9.80Hz,1H),3.00(s,1H),2.71-2.81(m,2H),2.42-2.58(m,2H),1.80-1.90(m, 1H). 13C NMR (75MHz, CDCl3) delta 174.0,159.0,143.0,135.5,134.0,130.6,129.6,129.0,128.8,126.9,125.4,125.3,114.0,67.1,59.0,57.0,56.0,55.2,50.9,39.5,36.7, 35.4. MS (ESI) [ M ] + 546.7.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- [2- (pyridin-4-yl) ethyl ] pyrrolidine-2-carboxamide (37) was prepared as a white solid (73%) according to general procedure A. 1H NMR (300MHz, CDCl3) δ 8.34(d, J ═ 5.84Hz,2H),7.65(t, J ═ 5.93Hz,1H),7.24-7.33(m,3H),7.20(d, J ═ 8.67Hz,2H),7.10-7.15(m,2H),7.05(d, J ═ 6.03Hz,2H),6.82(d, J ═ 8.67Hz,2H),3.75-3.86(m,2H),3.67(br.s.,5H),3.50(quin, J ═ 6.83Hz,2H),3.35-3.44(m,1H),3.10-3.20(m,2H),2.76 (m,2 dt, 7.06, 2.06H), 2.57, 7.7, 7, 14H), 14.7.7, 14(d, J ═ 8.67Hz, 2H). 13C NMR (75MHz, CDCl3) delta 172.7,160.3,149.4,148.3,134.5,133.6,131.2,130.2,129.6,128.7,126.9,124.2,114.5,67.1,56.2,55.2,54.8,49.1,38.9,34.6, 34.0. MS (ESI) [ M ] + 479.3.

(2S,4S) -N- [2- (4-acetylphenyl) ethyl ] -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (38) was prepared as a colorless liquid (35%) according to general procedure A. 1H NMR (300MHz, CDCl3) δ 7.65-7.72(m,2H),7.35-7.42(m,1H),7.18-7.24(m,3H),7.05-7.16(m,4H),6.83(d, J ═ 8.67Hz,2H),3.78-3.89(m,2H),3.66-3.74(m,4H),3.48-3.61(m,3H),3.37(d, J ═ 5.65Hz,1H),3.18(d, J ═ 8.10Hz,2H),2.74-2.87(m,2H),2.43-2.60(m,2H),2.18(d, J ═ 5.84Hz,3H),1.93-2.06(m, 1H). 13C NMR (75MHz, CDCl3) delta 172.4,160.5,157.6,140.4,136.6,134.4,133.7,131.5,130.5,129.5,128.7,128.6,127.0,126.7,121.7,114.6,66.9,55.5,55.2,54.7,54.2,48.6,40.0,35.1,33.4, 14.8. MS (ESI) [ M ] + 519.5.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-methanesulfonylphenyl) ethyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (39) was prepared according to general procedure A as a colorless oil (55%). 1H NMR (300MHz, CDCl3) δ 7.75(d, J ═ 8.29Hz,2H),7.64(t, J ═ 5.93Hz,1H),7.25-7.35(m,5H),7.16-7.22(m,4H),6.81-6.86(m,2H),3.76(s,3H),3.59-3.74(m,4H),3.42(q, J ═ 6.97Hz,2H),3.32-3.37(m,1H),3.23(dd, J ═ 5.84,9.61Hz,1H),3.01(d, J ═ 1.70Hz,1H),2.98(s,3H),2.79 (dd, J ═ 6.99,14.46, 2H),2.55(dd, J ═ 1.65, 1.55H), 1H), 2.55(dd, 1.81, 1H), 1H), 1.55H, 1H, 2.79 (dd, 1H). 13C NMR (75MHz, CDCl3) delta 174.0,159.2,145.6,138.7,135.4,133.9,130.7,129.9,129.7,129.6,128.9,127.5,127.0,114.1,67.0,58.6,56.7,55.9,55.3,50.7,44.5,39.5,36.3, 35.5. MS (ESI) [ M ] + 557.1.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N, N-diethyl-4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (40) was prepared according to general procedure A as a yellow oil (54%). 1H NMR (300MHz, CDCl3) δ 7.37-7.44(m,3H),7.31-7.35(m,1H),7.18-7.23(m,2H),6.87-6.93(m,2H),4.09(d, J ═ 2.07Hz,2H),3.91-4.00(m,3H),3.82-3.86(m,1H),3.81(s,3H),3.39-3.51(m,2H),3.18-3.29(m,1H),2.88-3.08(m,3H),2.36-2.48(m,1H),2.14(d, J ═ 14.69Hz,1H),1.04(t, J ═ 7.25Hz,3H),0.97(t, J ═ 7.16, 3H). 13C NMR (75MHz, CDCl3) delta 174.0,160.3,135.1,133.6,131.3,130.7,129.5,128.8,127.1,123.1,114.6,59.0,56.7,56.4,55.3,54.3,48.5,42.4,41.6,33.7,15.0, 12.8. MS (ESI) [ M ] + 430.4.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N, N-dipropylpyrrolidine-2-carboxamide (41) was prepared according to general procedure A as a colorless oil (63%). 1H NMR (300MHz, CDCl3) δ 7.55(dd, J ═ 1.70,7.54Hz,1H),7.13-7.34(m,6H),6.85(d, J ═ 8.48Hz,2H),3.73-3.92(m,7H),3.70(dd, J ═ 4.52,8.85Hz,1H),3.44(d, J ═ 5.84Hz,1H),2.99-3.35(m,6H),2.81(dd, J ═ 6.03,9.61Hz,1H),2.40(td, J ═ 8.34,13.28Hz,1H),1.80-1.89(m,1H),1.43-1.57(m,4H),0.86 (J ═ 4.52, J ═ 4.44, 44H). 13C NMR (75MHz, CDCl3) delta 173.4,158.9,136.4,133.6,130.8,129.7,129.2,128.1,126.8,113.9,60.9,58.3,56.3,55.3,53.9,50.7,49.4,48.3,36.3,22.8,20.9,11.4, 11.2. MS (ESI) [ M ] + 458.2.

(3S,5S) -1- [ (2-chlorophenyl) methyl ] -N- [ (4-methoxyphenyl) methyl ] -5- (4-phenylpiperazine-1-carbonyl) pyrrolidin-3-amine (42) was prepared as a colorless oil (23%) according to general procedure a. 1H NMR (300MHz, CDCl3) δ 7.45(dd, J ═ 1.79,7.44Hz,1H),7.27-7.39(m,5H),7.14-7.25(m,2H),6.84-6.95(m,5H),3.90-3.99(m,4H),3.85(dd, J ═ 3.49,9.14Hz,1H),3.80(s,3H),3.77(s,1H),3.54-3.71(m,3H),3.46(br.s.,1H),3.35(d, J ═ 10.55Hz,1H),2.88-3.10(m,5H),2.37-2.49(m,1H),2.06(d, J ═ 14.13, 1H). 13C NMR (75MHz, CDCl3) delta 172.6,159.7,150.7,135.4,133.7,130.9,130.6,129.6,129.3,128.8,127.0,120.7,116.6,114.3,57.5,56.4,55.3,54.6,49.7,49.6,49.3,45.6,42.2, 34.7. MS (ESI) [ M ] + 519.8.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N-ethyl-4- { [ (4- (methylthio) phenyl) methyl ] amino } pyrrolidine-2-carboxamide (43) was prepared according to general procedure A as a colorless oil (52%). 1H NMR (300MHz, CDCl3) d 7.45(br.s.,1H),7.37(dd, J ═ 3.67,5.56Hz,1H),7.29-7.33(m,1H),7.23(dd, J ═ 3.58,5.84Hz,2H),7.16-7.21(m,4H),3.83-3.90(m,1H),3.63-3.73(m,3H),3.29(d, J ═ 5.84Hz,1H),3.24(dd, J ═ 5.46,9.98Hz,1H),3.10-3.19(m,2H),2.98(d, J ═ 9.98Hz,1H),2.61(dd, J ═ 5.56,10.08, 1H), 2.91.49 (m,2H), 3.49H, 3.73 (dd, 3.73H), 3.73H, 3.46, 3.5.56H, 1H),3.73 (dd, 3.73H), 3.73H, 3.9.9.1H, 1H, 3.02 (dd, 3.73H). 13C NMR (75MHz, CDCl3) d173.5,136.6,136.3,135.3,133.8,130.7,129.3,128.5,128.3,126.5, 66.6,59.2,57.1,55.8,50.8,36.7,33.2,15.6, 14.1. MS (ESI) [ M ] + 418.3.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4- (methylthio) phenyl) methyl ] amino } -N-pentylpyrrolidine-2-carboxamide (44) was prepared according to general procedure A as a colorless oil (62%). 1H NMR (300MHz, CDCl3) d 7.47(br.s.,1H),7.31-7.39(m,2H),7.21-7.26(m,2H),7.17-7.20(m,4H),3.84-3.91(m,1H),3.59-3.75(m,3H),3.30(br.s.,1H),3.25(dd, J ═ 5.46,9.80Hz,1H),3.10(dt, J ═ 6.97,12.72Hz,2H),2.99(d, J ═ 10.17Hz,1H),2.60(dd, J ═ 5.46,9.98Hz,1H),2.48-2.54(m,1H),2.46(s,3H),1.91(d, J ═ 36.47, 1H), 1.84 (t, 1H),2.48-2.54(m,1H),2.46(s,3H),1.91(d, J ═ 10, 8H), 1H, 6H, 1. 13C NMR (75MHz, CDCl3) d174.0,137.1,136.7,135.8,134.2,131.0,129.7,128.9,128.7,126.9,67.1,59.6,57.5,56.3,51.3,38.9,37.1,29.2,29.1,22.3,16.1, 13.9. MS (ESI) [ M ] + 460.3.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4- (methylthio) phenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (45) was prepared according to general procedure A as a colorless oil (46%). 1H NMR (300MHz, CDCl3) d 7.40(br.s.,1H),7.33(dd, J ═ 2.92,4.43Hz,1H),7.09-7.24(m,12H),3.60-3.79(m,4H),3.37-3.50(m,2H),3.34(d, J ═ 6.03Hz,1H),3.21(dd, J ═ 5.75,9.51Hz,1H),2.98(d, J ═ 10.36Hz,1H),2.67-2.76(m,2H),2.47-2.58(m,2H),2.45(s,3H),1.86(d, J ═ 12.62Hz, 1H). 13C NMR (75MHz, CDCl3) d 173.8,138.8,137.8,135.5,134.7,134.0,130.7,129.6,129.1,128.7,128.6,128.5,126.9,126.8,126.4,67.0,58.6,56.6,55.9,50.7,39.9,36.4,35.4, 15.9. MS (ESI) [ M ] + 494.7.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4- (methylthio) phenyl) ethyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-2-carboxamide (46) was prepared according to general procedure A as a colorless oil (50%). 1H NMR (300MHz, CDCl3) d 7.47(br.s.,1H),7.32-7.38(m,1H),7.15-7.24(m,7H),7.02(d, J ═ 8.48Hz,2H),6.73(d, J ═ 8.67Hz,2H),3.71-3.80(m,4H),3.59-3.69(m,3H),3.42(dd, J ═ 6.69,13.28Hz,1H),3.32-3.38(m,1H),3.26(d, J ═ 4.14Hz,1H),3.19-3.24(m,1H),2.93(d, J ═ 9.98Hz,1H),2.61-2.71(m,2H),2.51(dd, 5.51, 5H), 2.04, 1H, 2.78 (br.78H, 1H), 2.45H, 1H, 2H, 1H, 2. 13C NMR (75MHz, CDCl3) d174.0,158.2,137.2,136.5,135.7,134.0,130.8,130.7,129.6,129.5,128.8,128.7,126.9,113.9, 67.2,59.3,57.1,56.1,55.2,51.2,40.0,37.1,34.6, 16.0. MS (ESI) [ M ] + 524.5.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4- (methylthio) phenyl) ethyl ] -4- { [ (4-nitrophenyl) methyl ] amino } pyrrolidine-2-carboxamide (47) was prepared as a colorless oil (44%) according to general procedure A. 1H NMR (300MHz, CDCl3) d 7.93(d, J ═ 8.67Hz,2H),7.53(t, J ═ 6.03Hz,1H),7.23-7.28(m,1H),7.06-7.18(m,8H),3.51-3.70(m,4H),3.37(q, J ═ 6.78Hz,2H),3.22(d, J ═ 3.20Hz,1H),3.17(dd, J ═ 5.65,9.80Hz,1H),2.88(d, J ═ 10.17Hz,1H),2.72(qd, J ═ 7.10,14.13Hz,2H),2.40-2.49(m,2H),2.39(s,3H),1.85(br, 1.70H), 1.79(m, 1H). 13C NMR (75MHz, CDCl3) d 173.3,145.7,145.6,136.4,135.4,134.6,132.9,129.4,128.7,128.4,127.8,127.7,125.9,125.8,122.6,66.1,58.4,56.3,55.3,50.3,38.2,36.1,34.5, 14.9. MS (ESI) [ M ] + 539.2.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (3, 4-difluorophenyl) ethyl ] -4- { [ (4- (methylthio) phenyl) methyl ] amino } pyrrolidine-2-carboxamide (48) was prepared as a colorless liquid (56%) according to general procedure A. 1H NMR (300MHz, CDCl3) d 7.58(br.s.,1H),7.34(d, J ═ 7.35Hz,1H),7.15-7.25(m,7H),6.88-7.00(m,2H),6.75-6.83(m,1H),3.70-3.80(m,1H),3.59-3.69(m,3H),3.35-3.45(m,1H),3.33(d, J ═ 6.97Hz,2H),3.24(dd, J ═ 5.65,9.61Hz,1H),2.96(d, J ═ 9.80Hz,2H),2.60-2.70(m,2H),2.54(dd, J ═ 5.56,10.46, 2H),2.41 (d, 4.85H), 49(d, 1H), 1H, 49H, 1H). 13C NMR (75MHz, CDCl3) d 174.1,137.5,135.9,135.6,134.0,130.6,129.7,128.8,128.8,126.9,126.9,124.5,124.5,124.4,124.4,117.5,117.3,117.2,117.0,67.1,59.2,57.1,56.1,51.2,39.6,36.9,34.7, 15.9. MS (ESI) [ M ] + 530.9.

