Solid-Phase Synthetic Route to Multiple Derivatives of a Fundamental Peptide Unit

Abstract

Amino acids are Nature’s combinatorial building blocks. When substituted on both the amino and carboxyl sides they become the basic scaffold present in all peptides and proteins. We report a solid-phase synthetic route to large combinatorial variations of this fundamental scaffold, extending the variety of substituted biomimetic molecules available to successfully implement the Distributed Drug Discovery (D3) project. In a single solid-phase sequence, compatible with basic amine substituents, three-point variation is performed at the amino acid α-carbon and the amino and carboxyl functionalities.

Introduction

The Distributed Drug Discovery (D3) project seeks to simultaneously educate and innovate while searching for drug leads for neglected diseases [1,2,3,4,5,6]. Central to this effort is the availability of simple, inexpensive and reproducible synthetic procedures providing access to large numbers of biomimetic molecules. To increase the diversity of molecule types available to D3 we decided to develop more flexible synthetic routes to derivatives of the peptide unit 1, one of the most fundamental and inherently biomimetic scaffolds in nature. There are many examples of drugs or drug leads based on this scaffold. They arise from natural or unnatural amino acids modified at both the amino and carboxylic acid functionalities. These include the recently approved drug lacosamide (Vimpat^®, 1a) to treat epilepsy [7]. Special cases of 1 are 2 and 3, in which R³ contains a basic amino group and n = 1 or 2. Compounds 3 have been shown to be selective binding agents for subclasses of 5-hydroxytryptamine (5-HT, serotonin) receptors [8]. This paper reports a flexible solid-phase synthetic route to many variations of R¹, R², and R³ on structures 2 and 3. It will be the basis for future synthetic laboratories compatible with the D3 process.

Figure 1. Important molecules containing the fundamental peptide unit 1.

Figure 1. Important molecules containing the fundamental peptide unit 1.

Compounds 1 can be readily synthesized on solid-phase using BAL type resins and naturally occurring amino acids [9,10]. For more extensive sets of derivatives, unnatural amino acid side chains R¹ can be introduced during the course of the solid-phase synthesis utilizing the recently described chemistry outlined in Scheme 1 [6].

Scheme 1. Synthetic route to amino- and carboxyl-substituted unnatural amino acids.

Scheme 1. Synthetic route to amino- and carboxyl-substituted unnatural amino acids.

Compounds 3 are usually constructed from naturally occurring amino acids in which the amino group is acylated with a variety of carboxylic acids and the carboxyl group is amidated with amines containing an additional basic amino group. The solid-phase synthesis of 3 utilizes BAL-type linkers, “lantern” technology, and either of two synthetic routes (Scheme 2, Path A or B). While Path A provides a direct route to product, Path B (through intermediate 17) permits greater flexibility by incorporation of many amines after attachment of the amino acid onto the BAL resin [11].

Scheme 2. Synthetic pathways A and B to compounds 3.

Scheme 2. Synthetic pathways A and B to compounds 3.

Results and Discussion

We sought to adapt the route shown in Scheme 1 to the synthesis of a more diverse set of compounds 2 and 3, where R¹ would possess both R and S configuration, not be restricted to naturally occurring amino acid side chains, and R³ in 2 contained basic amine functionality that could be readily varied. However, a key step in this synthesis would be the alkylation of intermediate 8 to 9 (Scheme 1), and any nucleophilic functionality present (such as the tertiary amines present in 3) would likely lead to side reactions. Therefore, we decided to modify our chemistry to perform the alkylation on a derivative of key intermediate imine 8 that would be compatible with this alkylation chemistry and permit subsequent simple conversion to multiple derivatives 2 even if R³ contained nucleophilic sites. Since it had already been shown that silyl protected intermediate 17 could be readily converted into 3 (Scheme 2), the silyl protected imine derivative 23 was chosen for this role (Scheme 3). It permitted alkylation to 24 without side reactions. This provided access to many additional derivatives 2 after deprotection of the silyl group (25 to 26), activation of the alcohol for displacement reactions (through either the mesylate 27a or the more reactive iodide 27b) and displacement with a variety of amines.

Model studies to 2 were performed with four alkylating agents R¹X: [R¹ = CH₂Ph, CH₂CH=CH₂; (CH₂)₇CH₃; CH₂Ph-4-CF₂-P(O)(OEt)₂] (Scheme 3). The acylating agent (R² = 4-NC-Ph), nucleophile R³ and chain length were kept constant. In addition these initial studies, which focused on the compatibility of the protected silyl alcohol with alkylation chemistry, utilized the alcohol activation and amine incorporation procedures (via mesylate 27a) employed in the earlier lantern-based work [11]. Products for this 11-step synthesis were obtained in moderate overall yields (Table 1). Structure 2d, which incorporates a stable, protected phosphotyrosine analog [6], gives a particular example of the interesting types of molecules available by this route.