(2S,4R) -1- [ (2-chlorophenyl) methyl ] -4-hydroxypyrrolidine-2-carboxylic acid (49). To a solution of 3(0.87g, 3.22mmol) in methanol (30ml) was added lithium hydroxide (0.386g, 16.12 mmol). After stirring at room temperature for 3h, the reaction mixture was concentrated in vacuo. The residue was dissolved in water and the pH was adjusted to 5 with 3N aqueous HCl. The reaction mixture was extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate and concentrated in vacuo to afford the product as a white solid (0.63g, 76%). 1H NMR (300MHz, CDCl3) d 7.74-7.79(m,2H),7.37-7.46(m,1H),7.28-7.35(m,3H),7.17-7.23(m,2H),5.01(d, J ═ 5.46Hz,1H),3.74-4.04(m,2H),3.69(s,1H),3.66(s,1H),3.29(dd, J ═ 6.03,11.11Hz,1H),3.12-3.17(m,1H),2.67-2.73(m,1H),2.44(s,3H),2.28(dd, J ═ 5.46,7.54Hz, 2H). MS (ESI) [ M ] +256.4, [ M-H ] -254.3.

(2S,4R) -1- [ (2-chlorophenyl) methyl ] -4-hydroxy-N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (50). To a solution of 49(0.825g, 3.2mmol) in DMF (10ml) was added 4-nitrophenylethylamine (0.981g, 4.8mmol), HBTU (1.346g, 3.5mmol) and diisopropylethylamine (2.8ml, 16.1 mmol). After stirring at room temperature for 24h, the reaction mixture was diluted with ethyl acetate and washed with water (100ml each, three times) and brine (100 ml). The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, methanol/dichloromethane) to afford the product as a white solid (0.55g, 42%). 1H NMR (300MHz, CDCl3) δ 8.02(d, J ═ 8.67Hz,2H),7.53(t, J ═ 6.03Hz,1H),7.16-7.35(m,6H),4.28-4.37(m,1H),3.72-3.87(m,2H),3.56(t, J ═ 8.10Hz,1H),3.44(q, J ═ 6.78Hz,2H),3.23(dd, J ═ 4.90,10.55Hz,1H),2.77-2.89(m,2H),2.53(dd, J ═ 4.14,10.55Hz,1H),2.28(ddd, J ═ 4.24,8.62,12.95, 1H),1.86-1.98(m, 1H). MS (ESI) [ M ] + 404.3.

(3R,5S) -1- [ (2-chlorophenyl) methyl ] -5- { [2- (4-nitrophenyl) ethyl ] carbamoyl } pyrrolidin-3-yl-4-methylbenzene-1-sulfonate (51). To a solution of 50(0.550g, 1.36mmol) in dichloromethane (5ml) was added tosyl chloride (0.519g, 2.72mmol) and pyridine (5 ml). After stirring at room temperature for 24h, the reaction mixture was concentrated in vacuo. The residue was dissolved in dichloromethane and washed twice with saturated aqueous copper sulfate solution. The combined organic fractions were pooled, washed with brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, ethyl acetate/hexanes) to provide the product as a white solid (0.44g, 58%). 1H NMR (300MHz, CDCl3) δ 7.76(d, J ═ 8.29Hz,2H),7.30-7.37(m,4H),7.15-7.25(m,4H),4.86-4.94(m, J ═ 5.65Hz,1H),3.75(d, J ═ 2.64Hz,2H),3.39-3.52(m,3H),3.17(dd, J ═ 4.90,11.87Hz,1H),2.73-2.88(m,3H),2.45(s,3H),2.32-2.42(m,1H),1.90-2.01(m, 1H).

General procedure B: to a solution of 45(0.12mmol, 0.068g) in THF (2ml) was added the corresponding amine (1.2mmol) and triethylamine (0.36mmol, 0.05 ml). The reaction was refluxed for 72 h. After cooling to room temperature, the solvent was evaporated in vacuo. The residue was purified by column chromatography (SiO2, MeOH/DCM) to give the desired product.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-nitrophenyl) ethyl ] -4-propylamino) pyrrolidine-2-carboxamide (52) was prepared as a yellow oil (11%) according to general procedure B. 1H NMR (300MHz, CDCl3) δ 8.02-8.08(m,2H),7.61(t, J ═ 6.03Hz,1H),7.32-7.36(m,1H),7.29(d, J ═ 8.67Hz,2H),7.16-7.25(m,3H),3.60-3.77(m,2H),3.47(dq, J ═ 3.77,6.72Hz,2H),3.21-3.29(m,2H),2.80-2.95(m,3H),2.38-2.59(m,4H),1.67-1.78(m,1H),1.40(qd, J ═ 7.29,14.67Hz,3H),0.87(t, J ═ 7.35, 3H). 13C NMR (75MHz, CDCl3) delta 174.4,146.8,146.7,135.7,133.9,130.4,129.7,129.4,128.8,126.9,123.7,67.4,59.8,57.4,56.9,49.9,39.3,37.6,35.6,23.3, 11.7. MS (ESI) [ M ] + 445.6.

(2S,4S) -4- (butylamino) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (53) was prepared according to general procedure B as a yellow oil (39%). 1H NMR (300MHz, CDCl3) δ 7.88-7.99(m,2H),7.79(t, J-5.93 Hz,1H),7.69(d, J-7.91 Hz,1H),7.29-7.36(m,2H),7.10-7.25(m,5H),3.61-3.66(m,2H),3.56(dd, J-6.69, 14.03Hz,1H),3.44-3.51(m,1H),3.34-3.43(m,1H),3.26(dd, J-6.78, 8.67Hz,1H),3.09(d, J-10.55 Hz,1H),2.82 (d, J-7.08, 13.99, 2H, 2.61, 2.70H), 2.09 (d, J-10.55 Hz,1H),2.82(q, H, J-7.08, 13.99, 2H, 70H, 2H, 19H, 1H, 27H, and 1H, 49H, 27H. 13C NMR (75MHz, CDCl3) delta 173.6,146.8,146.5,142.0,140.7,135.1,133.7,130.3,129.6,129.4,129.0,128.7,126.9,125.7,123.5,66.9,56.7,55.8,46.9,39.3,35.8,35.2,30.0,21.3,20.1, 13.6. MS (ESI) [ M ] + 459.3.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-nitrophenyl) ethyl ] -4- (pentylamino) pyrrolidine-2-carboxamide (54) was prepared according to general procedure B as a colorless oil (59%). 1H NMR (300MHz, CDCl3) δ 8.05(d, J ═ 8.67Hz,2H),7.62(t, J ═ 5.93Hz,1H),7.26-7.36(m,3H),7.16-7.25(m,2H),3.59-3.76(m,2H),3.47(td, J ═ 6.57,12.67Hz,2H),3.21-3.29(m,2H),2.81-2.96(m,3H),2.42-2.58(m,3H),1.72(ddd, J ═ 3.77,5.75,13.28Hz,1H),1.19-1.44(m,6H),0.87(t, J ═ 6.88Hz, 3H). 13C NMR (75MHz, CDCl3) delta 174.4,146.8,146.7,135.7,133.9,130.4,129.7,129.4,128.8,126.9,123.7,67.4,59.7,57.3,56.9,48.0,39.3,37.6,35.6,29.9,29.5,22.5, 14.0. MS (ESI) [ M ] + 473.8.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- (hexylamino) -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (55) was prepared according to general procedure B as a colorless oil (25%). 1H NMR (300MHz, CDCl3) δ 8.05(d, J ═ 8.67Hz,2H),7.54(t, J ═ 5.93Hz,1H),7.27-7.36(m,3H),7.15-7.25(m,3H),3.61-3.77(m,2H),3.46(td, J ═ 6.73,13.28Hz,2H),3.22-3.33(m,2H),2.75-2.99(m,3H),2.43-2.60(m,3H),1.64-1.80(m,4H),1.36-1.45(m,2H),1.21-1.32(m,6H),0.84-0.91(m, 3H). 13C NMR (75MHz, CDCl3) delta 174.4,146.7,135.6,133.9,130.4,129.7,129.4,128.8,126.9,123.7,67.2,59.5,57.2,57.0,48.0,39.3,37.4,35.5,31.6,30.0,27.0,22.6, 14.0. MS (ESI) [ M ] + 487.4.

(2S,4S) -4- (benzylamino) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (56) was prepared according to general procedure B as a colorless oil (81%). 1H NMR (300MHz, CDCl3) δ 7.96-8.03(m,2H),7.62(t, J ═ 5.75Hz,1H),7.15-7.37(m,11H),3.59-3.78(m,4H),3.45(q, J ═ 6.97Hz,2H),3.22-3.34(m,2H),2.96(d, J ═ 10.17Hz,1H),2.79(qd, J ═ 7.07,14.20Hz,2H),2.43-2.60(m,2H),1.82(td, J ═ 3.72,13.47Hz,1H),1.53(br.s, 2H). 13C NMR (75MHz, CDCl3) delta 174.4,146.7,146.6,140.0,135.7,134.0,130.5,129.7,129.4,128.8,128.5,128.1,127.2,126.9,123.6,67.2,59.6,57.4,56.4,52.0,39.2,37.4, 35.5. MS (ESI) [ M ] + 493.8.

(2S,4S) -4- (benzylamino) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (57) was prepared according to general procedure B as a colorless oil (13%). 1H NMR (300MHz, CDCl3) δ 8.00(d, J ═ 8.85Hz,1H),7.16-7.42(m,12H),3.62-3.75(m,1H),3.46-3.60(m,3H),3.32(t, J ═ 8.10Hz,1H),3.19-3.28(m,1H),3.14(dd, J ═ 4.24,10.64Hz,1H),2.86(dt, J ═ 4.43,6.83Hz,2H),2.54-2.64(m,1H),2.49(dd, J ═ 6.03,13.37Hz,1H),2.10-2.17(m,2H),1.88-2.00(m, 1H). 13C NMR (75MHz, CDCl3) d 173.7,146.7,146.6,135.5,133.8,130.2,129.7,129.4,129.3,128.8,128.5,127.7,127.0,125.5,123.7,67.8,62.7,59.5,57.6,56.9,39.3,38.6,35.4, 34.5. MS (ESI) [ M ] + 507.2.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-nitrophenyl) ethyl ] -4- [ (2- (phenylethyl) amino ] pyrrolidine-2-carboxamide (58) was prepared according to general procedure B as a yellow oil (30%). 1H NMR (300MHz, CDCl3) δ 7.99-8.06(m,1H),7.48(t, J ═ 5.93Hz,1H),7.28-7.36(m,3H),7.13-7.25(m,7H),3.60-3.71(m,2H),3.48(td, J ═ 6.90,13.89Hz,1H),3.28-3.35(m,1H),3.18-3.25(m,1H),2.88-3.02(m,2H),2.70-2.83(m,6H), 2.44-2H) (m,2H),1.85(d, J ═ 6.97Hz,1H),1.63-1.73(m, 1H). 13C NMR (75MHz, CDCl3) delta 174.2,146.7,139.7,135.6,133.9,130.3,129.7,129.4,128.8,128.8,128.6,128.6,128.5,128.5,126.9,126.3,123.6,67.3,59.2,57.2,56.7,48.9,39.2,37.6,36.2, 35.5. MS (ESI) [ M ] + 507.2.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (3-methoxyphenyl) methyl ] amino } -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (59) was prepared according to general procedure B as a yellow oil (20%). 1H NMR (300MHz, CDCl3) δ 8.01(d, J ═ 8.67Hz,2H),7.60(t, J ═ 5.93Hz,1H),7.31-7.35(m,1H),7.18-7.25(m,6H),6.74-6.88(m,4H),3.79(s,3H),3.69-3.72(m,1H),3.62-3.69(m,3H),3.45(q, J ═ 6.97Hz,2H),3.32(td, J ═ 2.99,5.51Hz,1H),3.25(dd, J ═ 5.37,9.89Hz,1H),2.97(d, J ═ 9.98Hz,1H),2.80(q, J ═ 7.35, 2H),2.81, 1H, 2.58H, 1H, 2.7.7.81, 1H. 13C NMR (75MHz, CDCl3) delta 174.5,159.8,146.7,146.7,141.1,135.6,134.0,130.5,129.7,129.5,129.4,128.8,126.9,123.6,120.4,114.1,112.4,67.1,59.4,57.2,56.5,55.2,51.8,39.3,37.2, 35.5. MS (ESI) [ M ] + 523.5.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (3, 4-dimethoxyphenyl) methyl ] amino } -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (60) was prepared as a colorless oil (41%) according to general procedure B. 1H NMR (300MHz, CDCl3) δ 8.00(d, J ═ 8.67Hz,2H),7.60(t, J ═ 6.03Hz,1H),7.30-7.36(m,1H),7.16-7.25(m,5H),6.76-6.84(m,4H),3.82-3.91(m,9H),3.64-3.72(m,2H),3.58-3.63(m,2H),3.45(q, J ═ 6.78Hz,2H),3.22-3.34(m,2H),2.95(d, J ═ 10.36Hz,1H),2.79(q, J ═ 7.10Hz,2H),2.45-2.59(m,2H),1.77-1.96(m, 2H). 13C NMR (75MHz, CDCl3) delta 174.3,149.1,148.3,146.7,135.7,134.0,132.5,130.5,129.7,129.4,128.9,126.9,123.6,120.2,111.6,111.3,111.1,67.2,59.6,57.5,56.2,55.9,51.8,39.2,37.4, 35.5. MS (ESI) [ M ] + 553.7.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-hydroxyphenyl) methyl ] amino } -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (61) was prepared as a colorless oil according to general procedure B (27%). 1H NMR (300MHz, CDCl3) δ 7.94(d, J ═ 8.67Hz,2H),7.69(t, J ═ 7.72Hz,1H),7.28-7.35(m,1H),7.14-7.25(m,4H),7.05(d, J ═ 8.48Hz,2H),6.61-6.74(m,2H),3.58-3.70(m,3H),3.46(td, J ═ 6.69,13.37Hz,1H),3.38(d, J ═ 6.78Hz,1H),3.24(dd, J ═ 6.12,9.32Hz,1H),3.01(d, J ═ 10.36Hz,1H),2.74-2.82(m,2H), 2.42-2.42 (m,2H), 1H (m, 84H). 13C NMR (75MHz, CDCl3) delta 174.3,155.7,146.7,135.4,133.8,130.4,129.8,129.6,129.4,129.0,128.8,126.9,125.8,123.6,115.6,67.2,58.8,56.9,56.3,51.3,39.3,36.8, 35.4. MS (ESI) [ M ] + 509.4.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -N- [2- (4-nitrophenyl) ethyl ] -4- ({ [4- (trifluoromethyl) phenyl ] methyl } amino) pyrrolidine-2-carboxamide (62) was prepared according to general procedure B as a colorless oil (9%). 1H NMR (300MHz, CDCl3) δ 8.02(d, J ═ 8.67Hz,2H),7.55(d, J ═ 8.10Hz,3H),7.32-7.38(m,3H),7.16-7.24(m,4H),3.61-3.79(m,4H),3.46(dd, J ═ 2.54,6.50Hz,2H),3.26(dd, J ═ 5.37,9.89Hz,2H),2.93(d, J ═ 10.17Hz,1H),2.76-2.85(m,2H),2.44-2.59(m,2H),1.84(dd, J ═ 4.14,13.38Hz, 1H). 13C NMR (75MHz, CDCl3) delta 174.2,146.6,144.1,135.5,134.0,130.5,129.8,129.4,129.0,128.2,126.9,125.3,123.6,67.1,59.6,57.6,56.5,51.4,39.2,37.3, 35.5. MS (ESI) [ M ] + 561.3.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-chlorophenyl) methyl ] amino } -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (63) was prepared as a colorless oil (14%) according to general procedure B. 1H NMR (300MHz, CDCl3) δ 8.02(d, J ═ 8.67Hz,2H),7.58(t, J ═ 6.03Hz,1H),7.14-7.36(m,10H),3.85(s,2H),3.59-3.77(m,5H),3.45(q, J ═ 6.84Hz,2H),3.25(dd, J ═ 5.37,9.89Hz,2H),2.92(d, J ═ 10.17Hz,1H),2.80(qd, J ═ 6.91,13.56Hz,2H),2.42-2.57(m, 2H). 13C NMR (75MHz, CDCl3) delta 174.3,146.6,138.5,135.6,134.0,132.9,130.5,129.7,129.6,129.4,128.9,128.6,128.5,126.9,123.7,67.1,59.6,57.5,56.4,51.3,39.2,37.3, 35.5. MS (ESI) [ M ] + 527.5.

(2S,4S) -1- [ (2-chlorophenyl) methyl ] -4- { [ (4-fluorophenyl) methyl ] amino } -N- [2- (4-nitrophenyl) ethyl ] pyrrolidine-2-carboxamide (64) was prepared as a colorless oil (55%) according to general procedure B. 1H NMR (300MHz, CDCl3) δ 8.01(d, J ═ 8.67Hz,2H),7.59(t, J ═ 6.12Hz,1H),7.31-7.36(m,1H),7.15-7.28(m,7H),6.95-7.02(m,2H),3.57-3.77(m,4H),3.45(q, J ═ 6.91Hz,2H),3.21-3.32(m,2H),2.93(d, J ═ 9.98Hz,1H),2.80(qd, J ═ 6.92,13.54Hz,2H),2.42-2.58(m,2H),1.76-1.86(m, 1H). 13C NMR (75MHz, CDCl3) delta 174.3,146.7,135.6,134.0,130.5,129.7,129.7,129.6,129.4,128.9,126.9,123.6,115.4,115.1,67.1,59.6,57.5,56.4,51.2,39.2,37.3, 35.5. MS (ESI) [ M ] + 511.3.