Scheme 3. Synthesis of multiply-substituted 2 where R³ contains a nucleophilic site.

Scheme 3. Synthesis of multiply-substituted 2 where R³ contains a nucleophilic site.

Table 1. Survey of alkylating agents with incorporation of a single amine.

Table 1. Survey of alkylating agents with incorporation of a single amine.

In this preliminary work a variety of by-products were formed during the alcohol to amine transformation (26 to 28 via 27a, Scheme 3). To minimize side-reactions and provide an alternative activation procedure, we developed a modified route through iodide intermediate 27b. A direct comparison of these two routes gave 2a in 65% crude purity via the mesylate intermediate 27a and 88% crude purity via the iodide 27b. We utilized this procedure (through the iodide) to create 32 new compounds and demonstrate the ability to introduce three points of variability (with basic functionality in R³) in structure 2. The results with purified yields are shown in Table 2.

Table 2. 32 compounds from 4 alkylating agents, 4 carboxylic acids and 2 amines.

Table 2. 32 compounds from 4 alkylating agents, 4 carboxylic acids and 2 amines.

Conclusions

This procedure provides ready access to the fundamental peptide scaffold 1 with multiple substitutions at positions R¹, R², and R³. The average overall purified yield to combinatorially prepare the 32 compounds 3a-3af using this 11-step synthetic sequence was 46%. The R³ site can contain basic residues. Alternatively, based on ample precedent [12], the key iodide intermediate 27b could provide access to many other interesting and valuable derivatives of 2 via simple nucleophilic displacement reactions.

Experimental

General

Organic solvents were of reagent grade and used directly without purification. Chloroform-d₁, 1-chloro-3-iodopropane, 1-(3-chlorophenyl)piperazine, cyclohexanecarboxylic acid, DIPCDI (N,N’-di-isopropylcarbodiimide), anhydrous DMF (N,N-dimethylformamide), 1-iodohexane and methane-sulfonyl chloride were purchased from Acros Organics. TFA (trifluoroacetic acid) and hydrochloric acid were obtained from Fisher Scientific. BTPP [tert-butylimino-tri(pyrrolidino)phosphorane] was purchased from Fluka. 3-Amino-1-propanol, benzoic acid, benzoyl peroxide, NBS (N-bromo-succinimide), tert-butylchlorodiphenylsilane, 1-chloro-4-iodobutane, 1-chloro-5-iodopentane, cyclo-propanecarboxylic acid, 4-cyanobenzoic acid, 3,4-dichlorobenzaldehyde, 1-(2,3-dichlorophenyl)-piperazine hydrochloride, α,α’-dichloro-o-xylene, DAST [(diethylamino)sulfur trifluoride], DIEA (N,N-diisopropylethylamine), HOBt (1-hydroxybenzotriazole hydrate), imidazole, 1-iodooctane, methanol-d₄, 1-(2-methoxyphenyl)piperazine, anhydrous NMP (1-methyl-2-pyrrolidinone), piperidine, anhydrous pyridine, quinaldic acid, 1,2,3,4-tetrahydroisoquinoline, TBAF (tetrabutylammonium fluoride), triethylamine, anhydrous TMOF (trimethyl orthoformate), and triphenylphosphine were obtained from Aldrich Chemical Co. Iodine was obtained from J. T. Baker. 4-Methylbenzhydrylamine hydrochloride resin (PL-MBHA·HCl, 1.6 mmol/g, 75–150 μm) was purchased from Polymer Laboratories. 4-(4-Formyl-3,5-dimethoxyphenoxy)butyric acid (BAL linker), Fmoc-Gly-OH (N-Fmoc-glycine), and HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate) were purchased from NovaBiochem. Diethyl [(4-α-bromomethyl)phenyldifluoromethyl]phosphonate [6] and O-tert-butyldiphenylsilyl-3-aminopropanol hydrochloride [13] were prepared according to literature procedures. Solution and solid-phase organic transformations and resin washes were carried out at ambient temperature, unless indicated otherwise. Ratios in all solvent and reagent mixtures prepared are volume to volume unless otherwise noted.