1-tert-butyl 2-methyl (2S,4R) -4-hydroxypyrrolidine-1, 2-dicarboxylate (65). Boc2O (30.3mmol, 6.6g) was added dropwise to a solution of methyl trans-4-hydroxy-L-proline 5(27.6mmol, 5.0g) in 1N NaOH solution (28.5ml) and 1, 4-dioxane (28.5ml) at 0 ℃. After stirring at room temperature for 8h, the solvent was removed in vacuo. The residue was dissolved in ethyl acetate and washed with water and brine. The organic layer was dried over anhydrous MgSO4, and concentrated in vacuo to give the desired product as a colorless liquid (6.1g, 90%). 1H NMR (300MHz, CDCl3) δ 4.51(m,1H),4.43(m,1H),3.74(s,3H),3.64(m,2H),2.29(d, J ═ 7.54Hz,1H),2.10(dd, J ═ 4.52,8.48Hz,1H),1.44(m, 9H). MS (ESI) [ M + H ] +246.3, [ M + Na ] + 268.1.

1-tert-butyl 2-methyl (2S,4R) -4- [ (4-methylbenzenesulfonyl) oxy ] pyrrolidine-1, 2-dicarboxylate (66). To a solution of 5(11g, 45mmol) in dichloromethane (35ml) was added tosyl chloride (10.27g, 53.9mmol) and pyridine (35ml) at 0 ℃. After stirring at reflux for 24h, the reaction mixture was concentrated in vacuo and redissolved in dichloromethane. Then washed with saturated aqueous copper sulfate and brine. The organic fraction was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, ethyl acetate/hexanes) to provide the product as a pale yellow liquid (12.6g, 70%). 1H NMR (300MHz, CDCl3) δ 7.79(d, J ═ 8.29Hz,2H),7.36(d, J ═ 8.10Hz,2H),4.99-5.09(m,1H),4.32-4.43(m,1H),3.72(s,3H),3.57-3.65(m,2H),2.46(s,4H),2.09-2.22(m, J ═ 8.70Hz,1H),1.36-1.44(m, 9H). MS (ESI) [ M + H ] +400.3, [ M + Na ] + 422.3.

1-tert-butyl 2-methyl (2S,4S) -4-azidopyrrolidine-1, 2-dicarboxylate (67). To a solution of 66(4.28g, 10.7mmol) in DMF (40ml) was added sodium azide (1.393g, 21.4 mmol). After heating at 70 ℃ for 16h, the reaction mixture was diluted with ethyl acetate, washed twice with water and once with brine. The organic fraction was dried over anhydrous magnesium sulfate and concentrated in vacuo to provide the desired product as a yellow liquid (2.87g, 99%). 1H NMR (300MHz, CDCl3) δ 4.30-4.47(m,1H),4.10-4.20(m,1H),3.66-3.80(m,4H),3.43-3.54(m,1H),2.47(ddt, J ═ 6.03,8.38,13.89Hz,1H),2.13-2.22(m,1H),1.40-1.51(m, 9H). MS (ESI) [ M + H ] +271.1, [ M + Na ] + 293.1.

1-tert-butyl 2-methyl (2S,4S) -4-aminopyrrolidine-1, 2-dicarboxylate (68). To a solution of azide 67(2.87g, 10.6mmol) in THF (46ml) under nitrogen was added PPh3(5.57g, 21.2mmol) and water (0.5 ml). The reaction mixture was refluxed for 6h with stirring. After removal of the solvent, the residue was dissolved in ether, treated with 0.1N HCl for 5min, and then extracted twice with ether. The aqueous layer was then treated with 1N NaOH until pH 10 and then extracted with dichloromethane. The combined dichloromethane fractions were dried over anhydrous magnesium sulfate and concentrated in vacuo to provide the desired product as a yellow liquid (2.08g, 80%). 1H NMR (300MHz, CDCl3) δ 4.20-4.37(m,1H),3.72-3.79(m,3H),3.63-3.72(m,1H),3.50-3.58(m,1H),3.26(dd, J ═ 4.99,10.64Hz,1H),2.38-2.53(m,1H),1.75-1.86(m,1H),1.39-1.48(m, 9H). MS (ESI) [ M + H ] + 245.3.

1-tert-butyl 2-methyl (2S,4S) -4- { [ (4-methoxyphenyl) methyl ] amino } pyrrolidine-1, 2-dicarboxylate (69). To a solution of 68(2.08g, 7.74mmol) in 1, 2-dichloroethane (26ml) was added 4-methoxybenzaldehyde (0.94ml, 7.74mmol), sodium triacetoxyborohydride (2.461g, 11.6mmol) and acetic acid (0.44ml, 7.74 mmol). After stirring at room temperature for 24h, the reaction mixture was quenched with saturated sodium bicarbonate solution and extracted three times with dichloromethane. The combined organic layers were dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, ethyl acetate/hexanes) to provide the product as a colorless liquid (2.193g, 73%). 1H NMR (300MHz, CDCl3) δ 7.21(d, J ═ 8.48Hz,2H),6.85(d, J ═ 8.67Hz,2H),4.20-4.35(m,1H),3.79(s,3H),3.58-3.75(m,6H),3.25-3.37(m,2H),2.29-2.45(m,1H),1.90-2.01(m,1H),1.39-1.48(m, 9H). MS (ESI) [ M + H ] + 365.9.

1-tert-butyl 2-methyl (2S,4S) -4- { [ (4-methoxyphenyl) methyl ] [ (2,2, 2-trichloroethoxy) carbonyl ] amino } pyrrolidine-1, 2-dicarboxylate (70). To a solution of 69(0.2g, 0.5mmol) in dichloromethane (2.5ml) was added Troccl (0.11ml, 0.77mmol) and triethylamine (0.14ml, 1.02mmol) at 0 ℃. The reaction mixture was allowed to reach room temperature and stirring was continued for 16 h. The reaction mixture was then washed with saturated aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate and concentrated in vacuo to give the product as a colorless liquid (0.23g, quantitative yield). 1H NMR (300MHz, CDCl3) δ 7.14(d, J ═ 8.29Hz,2H),6.85(d, J ═ 8.48Hz,2H),4.73-4.89(m,2H),4.40-4.60(m,3H),4.15-4.26(m,1H),3.79(s,3H),3.67-3.75(m,4H),3.37-3.51(m,1H),2.34-2.46(m,1H),2.06-2.23(m,1H),1.35-1.47(m, 9H). MS (ESI) [ M + H ] +539.3, [ M + Na ] + 563.3.

(2S,4S) -1- [ (tert-butoxy) carbonyl ] -4- { [ (4-methoxyphenyl) methyl ] [ (2,2, 2-trichloroethoxy) carbonyl ] amino } pyrrolidine-2-carboxylic acid (71). To a solution of 70(0.274g, 0.5mmol) in methanol (2ml) was added lithium hydroxide (0.048g, 2 mmol). After stirring at room temperature, methanol was removed under reduced pressure. The residue was suspended in water and the pH was adjusted to 5 by 1N aqueous HCl. The aqueous layer was extracted three times with dichloromethane. The combined organic fractions were dried over anhydrous magnesium sulfate and concentrated in vacuo to give the product as a white solid (0.225g, 84%). 1H NMR (300MHz, CDCl3) δ 9.02(br.s.,1H),7.14(d, J ═ 4.33Hz,2H),6.86(d, J ═ 7.16Hz,2H),4.72-4.91(m,2H),4.35-4.63(m,3H),4.16-4.33(m,1H),3.80(s,3H),3.70-3.76(m,1H),3.23-3.54(m,1H),2.20-2.52(m,2H),1.41(d, J ═ 17.52Hz, 9H). MS (ESI) [ M-H ] -523.1.

tert-butyl- (2S,4S) -4- { [ (4-methoxyphenyl) methyl ] [ (2,2, 2-trichloroethoxy) carbonyl ] amino } -2- [ (2-phenethyl) carbamoyl ] pyrrolidine-1-carboxylate (72). To a solution of 71(2.629g, 5mmol) in dry dichloromethane (17ml) was added phenethylamine hydrochloride (0.7ml, 5.5mmol), HBTU (2.086g, 5.5mmol) and diisopropylethylamine (3.2ml, 18 mmol). After stirring at room temperature for 16h, the reaction mixture was washed with 1N aqueous HCl. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, ethyl acetate/hexanes) to provide the product as a yellow solid (2g, 64%). 1H NMR (300MHz, CDCl3) δ 7.27(t, J ═ 3.67Hz,2H),7.12-7.21(m,5H),6.85(d, J ═ 8.67Hz,2H),4.78(br.s.,2H),4.33-4.64(m,3H),3.74-3.83(m,4H),3.50(d, J ═ 5.84Hz,2H),2.81-2.88(m,2H),2.17-2.55(m,2H),1.33-1.46(m, 11H).

2,2, 2-trichloroethyl-N- [ (4-methoxyphenyl) methyl ] -N- [ (3S,5S) -5- [ (2-phenylethyl) carbamoyl ] pyrrolidin-3-yl ] carbamate (73). To a 20% v/v solution of trifluoroacetic acid in dichloromethane (4.4ml) was added 72(2g, 3.2 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was then concentrated in vacuo to afford the product as a yellow liquid (1.60g, quantitative yield). MS (ESI) [ M ] + 528.7.

General procedure C. To a solution of 73(0.1g, 0.19mmol) in 1, 2-dichloroethane (4ml) was added the corresponding aldehyde (0.042g, 0.28mmol), sodium triacetoxyborohydride (0.12g, 0.57mmol) and acetic acid (1 drop). After stirring at room temperature for 48h, the reaction mixture was quenched with saturated aqueous sodium bicarbonate and extracted three times with dichloromethane. The organic extract was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was dissolved in methanol (8 ml). To the reaction mixture was added zinc (0.1g, 1.5mmol) and acetic acid (2 drops). After stirring at reflux for 1h, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by flash column chromatography (SiO2, MeOH/DCM) to afford the desired product.