Manual solid-phase organic syntheses were carried out in two types of reaction vessels. Peptide synthesis reaction vessels (50 mL) with coarse porosity fritted glass support and supplied with a GL thread and a Teflon-lined PBT screw cap (ChemGlass, CG-1860-03) were used for large scale (up to 3.7 mmol) reactions. Small scale reactions (typically 50 μmol) were performed in 3.5 mL fritted glass reaction vessels (Chemglass, IUP-0305-270H) equipped with polypropylene screw caps (Chemglass, CV-3730-G013) with Teflon-faced silicon septa (Chemglass, CV-4080-0013) on the Bill-Board set, which was designed by one of us (WLS) as inexpensive equipment [2,14] to simplify and expedite multiple, manual solid-phase syntheses. For agitation purpose, the large scale reactions in the peptide synthesizers were placed on an orbital shaker Roto Mix (Type 50800 by Thermolyne) while appropriate motor rotators were used as rotation apparatus for small scale reactions.

Depending on the number of reactions to be performed, the starting resin was distributed either by weight or as aliquots from an isopycnic suspension [2]. In the case of distribution by volume from an isopycnic suspension, the Bill-Boards were placed in their drain trays, and from a neutral buoyancy suspension in CH₂Cl₂–NMP, 50 μmols of the starting resin (with a known loading) was typically distributed, via repeated aliquots (1 mL), to each of the reaction vessels in a given Bill-Board (6-pack or 24-pack). During the distribution of the resin, the isopycnic solvent was allowed to drain through the frit in the reaction vessels. When distribution was complete, residual solvent was removed with an “air-push” from a disposable plastic pipet (Fisher, 13-711-23) fitted with a pierced septum (Aldrich, Z 12743-4). The resin was then washed with an appropriate solvent (this solvent wash was also carried out when the resin was weighed into the reaction vessels). The bottom of each reaction vessel was then capped, and a new calibrated pipet (Fisher, 13-711-24) was used for adding each reagent in the following step. The tops of all reaction vessels were capped and the Bill-Board was placed on an appropriate rotation apparatus. Following the reaction the reagents and solvents were drained and the resin product was then washed with the indicated solvents. Resin-bound intermediates were air-dried after the final CH₂Cl₂ washes, unless re-weighing was necessary, in which case overnight drying was carried out under high vacuum (≤ 0.2 mm Hg) or under low vacuum (house vacuum) for 24–36 h in a vacuum desiccator. During resin washing with solvents for large scale reactions, at least 3 min of solvent contact with the resin in the reaction vessels (bottom closed) was performed, then the resin was drained, followed by an air-push. For washing of small scale reactions, at least 30 sec was normally used after addition of solvents to the reaction vessels (with bottom end open for draining) followed by an air-push. Solid-phase reactions at elevated temperatures (50 °C, 60 °C and/or 80 °C) were carried out in an Isotemp® Oven Model 280A (Fisher Scientific) with the reaction vessels capped to finger tightness.

Analytical thin layer chromatography (TLC) was performed with EM Science silica gel 60 F₂₅₄, 0.25 mm pre-coated glass plates (EMD Chemical Inc., 5715-7). TLC plates were visualized using UV₂₅₄. Column chromatography was performed on HyperSep SI® 3 mL cartridges (60108-315) pre-loaded with 500 mg of silica gel 60 (irregular particles 40–63 μm) from Thermo Electron Corporation. The yields of the final compounds, after chromatographic purification, were calculated on the basis of the initial loading of the starting resins and are the overall yields for all reaction steps starting from these resins. ¹H-NMR (500 MHz) and ¹³C -NMR (125 MHz) spectra were recorded on a Bruker Avance III 500 spectrometer. Chemical shifts are reported in parts per million (ppm) and are referenced to the centerline of chloroform-d₁ (δ 7.26 ppm ¹H-NMR, 77.0 ppm ¹³C-NMR) using TMS (0.00 ppm), or chloroform-d₁ mixed with methanol-d₄ (2–10%). Coupling constants are given in Hertz (Hz).