(2S,4S) -1-hexyl-4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (74) was prepared as a colorless oil according to general procedure C (27%). 1H NMR (300MHz, CDCl3) δ 7.50(t, J ═ 6.03Hz,2H),7.27-7.30(m,1H),7.24(s,1H),7.14-7.22(m,6H),6.83-6.87(m,2H),3.79(s,3H),3.61(d, J ═ 3.77Hz,2H),3.41-3.59(m,2H),3.23(dt, J ═ 3.01,5.84Hz,1H),2.97-3.04(m,2H),2.75-2.82(m,2H),2.33-2.52(m,3H),2.21-2.31(m,1H),1.69-1.78(m,1H),1.07-1.38(m,9H), t, 0.89(t, 6H), 3H). 13C NMR (75MHz, CDCl3) delta 174.8,158.7,139.0,132.3,129.3,128.7,128.5,126.4,113.8,67.6,59.3,56.2,56.0,55.3,51.3,39.8,37.1,35.6,31.7,28.8,27.0,22.6, 14.0. MS (ESI) [ M ] + 438.5.

(2S,4S) -1- (cyclohexylmethyl) -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (75) was prepared as a colorless oil (24%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.41(t, J ═ 5.84Hz,1H),7.14-7.25(m,6H),6.83-6.89(m,2H),3.76-3.82(m,3H),3.61-3.70(m,2H),3.50-3.59(m,2H),3.24(td, J ═ 2.76,5.98Hz,1H),2.94-3.01(m,2H),2.80(qd, J ═ 6.98,19.00Hz,2H),2.31-2.45(m,2H),2.08-2.25(m,2H),1.48-1.81(m,8H),1.23-1.37(m,1H),1.04-1.21(m,3H), 0.68 (m, 0.68, 7H), 1.11H, 11H, 1H, 11H). 13C NMR (75MHz, CDCl3) delta 173.6,157.6,137.7,131.1,128.2,127.6,127.5,127.4,125.3,112.7,66.9,61.9,58.1,55.2,54.1,50.2,38.5,35.8,35.6,34.4,30.6,29.6,25.5,24.9, 24.8. MS (ESI) [ M ] + 450.6.

(2S,4S) -1-benzyl-4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (76) was prepared as a colorless oil (29%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.44-7.53(m,1H),7.30(m,1H),7.12-7.26(m,9H),7.03-7.09(m,2H),6.83(d, J ═ 8.48Hz,2H),3.73-3.81(m,4H),3.46-3.60(m,4H),3.36(d, J ═ 13.00Hz,1H),3.15-3.26(m,2H),2.87(d, J ═ 10.36Hz,1H),2.72-2.81(m,2H),2.39-2.52(m,2H),1.74-1.83(m, 1H). 13C NMR (75MHz, CDCl3) delta 174.3,158.7,138.9,138.3,132.2,129.3,128.7,128.6,128.5,128.4,127.2,126.4,113.8,67.1,59.6,59.2,56.0,55.3,51.3,39.7,37.4, 35.6. MS (ESI) [ M ] + 444.5.

(2S,4S) -1- [ (3-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (77) was prepared as a colorless oil (21%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.45(t, J ═ 5.18Hz,1H),7.11-7.25(m,10H),6.94(d, J ═ 6.97Hz,1H),6.80-6.86(m,2H),3.78(s,3H),3.73(d, J ═ 13.37Hz,1H),3.60(d, J ═ 5.65Hz,2H),3.51-3.57(m,1H),3.41-3.50(m,1H),3.30(d, J ═ 13.56Hz,1H),3.22-3.27(m,1H),3.16(dd, J ═ 6.12,9.51Hz,1H),2.87(d, J ═ 10.36, 1H),2.92, 2.78 (m, 2.83H, 2.83, 2H), 2.83-3.52H, 2H, 2.83(d, J ═ 13.56Hz, 2H). 13C NMR (75MHz, CDCl3) delta 173.9,158.9,140.3,138.8,134.3,131.3,129.7,129.5,128.6,128.6,128.5,127.4,126.6,126.5,113.9,67.3,58.9,55.8,55.3,51.1,39.7,37.2, 35.5. MS (ESI) [ M ] + 478.3.

(2S,4S) -1- [ (4-chlorophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (78) was prepared according to general procedure C as a colorless oil (19%). 1H NMR (300MHz, CDCl3) δ 7.43(t, J ═ 5.75Hz,1H),7.10-7.26(m,10H),6.94(d, J ═ 8.29Hz,2H),6.81-6.86(m,2H),3.75-3.82(m,5H),3.60-3.74(m,3H),3.48-3.60(m,4H),3.26-3.33(m,1H),3.19-3.25(m,1H),3.13-3.18(m,1H),2.67-2.90(m,4H),2.35-2.50(m,2H),1.77(ddd, J ═ 3.49,5.13,13.42Hz, 1H). 13C NMR (75MHz, CDCl3) delta 174.1,158.8,138.8,136.7,132.9,132.1,130.1,129.8,129.3,129.2,129.1,128.9,128.8,128.7,128.6,128.6,128.5,126.5,113.9,113.8,67.2,59.1,58.8,56.0,55.3,51.3,39.6,37.4, 35.5. MS (ESI) [ M ] + 478.5.

(2S,4S) -1- [ (2-methoxyphenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (79) was prepared as a colorless oil (25%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.82(t, J ═ 5.84Hz,1H),7.28(d, J ═ 1.70Hz,1H),7.13-7.24(m,7H),7.10(dd, J ═ 1.51,7.35Hz,1H),6.80-6.92(m,4H),3.87(d, J ═ 12.81Hz,1H),3.77(s,3H),3.72(s,3H),3.51-3.65(m,3H),3.32-3.44(m,2H),3.15-3.24(m,2H),2.85(d, J ═ 10.17Hz,1H),2.77(dt, J ═ 1.98,7.21Hz,2H), 2.42-2.42 (m,2H),2.54(d, 13.75H), 1H, 13H, 5J ═ 1H. 13C NMR (75MHz, CDCl3) delta 173.4,157.7,156.6,138.1,131.2,129.6,128.3,127.7,127.6,127.5,125.4,125.3,119.5,112.8,109.5,65.8,58.2,54.8,54.3,54.2,53.6,50.2,39.2,36.4, 34.9. MS (ESI) [ M ] + 474.6.

(2S,4S) -1- [ (3-methoxyphenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (80) was prepared as a colorless oil (45%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.54(br.s.,1H),7.31(d, J ═ 8.48Hz,2H),7.10-7.24(m,6H),6.69-6.87(m,4H),3.80-3.90(m,2H),3.75(d, J ═ 15.82Hz,0H),3.01-3.65(m,6H),2.74-2.87(m,2H),2.34-2.52(m,2H),1.87-2.04(m, 2H). 13C NMR (75MHz, CDCl3) d 171.3,157.7,157.5,136.5,128.8,127.3,126.6,126.3,124.2,122.9,122.6,118.9,112.2,110.9,107.3,64.1,56.0,54.2,53.1,53.1,52.7,47.2,38.1,33.1, 32.5. MS (ESI) [ M ] + 474.9.

(2S,4S) -1- [ (3, 4-dimethoxyphenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (81) was prepared as a colorless oil (55%) according to general procedure C. 1H NMR (300MHz, CDCl 3). delta.7.02-7.26 (m,3H),6.80-6.94(m,6H),6.46-6.79(m,2H),3.83-3.91(m,12H),3.75-3.79(m,2H),3.43-3.73(m,2H),2.95-3.33(m,1H),2.68-2.91(m,2H),1.68-2.50(m, 4H). 13C NMR (75MHz, CDCl3) d 172.1,146.8,146.2,131.4,127.0,126.8,126.4,126.3,126.2,126.1,124.0,118.4,117.0,111.5,111.5,108.8,108.2,62.7,56.6,53.6,53.5,52.9,49.6,48.9,37.6,35.0,33.2, 30.7. MS (ESI) [ M ] + 504.7.

(2S,4S) -4- { [ (4-methoxyphenyl) methyl ] amino } -1- [ (2-methylphenyl) methyl ] -N- (2-phenylethyl) pyrrolidine-2-carboxamide (82) was prepared as a colorless oil (19%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.36(t, J ═ 5.75Hz,1H),7.12-7.19(m,7H),7.07-7.11(m,2H),6.84(d, J ═ 8.48Hz,2H),3.75-3.80(m,4H),3.51-3.70(m,5H),3.45-3.50(m,1H),3.29-3.38(m,1H),3.17-3.28(m,2H),2.95(d, J ═ 9.98Hz,1H),2.67(dt, J ═ 2.73,7.02Hz,2H),2.42-2.51(m,2H),2.21-2.27(m,3H),2.19(d, J ═ 3.01, 1H), 1.74H, 1.82H, 1H). 13C NMR (75MHz, CDCl3) delta 172.2,156.5,136.6,134.3,134.0,129.9,128.0,127.1,126.9,126.5,126.4,126.3,124.9,124.1,123.8,111.6,111.6,65.6,57.4,55.4,53.9,53.0,49.1,37.5,35.2,33.3, 16.9. MS (ESI) [ M ] + 458.4.

(2S,4S) -4- { [ (4-methoxyphenyl) methyl ] amino } -1- [ (4-methylphenyl) methyl ] -N- (2-phenylethyl) pyrrolidine-2-carboxamide (83) was prepared as a colorless oil (26%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.48(t, J ═ 5.84Hz,1H),7.13-7.25(m,6H),7.07(d, J ═ 7.91Hz,2H),6.92-6.98(m,2H),6.81-6.86(m,2H),3.78(s,3H),3.72(d, J ═ 13.00Hz,1H),3.57(d, J ═ 3.77Hz,2H),3.46-3.54(m,2H),3.33(d, J ═ 13.00Hz,1H),3.22(d, J ═ 3.20Hz,1H),3.17(dd, J ═ 6.03,9.61Hz,1H),2.87(d, J ═ 10.92, 1H, 2.76, 2H, 3.73H), 3.7 (d, J ═ 6.03,9.61Hz,1H),2.87(d, J ═ 10, 2.76, 2H, 2.73H), 2.73H, 3.73H, 2.73H, 2H, 3.73H, 2H, 3.7H, 2H. 13C NMR (75MHz, CDCl3) d 173.1,157.5,137.7,135.6,133.9,130.8,128.1,127.8,127.4,127.3,127.2,125.2,112.6,65.8,58.0,57.8,54.8,54.0,50.0,38.5,36.1,34.4, 19.8. MS (ESI) [ M ] + 458.4.