Electrospray ionization mass spectrometry was conducted using a PESciex API III triple stage quadrupole mass spectrometer operated in either positive-ion or negative-ion detection mode. LC/MS analyses were conducted using an Agilent system, consisting of a 1100 series HPLC connected to a diode array detector and a 1946D mass spectrometer configured for positive-ion/negative-ion electro-spray ionization. The LC/MS samples were analyzed as solutions in CH₃CN, prepared at 0.08–0.12 mg/mL concentration. The LC/MS derived composition of mixtures was determined based on UV integration at 210 nm. The LC/MS chromatography was carried out on an Agilent Zorbax SB–C₈ column (PN835975-906; 4.6 × 50 mm, 3.5 μm) with linear gradients of 0.1% TFA in CH₃CN and 0.1% aqueous TFA and were run at 1.0 mL/min flow rate from 20:80 to 90:10 for 25 min. The composition of reaction mixtures was determined based on the integration of NMR spectra as well as LC/MS results. High-resolution mass spectrometry was obtained on a MAT 95XP (Thermo Electron Corp.) with chemical ionization (CI), electron impact (EI), or fast-atom bombardment (FAB) mode.

General procedures for manual solid-phase organic syntheses

Preparation of MBHA-BAL Resin 4: PL-MBHA·HCl resin (1.03 g, 1.65 mmol, 1.60 mmol/g) loaded in a 50 mL peptide synthesis reaction vessel was sequentially washed with DMF (3 × 17 mL), CH₂Cl₂ (2 × 17 mL), 10% DIEA/CH₂Cl₂ (5 × 17 mL) and CH₂Cl₂ (2 × 17 mL). A solution of 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid (1.77 g, 6.60 mmol, 4 equiv), HBTU (2.50 g, 6.60 mmol, 4 equiv) and DIEA (2.30 mL, 13.2 mmol, 8 equiv) in anhydrous DMF (23 mL) prepared immediately prior to the reaction was then added to the washed resin. The reaction mixture was agitated on an orbital shaker for 18 h. The completion of the reaction was confirmed by a negative chloranil test [15]. The resultant orange yellow MBHA-BAL resin 4 was washed with DMF (3 × 17 mL) and CH₂Cl₂ (4 × 17 mL), and dried under low vacuum for 24 h.

Preparation of the Secondary Amine-Bound Resin 21 by Reductive Amination: The dried MBHA-BAL resin 4 (1.51 g, 1.65 mmol) was swelled in CH₂Cl₂ (17 mL) for 1 h, followed by DMF washes (3 × 17 mL). O-tert-Butyldiphenylsilyl-3-aminopropanol hydrochloride [13] (8.66 g, 24.75 mmol, 15 equiv) was added as a solid followed by addition of DMF–TMOF (1:2, 28 mL) to the resin. Theresulting reaction mixture was heated to 60 °C for 24 h. The resin-bound Schiff base product was washed with DMF (4 × 17 mL), THF (5 × 17 mL), MeOH (2 × 17 mL), CH₂Cl₂ (2 × 17 mL), and MeOH (3 × 17 mL). After the resulting imine resin was swelled in CH₂Cl₂ (17 mL) for 30 min followed by THF (17 mL) wash, it was treated with a solution of NaBH₃CN (1.04 g, 16.5 mmol, 10 equiv) in 5% HOAc in THF–MeOH (1:1, 28 mL). The reaction mixture was agitated on an orbital shaker for 18 h. The resin was drained and washed with THF–MeOH (1:1, 3 × 17 mL), MeOH (2 × 17 mL), THF (3 × 17 mL) and CH₂Cl₂ (3 × 17 mL). The amine resin product 21 was air-dried and was then used directly for subsequent coupling reactions.

Coupling of the Amine Resin 21 with N-Fmoc-Glycine using HBTU to Prepare Resin-Bound 22: The amine resin 21 (1.65 mmol) was swelled in CH₂Cl₂ (17 mL) for 30 min, and then it was washed with CH₂Cl₂–DMF (85:15, 17 mL). Fmoc-Gly-OH (2.45 g, 8.25 mmol, 5 equiv), HBTU (3.13 g, 8.25 mmol, 5 equiv), CH₂Cl₂–DMF (85:15, 39 mL) and DIEA (2.87 mL, 16.5 mmol, 10 equiv) were then added to the reaction vessel. The reaction mixture was agitated on an orbital shaker for 42 h. The completion of the reaction was indicated by a negative chloranil test [15]. The resulting Fmoc-Gly-BAL resin product 22 was then washed with DMF (3 × 17 mL), THF–MeOH (1:1, 3 × 17 mL), THF (3 × 17 mL), and CH₂Cl₂ (3 × 17 mL). The washed resin 22 was dried under low vacuum for 24–36 h.

Preparation of the 3,4-Dichlorobenzaldehyde Imine of Glycine on BAL Resin 23: The resin 22 (1.65 mmol) was swelled in CH₂Cl₂ (17 mL) for 30 min, and washed with DMF (5 × 17 mL), then it was treated with 20% piperidine/DMF (23 mL, 20 min) and subsequently washed with DMF (6 × 17 mL). The resin was swelled in CH₂Cl₂ (17 mL) for 30 min and washed with NMP (4 × 17 mL). A solution of 3,4-dichlorobenzaldehyde (1.25 M, 19.8 mL, 24.75 mmol, 15 equiv) in NMP–TMOF (1:2) was then added to the reaction vessel and the reaction mixture was rotated for 24 h. The resulting resin-bound Schiff base product 23 was sequentially washed with NMP (4 × 17 mL), THF (3 × 17 mL), CH₂Cl₂ (3 × 17 mL) and dried under low vacuum for 24 h.

Alkylation of the Aldimine of Glycine on BAL Resin 23 with Alkyl Halides, Subsequent Imine Hydrolysis, and Acylation with Carboxylic Acids: After resin-bound Schiff base 23 (50 μmol) was pre-swelled in CH₂Cl₂ for 1 h, it was washed with NMP (4 × 1 mL). To the resin was then added a solution of R¹X in NMP (2.0 M, 0.25 mL, 0.5 mmol, 10 equiv), followed by addition of BTPP solution in NMP (2.0 M, 0.25 mL, 0.5 mmol, 10 equiv). Alkylation was allowed to proceed at ambient temperature for 24 h with rotation. The alkylated resin product was filtered and washed with NMP (4 × 1 mL), CH₂Cl₂ (4 × 1 mL) and THF (3 × 1 mL). The resin was then treated with 1N HCl–THF (1:2, 1 mL) for 20 min. The resulting resin was filtered and washed with THF (3 × 1 mL), 10% DIEA/CH₂Cl₂ (5 × 1 mL), CH₂Cl₂ (2 × 1 mL). After the resin was swelled in CH₂Cl₂ (1 mL) for 1 h, and washed with DMF (3 × 1 mL), to the resin was added a solution of carboxylic acid R²COOH and HOBt in DMF (0.38 M, 0.66 mL, 0.25 mmol, 5 equiv), which was pre-mixed (6 - 10 min before addition) with DIPCDI (neat, 32 mg, 0.25 mmol, 5 equiv). The reaction mixture was allowed to rotate for 18 h. The completion of the reaction was confirmed by a negative chloranil test [15], and the filtered resin product 25 was washed with DMF (4 × 1 mL), THF (3 × 1 mL), CH₂Cl₂ (3 × 1 mL), and dried in air.

Model Studies for the Synthesis of 2a–2d via Deprotection of Silyl Ether 25 with TBAF, Subsequent Mesylation of Alcohol 26 using Mesyl Chloride in Pyridine, and N-Alkylation with Tetrahydroisoquinoline: The acylated resin product 25 (50 μmol) was washed with THF (3 × 1 mL), and swelled in THF for 1 h. TBAF (1 M in THF, 1.0 mL, 1.0 mmol, 20 equiv) was added to the drained resin, and the reaction mixture was allowed to rotate for 18 h. The drained resin was then washed with THF (5 × 1 mL) and CH₂Cl₂ (3 × 1 mL). After the alcohol resin 26 was swelled in CH₂Cl₂ for 1 h, a suspension of anhydrous pyridine with MsCl (1 M, 1.0 mL, 0.5 mmol, 20 equiv) was added to the resin, and the resulting mixture was rotated for 1 h. The drained resin product 27a was washed with DMF (3 × 1 mL), H₂O (1 × 1 mL), DMF (2 × 1 mL) and CH₂Cl₂ (5 × 1 mL). After the air-dried resin was swelled in CH₂Cl₂ for 30 min, to the resin was added a solution of tetrahydroisoquinoline in DMSO (1 M, 0.75 mL, 0.75 mmol, 15 equiv), and the reaction mixture was heated to 80 °C for 5 h. The resulting resin product was drained, washed with DMF (2 × 1 mL), H₂O (2 × 1 mL), DMF (3 × 1 mL), CH₂Cl₂ (4 × 1 mL), and air-dried. The resin product was then cleaved with 50% TFA/CH₂Cl₂ (1 mL) over 1.5 h, and the filtrate of the reaction mixture was collected and combined with washes of CH₂Cl₂ (2 × 1 mL) of the resin. A 100 μL sample of the combined solution was analyzed for crude purity by LC/MS. The cleavage solution was evaporated with a stream of nitrogen in a contained system with trapping of the evaporated TFA in 2N NaOH. The crude residue was re-dissolved in CH₂Cl₂, or CH₂Cl₂ with MeOH, if needed (total solution volume ≤0.5 mL), and purified using a pre-loaded silica gel cartridge with CH₂Cl₂–MeOH (95:5 or 93:7) to elute the purified product. Following solvent removal under N₂ flow, the purified product 2a–2d was normally obtained as an amorphous white solid or light yellow oil.