(2S,4S) -1- [ (4-hydroxyphenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (84) was prepared as a colorless oil (9%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.50(t, J ═ 5.56Hz,1H),7.12-7.23(m,7H),6.80-6.89(m,4H),6.60(d, J ═ 8.29Hz,2H),3.74(s,3H),3.61-3.70(m,3H),3.50(td, J ═ 7.18,14.46Hz,2H),3.31(s,1H),3.21(d, J ═ 13.00Hz,1H),3.09(dd, J ═ 6.40,9.42Hz,1H),2.92(d, J ═ 10.36Hz,1H),2.69-2.85(m,3H),2.36-2.48(m,2H),1.83(dd, 24, 13.47H), 1H. 13C NMR (75MHz, CDCl3) delta 174.1,159.5,155.7,138.7,130.2,129.9,129.0,128.7,128.6,128.6,126.5,115.4,114.2,66.5,58.2,57.8,55.4,55.2,50.3,40.0,35.6, 35.4. MS (ESI) [ M ] + 460.5.

(2S,4S) -1- { [4- (dimethylamino) phenyl ] methyl } -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (85) was prepared as a colorless oil (3%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.37(br.s.,1H),7.28(d, J ═ 1.88Hz,0H),7.15-7.22(m,4H),6.95(d, J ═ 8.67Hz,1H),6.82-6.86(m,1H),6.63(d, J ═ 8.67Hz,1H),3.78(s,2H),3.61-3.69(m,2H),3.44-3.52(m,1H),3.32(d, J ═ 12.81Hz,1H),3.25(d, J ═ 5.09Hz,1H),3.15(dd, J ═ 5.84,9.61Hz,1H),2.89-2.97(m,5H),2.76 (J ═ 76, 1H), 49.49.49H, 1H, 49.49H, 1H, 49H, 1H, 49.53H, 1H, and 1H. 13C NMR (75MHz, CDCl3) delta 172.6,148.1,137.1,127.8,127.7,126.9,126.7,124.6,124.0,112.1,110.7,64.7,56.9,54.1,53.4,49.1,38.8,38.1,35.0,33.8, 29.0. MS (ESI) [ M ] + 487.6.

(2S,4S) -1- [ (4-cyanophenyl) methyl ] -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (86) was prepared as a colorless oil (27%) according to general procedure C. 1H NMR (300MHz, CDCl3) d 7.52(d, J ═ 8.29Hz,2H),7.44(t, J ═ 5.75Hz,1H),7.08-7.25(m,9H),6.84(d, J ═ 8.67Hz,2H),3.75-3.82(m,4H),3.50-3.65(m,4H),3.38(d, J ═ 13.75Hz,1H),3.23-3.29(m,1H),3.20(dd, J ═ 5.93,9.70Hz,1H),2.70-2.89(m,3H),2.42-2.51(m,1H),2.37(dd, J ═ 5.65,9.98, 1H),1.73-1.83(m, 1H). 13C NMR (75MHz, CDCl3) d228.8,173.8,158.8,143.8,138.8,132.2,129.3,128.9,128.7,128.6,126.5,113.9,67.5,59.2,59.1,56.1,55.3,51.4,39.5,37.4, 35.4. MS (ESI) [ M ] + 469.4.

(2S,4S) -4- { [ (4-methoxyphenyl) methyl ] amino } -1- (naphthalen-2-ylmethyl) -N- (2-phenylethyl) pyrrolidine-2-carboxamide (87) was prepared as a colorless oil (23%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.80-7.84(m,1H),7.72-7.79(m,2H),7.58(s,1H),7.43-7.55(m,3H),7.10-7.24(m,8H),6.82(d, J ═ 8.67Hz,2H),3.93(d, J ═ 13.00Hz,1H),3.77(s,3H),3.43-3.59(m,5H),3.21-3.29(m,2H),2.89(d, J ═ 10.17Hz,1H),2.76(dt, J ═ 3.20,6.88Hz,2H),2.42-2.55(m,2H),1.80(ddd, J ═ 3.49,5.65,13.47, 1H). 13C NMR (75MHz, CDCl3) delta 172.5,157.1,137.2,134.1,131.7,131.1,130.1,127.7,127.0,126.9,126.5,126.1,126.0,125.4,125.0,124.8,124.5,124.1,112.2,65.6,58.1,57.4,54.2,53.6,49.5,38.1,35.7, 33.9. MS (ESI) [ M ] + 494.4.

(2S,4S) -1- (2H-1, 3-benzodioxol-5-ylmethyl) -4- { [ (4-methoxyphenyl) methyl ] amino } -N- (2-phenylethyl) pyrrolidine-2-carboxamide (88) was prepared as a colorless oil (11%) according to general procedure C. 1H NMR (300MHz, CDCl3) δ 7.35(br.s.,1H),7.14-7.25(m,7H),6.84(d, J ═ 8.67Hz,2H),6.69(d, J ═ 7.91Hz,1H),6.59(s,1H),6.53(d, J ═ 7.72Hz,1H),5.92(d, J ═ 2.07Hz,2H),3.78(s,3H),3.61-3.70(m,3H),3.51(td, J ═ 6.64,13.09Hz,2H),3.27(d, J ═ 13.00Hz,2H),3.13(dd, J ═ 5.84,9.61Hz,1H),2.92(d, J ═ 10.93, 1H),2.79(d, J ═ 2H),3.79 (dt, 2H), 49H, 2.79(d, J ═ 6.7, 2H). 13C NMR (75MHz, CDCl3) delta 172.7,146.3,145.4,137.4,130.5,128.3,127.3,127.2,125.1,120.3,112.6,107.6,106.7,99.5,97.0,65.4,57.6,57.1,54.4,53.9,49.4,38.4,35.2, 34.1. MS (ESI) [ M ] + 488.6.

Calcium mobilization assay. Two separate stable cell lines were created by overexpressing the human NPFFR1 receptor and the human NPFFR2 receptor in CHO-RD-HGA16(Molecular Devices) cells. One day prior to assay, cells were plated at 30,000 cells/well (100 μ L volume) into Ham's F12 supplemented with 10% fetal bovine serum, 100 units of penicillin/streptomycin, and 100 μ g/mL normocin (TM) in 96-well black-walled assay plates.

cells were incubated overnight at 37 ℃ with 5% CO 2. Prior to the assay, Calcium 5dye (Calcium 5 dye; Molecular Devices) was reconstituted according to the manufacturer's instructions. Reconstituted dye was diluted 1:40 in warm assay buffer (1 × HBSS, 20mM HEPES, 2.5mM probenecid, pH 7.4, at 37 ℃). Growth medium was removed and cells were gently washed with 100 μ Ι _ of warm assay buffer. Cells were incubated at 37 ℃ in 200 μ L of diluted calcium 5dye with 5% CO2 for 45 minutes. A single concentration of each test compound was prepared in 2.25% BSA/8% DMSO/assay buffer at 10x the desired final concentration. Serial dilutions of NPFF were prepared in 0.25% BSA/1% DMSO/assay buffer at 10x the desired final concentration, aliquoted in 96-well polypropylene plates, and warmed to 37 ℃. After the dye loading incubation period, cells were pretreated with 25 μ Ι _ of test compound and incubated at 37 ℃ for 15 min. After the pretreatment incubation period, plates were read with II (molecular devices). Calcium-mediated changes in fluorescence were monitored every 1.52 seconds over a 60 second period, with II addition of 25 μ Ι _ of NPFF serial dilutions (excitation at 485nm, detection at 525 nm) at the 19 second time point. Peak kinetic reduction (SoftMax, Molecular Devices) was plotted against the log of fluorescence units (RFU) versus compound concentration.

Data were fitted to a three-parameter logistic curve to generate EC50 values (GraphPad Prism, GraphPad Software, inc., San Diego, CA). The apparent Ke value was calculated using the equation Ke ═ L ]/((EC50+/EC50-) -1), where [ L ] is the concentration of the test compound, EC50+ is EC50 for NPFF with the test compound, and EC 50-is EC50 for NPFF alone. Ke values are considered to be valid when the EC50+/EC 50-ratio is at least 4.

384 well high throughput screening

Stable human NPFFR1 CHO-RD-HGA16 cells were plated at 5,000 cells/well in 30 μ L/well volume into Ham's F12 medium supplemented with 1% FBS and 100 units penicillin/streptomycin in 384 well Greiner black-wall microplates using a microflo (tm) selection dispenser equipped with a 5 μ L cassette (BioTek). The plated cells were incubated overnight at 37 ℃ with 5% CO2 and 95% relative humidity. The following day, compound test plates were prepared by: the previously replicated library subplates were diluted with assay buffer to obtain 100 μ M (10 × desired final concentration) working solution and columns 1,2, 23 and 24 were filled with positive and negative controls. Additional compound test plates containing NPFF EC60 concentrations (250nM, prepared at 10x the desired final concentration in 1% DMSO/assay buffer) were prepared for the antagonist portion of the screen. Calcium 5dye (Bulk Kit, Molecular Devices) reconstituted according to the manufacturer's instructions was diluted 1:20 in pre-warmed (37 ℃) assay buffer (1X HBSS, 20mM HEPES, 2.5mM probenecid, pH 7.4, at 37 ℃) and 30 μ Ι _ was added to the plate with Biomek NX followed by incubation at 37 ℃, 5% CO2, 95% relative humidity for 45 minutes. Dye loaded plates were pretreated with 8.5 μ L of 8% DMSO/assay buffer using Biomek NX and incubated at 37 ℃, 5% CO2, 95% relative humidity for 15 minutes. After this incubation period, plates were read with Tetra to assess agonist activity. Calcium mediated changes in fluorescence were monitored every 1 second over a 60 second period, with Tetra adding 8.5 μ Ι _ from compound plate at the 10 second time point (excitation at 470-495 nm, detection at 515-575 nm). The cell plates were then incubated at 37 ℃, 5% CO2, 95% relative humidity for an additional 15 minutes before being read with Tetra to assess antagonist activity. Calcium-mediated changes in fluorescence were monitored every 1 second over a 60 second period, with Tetra adding 8.5 μ Ι _ from NPFF EC60 plate (excitation at 470-495 nm, detection at 515-575 nm) at the 10 second time point. Data was derived from Screen works (molecular devices) using statistical analysis of the response to baseline (ROB), which presented the data as fold-response compared to baseline samples. Percent inhibition was calculated using the equation (1- (cmpd ROB/NPFF EC60 ROB)). times.100.

Measurement of cAMP. Stable human NPFFR1-CHO (ES-491-C) and NPFFR2-CHO (ES-490-C) cell lines were purchased from Perkinelmer and used with the Ultra kit (TRF0262) to detect cAMP accumulation in low volume 96-well plates. Stimulation buffer containing 1X HBSS, 5mM HEPES, 0.1% BSA stabilizer, and 0.5mM IBMX was prepared at room temperature and titrated to 7.4. Serial dilutions of agonist control NPFF were prepared in 2% DMSO/stimulation buffer at 8X the desired final concentration and 2.5 μ Ι _ was added to the assay plates.

a single concentration of each test compound was prepared at 4X the desired final concentration in 2% DMSO/stimulation buffer, and 5 μ Ι _ was added to the assay plate. Forskolin (forskolin) (1 μ M) at EC80 concentration was prepared at 8X in 2% DMSO/stimulation buffer and 2.5 μ L was added to the assay plate.