α-[(4-Cyanobenzoyl)amino]-N-[(3,4-dihydroisoquinolin-2(1H)-yl)propyl]-benzenepropanamide (2a): Product 2a was obtained from resin 23, benzyl bromide, 4-cyanobenzoic acid, and tetrahydroisoquinoline as an amorphous white solid (6 mg, 26% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 65%, t_R = 7.3 min; ¹H-NMR (CD₃OD/CDCl₃): δ 1.67–1.78 (m, 2H), 2.49–2.61 (m, 3H), 2.74–2.86 (m, 2H), 2.88–2.93 (m, 1H), 2.96 (dd, J = 8.0 Hz, J = 13.5 Hz, 1H), 3.03 (dd, J = 6.0 Hz, J = 13.5 Hz, 1H), 3.24–3.32 (m, 1H), 3.37–3.44 (m, 1H), 3.51–3.61 (m, 2H), 4.53 (dd, J = 7.7 Hz, J = 13.8 Hz, 1H), 6.95–7.01 (m, 2H), 7.02–7.06 (m, 2H), 7.12–7.18 (m, 3H), 7.19–7.23 (m, 3H), 7.32–7.36 (m, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.5 Hz, 2H). ¹³C-NMR (CD₃OD/CDCl₃): δ 23.7, 24.2, 35.8, 37.8, 49.1, 51.9, 52.5, 55.8, 115.2, 117.9, 126.1, 126.8, 126.9, 127.6, 128.1, 128.6, 128.7, 128.9, 129.2, 130.1, 132.2, 136.6, 137.3, 166.2, 172.6. HRMS calcd. for (M + H)⁺: C₂₉H₃₁N₄O₂ 467.2447, found 467.2431.

2-[(4-Cyanobenzoyl)amino]-N-[(3,4-dihydroisoquinolin-2(1H)-yl)propyl]-4-pentenamide (2b): This product was obtained from resin 23, allyl bromide, 4-cyanobenzoic acid, and tetrahydroisoquinoline as a light yellow oil (4 mg, 19% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (92:8). Initial LC/MS purity 60%, t_R = 5.4 min; ¹H-NMR (CD₃OD/CDCl₃): δ 1.97–2.04 (m, 2H), 2.49–2.58 (m, 1H), 2.60–2.68 (m, 1H), 3.06–3.11 (m, 3H), 3.24–3.31 (m, 1H), 3.32–3.38 (m, 1H), 3.40–3.48 (m, 2H), 4.03–4.19 (m, 2H), 4.29 (s, 1H), 4.61 (dd, J = 6.0 Hz, J = 7.7 Hz, 1H), 5.05–5.21 (m, 2H), 5.65–5.82 (m, 1H), 7.06 (d, J = 7.5 Hz, 1H), 7.08–7.14 (m, 1H), 7.18 (d, J = 7.1 Hz, 1H), 7.21–7.28 (m, 3H), 7.70 (d, J = 8.3 Hz, 2H), 7.97 (d, J = 8.5 Hz, 2H). ¹³C-NMR (CD₃OD/CDCl₃): δ 24.2, 25.3, 36.4, 36.7, 41.5, 44.4, 53.5, 53.8, 115.3, 118.0, 118.9, 126.6, 126.7, 127.2, 127.3, 128.1, 128.9, 129.0, 132.3, 132.8, 137.4, 166.1, 172.3. HRMS calcd. for (M + H)⁺: C₂₅H₂₉N₄O₂ 417.2212, found 417.2200.

2-[(4-Cyanobenzoyl)amino]-N-[(3,4-dihydroisoquinolin-2(1H)-yl)propyl]decanamide (2c): Product 2c was obtained from resin 23, 1-iodooctane, 4-cyanobenzoic acid, and tetrahydroisoquinoline as a light yellow oil (7 mg, 29% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (95:5). Initial LC/MS purity 65%, t_R = 11.8 min; ¹H-NMR (CD₃OD/CDCl₃): δ 0.86 (t, J = 7.0 Hz, 3H), 1.17–1.48 (m, 12H), 1.71–1.95 (m, 2H), 1.99–21.6 (m, 2H), 2.99–3.52 (m, 7H), 3.55–3.79 (m, 1H), 3.92–4.25 (m, 1H), 4.30–4.69 (m, 2H), 7.03-7.15 (m, 1H), 7.20 (d, J = 7.1 Hz, 1H), 7.24–7.34 (m, 3H), 7.71 (d, J = 8.5 Hz, 2H), 8.00 (d, J = 8.7 Hz, 2H). ¹³C-NMR (CD₃OD/CDCl₃): δ 14.0, 22.6, 23.8, 24.3, 25.9, 29.1, 29.2, 29.3, 31.7, 32.0, 35.7, 49.3, 52.2, 52.6, 55.0, 115.2, 118.0, 126.1, 126.8, 127.6, 128.2, 128.7, 128.9, 130.1, 132.2, 137.4, 166.5, 171.1. HRMS calcd. for (M + H)⁺: C₃₀H₄₁N₄O₂ 489.3151, found 489.3138.

α -[(4-Cyanobenzoyl)amino]-4-[(ethoxyhydroxyphosphinyl)difluoromethyl]-N-[(3,4-dihydroisoquinolin-2(1H)-yl)propyl]benzenepropanamide (2d): Product 2d was obtained from resin 23, diethyl [(4-α-bromomethyl)phenyldifluoromethyl]-phosphonate, 4-cyanobenzoic acid, and tetrahydroisoquinoline as a light yellow oil (6 mg, 19% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (92:8). Initial LC/MS purity 47%, t_R = 5.9 min; ¹H-NMR (CD₃OD/CDCl₃): δ 1.05–1.19 (m, 3H), 1.30–1.41 (m, 2H), 2.57–2.80 (m, 3H), 3.03–3.43 (m, 10H), 4.04–4.16 (1H), 4.25 (s, 1H), 4.70–4.83 (m, 1H), 7.08–7.36 (m, 8H), 7.56 (d, J = 7.3 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 8.01 (d, J = 8.2 Hz, 2H). ¹³C-NMR (125 MHz, CD₃OD/CDCl₃): δ 16.3, 22.9, 24.7, 36.1, 38.9, 41.6, 52.6, 52.8, 55.5, 62.6, 115.1, 117.9, 126.5, 126.7, 126.9, 127.1, 127.7, 128.0, 128.2, 128.5, 128.7, 128.9, 130.5, 132.2, 137.7, 165.6, 170.8. HRMS calcd. for (M + H)⁺: C₃₂H₃₇F₂N₄O₅P 625.2313, found 625.2326.

Direct Comparison of Activation of Alcohol Resin 26 via Mesylation (to 27a) with that via Iodination (to 27b) using Optimal N-Alkylation and Cleavage Conditions: The mesylation of the alcohol resin 26 was the same as described above. Iodination of the resin 26 was performed as follows. After the free alcohol resin 26 was swelled in CH₂Cl₂ for 1 h and washed with DMF (3 × 1 mL), a pre-mixed solution of iodine (63 mg, 0.25 mmol, 5 equiv), PPh₃ (66 mg, 0.25 mmol, 5 equiv), and imidazole (17 mg, 0.25 mmol, 5 equiv) in DMF (1.0 mL) was added. After 18 h, the filtered resin was washed with DMF (3 × 1 mL), MeOH (3 × 1 mL), DMF (2 × 1 mL), and CH₂Cl₂ (3 × 1 mL). After mesylated resin 27a and iodinated resin 27b were swelled in CH₂Cl₂ for 40 min, to each resin was added a solution of tetrahydroisoquinoline in DMSO (1 M, 0.75 mL, 0.75 mmol, 15 equiv). The reaction was heated to 80 °C over 3 h for mesylated resin or 50 °C over 6 h for iodinated resin. The resulting resin products were drained, sequentially washed with DMF (2 × 1 mL), MeOH (2 × 1 mL), DMF (3 × 1 mL), CH₂Cl₂ (4 × 1 mL), and cleaved with 90% TFA/CH₂Cl₂ (1 mL) over 1.5 h. The filtrate of the reaction mixture was collected and combined with washes of 50% TFA/CH₂Cl₂ (1 × 1 mL) and CH₂Cl₂ (1 × 1 mL) of the resin. A 100 μL sample of the combined solution was analyzed for crude purity by LC/MS. The crude purity for the product 2 was found to be 65% for the product through the mesylation and 88% for the product through the iodination process.