Cells were dispersed (lift) with versene and spun at 270g for 10 minutes. The cell pellet was resuspended in stimulation buffer and 4,000 cells (10 μ Ι _) were added to each well. After incubation at room temperature for 30min, Eu-cAMP tracer and uLIGHT-anti-cAMP working solution were added according to the manufacturer's instructions. After incubation at room temperature for 1 hour, the TR-FRET signal (ex 337nm) was read on a CLARIOstar multi-mode plate reader (BMG Biotech, Cary NC).

Fluorescence values at 665nm were plotted against the log of compound concentration using non-linear regression analysis to generate EC50 values (GraphPad Prism, GraphPad Software, inc., San Diego CA). The Ke value was calculated using the same equation as described in the calcium mobilization method.

Radioligand binding assay. Binding assays were performed according to the protocol of Perkinelmer in a final volume of 500. mu.L of assay buffer (50mM Tris-HCl, 1mM MgCl2, 60mM NaCl, 0.5% BSA, pH 7.4). For NPFF1, the assay mixture contained 25 μ L of 0.065nM [125I ] NPFF (PerkinElmer, KD 0.11nM, NEX381), 25 μ L of test compound (prepared at 8X final desired concentration in 8% DMSO/assay buffer), and 150 μ L of CHO-hNPFFR1 membrane (1 μ g protein/well, PerkinElmer, RBHNF1M400 UA). Specific binding is defined as the difference between [125I ] NPFF binding in the absence and presence of 100nM final non-radiolabeled NPFF. After incubation at 27 ℃ for 120 minutes, the binding assay was terminated by: vacuum filtered onto a Unifilter GF/C glass fiber filter (pre-soaked in 0.1% PEI) using a Brandel (Gaithersburg, Md., USA) 96-well harvester followed by three washes with ice cold wash buffer (50mM Tris-HCl, 0.1% BSA, pH 7.4). The filter plate was dried at 55 ℃ for 1 hour. Microscint 20(50 μ L) was added to each well and the filter-bound radioactivity was counted on a Packard TopCount NXT microplate scintillation and luminescence counter. The percentage of specific [125I ] NPFF binding was plotted against the log of compound concentration.

Data were fitted to a site (fitting Ki) competitive binding model using GraphPad Prism (GraphPad Software, inc., San Diego CA), logEC50 ═ log (10^ logKi ^ 1+ radioligand NM/HotKdNM), to generate Ki values for test compounds. NPFF2 binding assay was performed using the same protocol except using 0.1nM [125I ] NPFF (PerkinElmer, KD ═ 0.15nM, NEX381) and CHO-hNPFFR2 membrane (1 μ g protein/well, PerkinElmer, RBHNF2M400 UA).

Kinetic solubility determination. mu.L of a stock solution of test compound (10mM DMSO) was combined with 490. mu.L of PBS (potassium dihydrogen phosphate 1mM, disodium hydrogen phosphate 3mM, and sodium chloride 155mM buffer). The solution was stirred at room temperature on a VX-2500 multi-tube vortex mixer (VWR) for 2 hours. After stirring, the sample was filtered on a glass fiber filter (1 μm) and the eluate was diluted 200-fold with a mixture of acetonitrile: water (1: 1). Nicardipine (nicardipine) and imipramine (imipramine) were used as reference compounds at each experimental occasion, and low solubility and high solubility were evaluated, respectively. All samples were evaluated in triplicate and analyzed by LC-MS/MS for standards prepared in the same matrix using electrospray ionization.

Two-way MDCK-MDR1 permeability assay. MDCK-mdr1 cells at passage 5 were seeded on a permeable polycarbonate support in 12-well plates and allowed to grow and differentiate for 3 days. On day 3, media (DMEM supplemented with 10% FBS) was removed from both sides of the transwell insert and the cells were washed with warm HBSS. After the washing step, the chamber was filled with warm transport buffer (HBSS containing 10mM HEPES, 0.25% BSA, pH 7.4) and the plates were incubated at 37 ℃ for 30min before TEER (transepithelial resistance) measurements were performed.

The buffer in the donor compartment (top side of the a-to-B assay, substrate side of the B-to-a assay) was removed and replaced with working solution (10 μ M test article in transfer buffer). The plate was then placed at 37 ℃ with gentle stirring. At the indicated time points (30min, 60min and 90min), aliquots of the transfer buffer were removed from the receiving chamber (receiver chamber) and replenished with fresh transfer buffer. The samples were quenched with ice-cold ACN containing an internal standard and then centrifuged to pellet the proteins. The resulting supernatant was further diluted with 50/50ACN/H2O (H2O only for Atenolol (Atenolol)), and submitted for LC-MS/MS analysis. The reported apparent permeability (Papp) values were calculated from a single assay. Atenolol and propranolol (propranolol) were tested as low and medium permeability references. Bidirectional transport of digoxin (digoxin) was evaluated to demonstrate Pgp activity/expression.

The apparent permeability (Papp, measured in cm/s) of a compound is determined according to the following formula:

dQ/dt is the net apparent rate in the receiving compartment

A is the area of the Transwell measured in cm2 (1.12cm2)

Ci is the initial concentration of compound added to the donor compartment

And 60 is the conversion factor from minutes to seconds.

In vivo pharmacology.

An animal. Adult male (n-18) Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 225g-300g were individually housed IN a light/dark cycle of 12/12 hours, with behavioral experiments performed during the light period. Except during the testing period, the rats were freely available for food and water and were maintained and used by the National Academy of Sciences, Washington, D.C. according to the guidelines of the International Association for the Study of Panel (Zimmermann, M., Ethical guidelines for the administration of experimental patients in the relevant industries and Animals, Panel, 16,109, 110,1983) and the guidelines of the 2011 Laboratory Animal Care and Use of Laboratory Animals (the Institute of Life Sciences Laboratory Resources Institute, the National Research Committee, the National Academy of Sciences, Washington, D.C. (Institute of Laboratory Resources Life Sciences, National Research, National Institute of Laboratory Resources, Life Sciences, National Research, maintenance and Use of Laboratory Sciences, National Research and institutes, the Committee of health of Sciences, Washington, D.C. and maintenance and Use of Laboratory Research, and institutes, the Committee of health Care and Use of health Care, Committee, United nationality and institutes of Research.

A medicine is provided. Fentanyl was purchased from Sigma-Aldrich (st. louis, MO), dissolved in 0.9% saline, and injected subcutaneously in a volume of 1 ml/kg. Compound 16 and compound 33 were dissolved in a vehicle of 20% dimethyl sulfoxide in saline and injected intraperitoneally in a volume of 1 ml/kg.

Fentanyl-induced hyperalgesia. Pain thresholds were measured using calibrated von Frey filaments (1.4g-26 g; North Coast Medical, Morgan Hill, Calif.). Rats (n-6/group) were placed in an overhead plastic chamber with wire mesh bottom (IITC Life Science inc., Woodland Hills, CA) and allowed to habituate before testing. The filaments are applied in ascending order of filament force (filament force), starting with the lowest filament, from below the mesh base, perpendicular to the plantar medial surface of the hindfoot. The wire is applied until buckling occurs for about two seconds. The mechanical rearfoot threshold (PWT) corresponds to the lowest force that causes rearfoot to be rearfoot in at least two of the three applications. A force greater than 26g will physically lift the foot without CFA treatment and not reflect pain-like behavior.

On the day before and on the day of fentanyl treatment (D-1 and D0), a baseline of pain was established for each rat, followed by 4 subcutaneous injections of 0.06mg/kg fentanyl, each at 15min intervals, for a total dose of 0.24 mg/kg. In a group of rats, PWT measurements were taken on days 1-5 to monitor the onset and recovery from fentanyl-induced hyperalgesia. In the other two groups of rats, the anti-nociceptive dose-response curves for compound 16 and compound 33 were established on day 1 using a multi-cycle cumulative dosing procedure, with measurements taken immediately prior to drug administration, then 30min after drug administration before the next injection, and continued for doses in the range from 3.2mg/kg-32 mg/kg.

And (6) analyzing the data. PWT in each group was averaged and plotted as a function of dose. Repeated measures one-way ANOVA was used with time or treatment as the in-subject factor input followed by Bonferroni post-hoc test to determine statistical significance. For all tests, P <0.05 was considered statistically significant.

Pharmacological evaluation of Compound 16 and Compound 33

Compound 16 and compound 33 were identified as potent NPFF1 antagonists and were selected for further characterization and evaluation.

The concentration-response curves for compound 16 in NPFF1 and NPFF2 calcium mobilization assays are shown in fig. 4, where a panel shows antagonist activity of compound 16 in the NPFF1 calcium mobilization functional Ke assay, and B panel shows antagonist activity of compound 16 in the NPFF2 calcium mobilization functional Ke assay. A shows concentration-response curves for NPFF (°) alone and NPFF +5 μ M final 16(□) in stable NPFF1-RD-HGA16 cells. Panel B shows concentration-response curves for NPFF alone (o) and NPFF +10 μ M final 16(□) in stable NPFF2-RD-HGA16 cells. A right shift of the NPFF curve in the presence of the test compound was used to calculate the Ke value. Representative data from one experiment is shown, and each data point is the mean ± SD of duplicate determinations.

concentration-response curves for compound 16 and compound 33 in NPFF1 and NPFF2 cAMP assays are shown in fig. 5, where a panel shows antagonist activity of compound 16 and compound 33 in NPFF1 cAMP functional Ke assay, and B panel shows antagonist activity of compound 16 and compound 33 in NPFF2 cAMP functional Ke assay. The A-diagram shows the concentration-response curves for NPFF alone (. smallcircle.), NPFF + 4. mu.M final 16(□) and NPFF + 2. mu.M final 33 (. diamond.) in stable NPFF1-CHO cells. The B plot shows the concentration-response curves for NPFF alone (. smallcircle.), NPFF + 10. mu.M final 16(□) and NPFF + 10. mu.M final 33 (. diamond.) in stable NPFF2-CHO cells. A right shift of the NPFF curve in the presence of the test compound was used to calculate the Ke value. Each data point is the mean ± SEM of at least N-3 performed in duplicate.

In the cAMP assay, the NPFF +16 curve had a Hill slope (Hill slope) of 1.3 in both NPFF1 cells and NPFF2 cells, while the NPFF +33 curve had Hill slopes of 1.4 and 1 in NPFF1 cells and NPFF2 cells, respectively, thus indicating that the activity of these compounds is not due to aggregate formation. These results correlate with results from calcium mobilization assays, where the NPFF +16 curve has Hill slopes of 1.7 and 1.1 in NPFF1 and NPFF2 cells, respectively, and the NPFF +33 curve has a Hill slope of 1.4 in both cell lines.

Table 6 below lists selected physicochemical and preliminary ADME (absorption, distribution, metabolism and excretion) properties determined for compound 1, compound 16 and compound 33.

TABLE 6

HBD: an H bond donor; HBA: an H bond receptor. N.D.: it is not determined.

Compound 16 and compound 33 were tested as free base forms in kinetic solubility and two-way MDCK-MDR1 permeability assays. As can be seen in table 6, compound 16 was soluble in aqueous solution, showing a kinetic solubility of 146.8 ± 6.9 μ M (mean ±% CV), compound 16 falling within the range of compounds with good solubility, while compound 33 had a lower solubility of 45.9 ± 7.7 μ M, which is expected for larger molecules with higher molecular weights. In addition, these compounds contain more than one protonatable nitrogen atom, which can be converted to salt form to improve solubility and bioavailability.

One of the major challenges of Central Nervous System (CNS) drugs is their ability to cross the Blood Brain Barrier (BBB) and reach the CNS. For most drugs, BBB permeability is affected by two factors: the ability to passively penetrate through the BBB and avoid efflux via transport proteins such as P-glycoprotein. Compound 16 and compound 33 were evaluated in a bidirectional transport assay using MDCK-MDR1 cells, which were stably transfected with human MDR1 cDNA and expressed higher levels of P-glycoprotein (Pgp) compared to wild-type. Table 6 shows that compound 16 crossed the cellular barrier from apical (A) to basolateral (B) at a rate of 7.6X 10-6cm/s and in the reverse direction B to A at a rate of 6.7X 10-6cm/s, demonstrating moderate BBB permeability (in the range of 3X 10-6 cm/s-6X 10-6 cm/s). Compound 33 was also able to penetrate the BBB, although less permeable than compound 16. Neither compound is a Pgp substrate, as indicated by the efflux ratio (PB → a/PA → B). Taken together, the data demonstrate the properties of these compounds as CNS positive ligands.

Anti-hyperalgesic Effect of Compound 16 and Compound 33 in rats

Compound 16 and compound 33 were tested in the rat fentanyl-induced hyperalgesia model and the results are shown in figure 6, with a in figure 6 showing the results of fentanyl-induced mechanical hyperalgesia and B in figure 6 showing the anti-hyperalgesic effects of compound 16 and compound 33 (N ═ 6/group). The ordinate in the figure is the let-down threshold in g, and the abscissa is time. P <0.05 compared to before fentanyl treatment (day 0) or compared to "V" (vehicle) treatment.

Prior to fentanyl treatment, the test rats showed a mean withdrawal threshold (PWT) of 24.2 ± 1.8g, administered i.p., which decreased to 5.7 ± 0.8g on day 1 after fentanyl administration. One-way repeated measures ANOVA, with time as the repeated measures factor input, revealed a significant major effect of fentanyl treatment on withdrawal thresholds (F (6,35) ═ 23.42, p < 0.0001). Bonferroni post-hoc tests revealed significant differences between day 1-day 3 compared to day 0.

As shown in figure 6, both compound 16 and compound 33 increased PWT dose-dependently over the dose range of 3.2mg/kg-32mg/kg when tested on day 1. Treatment with compound 16 produced a significant primary effect as determined by one-way repeated measures ANOVA, with treatment as an in-subject factor input: f (3,20) ═ 15.10, p < 0.0001. Furthermore, Bonferroni post hoc tests revealed significant differences between 16 at 10mg/kg and 32mg/kg compared to vehicle. Similarly, treatment with compound 33 produced a significant primary effect (F (3,20) ═ 12.45, p <0.001), and Bonferroni post hoc testing revealed a significant difference of 32mg/kg of 33 compared to vehicle.

The foregoing results demonstrate the effectiveness of compound 16 and compound 33 in reversing opioid-induced hyperalgesia in rats as model systems for human response to such compounds.

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Accordingly, the present disclosure contemplates neuropeptide FF receptor modulators comprising a compound according to formula (I) herein, wherein R2 is selected from-N- (C2-C5 alkyl) 2 and NH-R1, wherein R1 is selected from C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; r3 is selected from the group consisting of C3-C9 alkyl, aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocycloalkyl, and arylalkyl; r4 is selected from H and C1-C2 alkyl; and R5 is selected from C3-C9 alkyl, heteroarylalkyl, heteroaryl, heterocycloalkyl, heterocycle, cycloalkylalkyl, and arylalkyl; or a pharmaceutically acceptable salt thereof. Such neuropeptide FF receptor modulators may be comprised of: r2 is NH-R1 wherein R1 is selected from C3-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; for example, R1 can be C3-C6 alkyl, or alternatively, R1 can be phenethyl substituted with lower alkoxy, nitro, lower alkyl, halogen, or halogenated lower alkyl.

Neuropeptide FF receptor modulators may be configured to include compounds of formula II, wherein R2 is selected from-N- (C2-C5 alkyl) 2; and X is S, SO2, O, NH, or CH 2.

Alternatively, neuropeptide FF receptor modulators may be constructed comprising a compound of formula IIA, wherein R1 is selected from the group consisting of C2-C9 alkyl, heterocycloalkyl, cycloalkylalkyl, aminoalkyl and arylalkyl; and X is S, SO2, O, NH, or CH 2; for example, R1 may be substituted or unsubstituted C3-C6 alkyl, benzyl, or phenethyl, and X may be oxygen. More specifically, R1 may be C3-C6 alkyl, or R1 may be phenethyl substituted with lower alkoxy, nitro, lower alkyl, halogen or halogenated lower alkyl.

Neuropeptide FF receptor modulators can be configured to include compounds of formula III, wherein R3 is selected from the group consisting of C3-C9 alkyl, aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocycloalkyl, and arylalkyl; and R4 is selected from H and C1-C2 alkyl. Modulators may be constructed wherein R3 is benzyl or substituted benzyl, phenethyl or substituted phenethyl, and R4 is H. Alternatively, modulators may be constructed wherein R3 is benzyl monosubstituted with methoxy; and R4 is H. Alternatively, modulators may be constructed wherein R3 is benzyl or substituted benzyl, and R4 is methyl. As still further options, modulators may be constructed wherein R3 is a C3-C6 alkyl.

Neuropeptide FF receptor modulators can be configured to include compounds of formula IV, wherein R5 is selected from C3-C9 alkyl, heteroarylalkyl, heteroaryl, heterocycloalkyl, heterocycle, cycloalkylalkyl, and arylalkyl; and X is S, SO2, O, NH, or CH 2. Modulators may be constituted wherein R5 is benzyl or substituted benzyl, for example wherein R5 is mono-substituted benzyl and the substituent is halogen or methoxy in the 2 or 3 position.

In another aspect, a neuropeptide FF receptor modulator may be comprised of a compound having the structure listed in table 1 as such, wherein R2 is selected from the group consisting of the R2 species listed in such table.

In further aspects, the neuropeptide FF receptor modulator may be comprised of a compound having the structure listed thus far in table 2, wherein R1 is selected from the group consisting of the R1 species listed in such table.

In further aspects, the neuropeptide FF receptor modulator may be comprised of a compound having the structure listed thus far in table 3, wherein R3 and R4 are selected from the group consisting of the R3 and R4 species listed in such table.

In yet another aspect, a neuropeptide FF receptor modulator may be comprised of a compound having the structure listed in table 4 as such, wherein R5 is selected from the group consisting of the R5 species listed in such table.

In further aspects, the neuropeptide FF receptor modulator may be comprised of the following compounds:

In still further aspects, the neuropeptide FF receptor modulator may be comprised of a compound of:

The present disclosure also contemplates pharmaceutical compositions comprising a neuropeptide FF receptor modulator as variously described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may also comprise an opioid, for example, at least one selected from the group consisting of: fentanyl, morphine, oxycodone, hydrocodone, and buprenorphine. Alternatively, the pharmaceutical composition may further comprise an antipsychotic drug, for example, at least one selected from the group consisting of haloperidol and aripiprazole. As still further options, the pharmaceutical composition may further comprise a monoamine reuptake inhibitor, e.g. at least one selected from the group consisting of fluoxetine and sertraline.

The present disclosure also contemplates a method for treating a subject suffering from or susceptible to a condition or disorder in which modulation of neuropeptide FF receptor activity is of therapeutic benefit, comprising administering to a subject suffering from or susceptible to such condition or disorder a therapeutically effective amount of a neuropeptide FF receptor modulator as variously described herein. In such methods, the condition or disorder may include the use or abuse of one or more opioids, for example, fentanyl, morphine, oxycodone, hydrocodone, buprenorphine, heroin, and opioid derivatives of the foregoing. In the methods broadly described above, the therapeutic benefit may include at least partial reduction of opioid-induced hyperalgesia, e.g., wherein the opioid-induced hyperalgesia is induced by an opioid comprising at least one selected from the group consisting of: fentanyl, morphine, oxycodone, hydrocodone, and buprenorphine. The methods broadly described above can be performed wherein the administration of a neuropeptide FF receptor modulator comprised as variously described herein is performed in a therapeutic intervention comprising co-administering an agent that reduces a side effect of the neuropeptide FF receptor modulator, e.g., wherein the agent that reduces a side effect of the neuropeptide FF receptor modulator is an agent that produces tolerance or hyperalgesia as a side effect.

The methods broadly described above can be carried out in specific embodiments, further comprising administering an effective amount of a second therapeutically effective agent. Alternatively, the methods broadly described above may be carried out in other embodiments, wherein the condition or disorder in which modulation of neuropeptide FF receptor activity is of therapeutic benefit is selected from the group consisting of: reduction of opioid tolerance and reduction of hyperalgesia.

Thus, it will be understood that the present disclosure contemplates a wide variety of compounds, neuropeptide FF receptor modulators, pharmaceutical and therapeutic compositions and formulations, and methods of making and using the foregoing.

Thus, while the present invention has been described herein with reference to particular aspects, features and illustrative embodiments thereof, it will be understood that the utility of the present invention is not thus limited, but rather extends to and encompasses numerous other variations, modifications and alternative embodiments, as will occur to those of ordinary skill in the art to which the present disclosure pertains based on the description herein. Accordingly, the subject matter as claimed in the claims is intended to be broadly construed and interpreted, as including all such variations, modifications and alternative embodiments, within its spirit and scope.