Synthesis of 32 New Resin Products 25 (Targeting Piperazine Derivatives 3 ) from the Same Aldimine of Glycine on BAL Resin 23 through Alkylation, Hydrolysis, and Acylation: Resin-bound Schiff base 23 (1.65 mmol) pre-swelled in CH₂Cl₂ for 1 h was evenly distributed to 33 of the reaction vessels in two separate 24-pack BillBoards via an isopycnic solution in CH₂Cl₂–NMP (9:5, v/v). 32 of the reaction vessels were arranged as two 4 × 4 grids on the BillBoards, and the 33^rd reaction vessel was put at position on A5 on one of the BillBoards for the quality control experiment for the resin 23. After the drained resin (50 μmol) was washed with NMP (4 × 1 mL), to the four reaction vessels down the first column positions (i.e. A1, B1, C1 and D1) on both BillBoards were added 1-iodohexane (2.0 M, 0.25 mL, 0.50 mmol, 10 equiv). To the four reaction vessels down the 2^nd column positions on both BillBoards were added 4-methoxybenzyl chloride (2.0 M, 0.25 mL, 0.50 mmol, 10 equiv). To the four reaction vessels down the 3^rd column positions on both BillBoards were added 3-fluorobenzyl chloride (2.0 M, 0.25 mL, 0.50 mmol, 10 equiv). To the four reaction vessels down the 4^th column positions on both BillBoards were added 4-fluorobenzyl bromide (2.0 M, 0.25 mL, 0.50 mmol, 10 equiv). To the control reaction vessel was added a solution of BnBr in NMP (2.0 M, 0.25 mL, 0.50 mmol, 10 equiv) for quality control of the starting resin 23.

Then a solution of BTPP in NMP (2.0 M, 0.25 mL, 0.50 mmol, 10 equiv) was added to each of the 33 reaction vessels. Alkylation was allowed to proceed at ambient temperature for 24 h with rotation. The alkylated resin product was filtered and washed with NMP (4 × 1 mL), CH₂Cl₂ (4 × 1 mL) and THF (3 × 1 mL). The resin was then treated with 1N HCl–THF (1:2, 1 mL) for 20 min. The resulting resin was filtered and washed with THF (3 × 1 mL), 10% DIEA/CH₂Cl₂ (5 × 1 mL), CH₂Cl₂ (2 × 1 mL). After the resin was swelled in CH₂Cl₂ (1 mL) for 1 h, and washed with DMF (3 × 1 mL), to the resins across row A positions (i.e. A1, A2, A3, A4) on both BillBoards were added a solution of cyclopropanecarboxylic acid and HOBt in DMF (0.38 M, 0.66 mL, 0.25 mmol, 5 equiv) which was pre-mixed (6 - 10 min before addition) with DIPCDI (32 mg, 0.25 mmol, 5 equiv). To the resins across row B positions on both BillBoards were added a solution of cyclohexanecarboxylic acid and HOBt in DMF (0.38 M, 0.66 mL, 0.25 mmol, 5 equiv) which was pre-mixed (6 - 10 min before addition) with DIPCDI (32 mg, 0.25 mmol, 5 equiv). To the resins across row C positions on both BillBoards were added a solution of benzoic acid and HOBt in DMF (0.38 M, 0.66 mL, 0.25 mmol, 5 equiv) which was pre-mixed (6 - 10 min before addition) with DIPCDI (32 mg, 0.25 mmol, 5 equiv). To the resins across row D positions on both BillBoards were added a solution of quinaldic acid and HOBt in DMF (0.38 M, 0.66 mL, 0.25 mmol, 5 equiv) which was pre-mixed (6 min before addition) with DIPCDI (32 mg, 0.25 mmol, 5 equiv; and it was observed that the colorless clear solution turned purple 2 min after DIPCDI was added). To the quality control reaction vial was added a solution of benzoic acid and HOBt in DMF (0.38 M, 0.66 mL, 0.25 mmol, 5 equiv) which was pre-mixed (6 - 10 min before addition) with DIPCDI (neat, 32 mg, 0.25 mmol, 5 equiv). The reaction mixture was rotated for 18 h. The completion of the reaction was confirmed by a negative chloranil test [15], and the filtered resin product 25 was washed with DMF (4 × 1 mL), THF (3 × 1 mL), CH₂Cl₂ (3 × 1 mL), and dried in air.

Deprotection of the Silyl Ether on Resin 25 with TBAF and Subsequent Iodination using Triphenylphosphine, Iodine and Imidazole to Form Resin-Bound 27b: The resin product 25 was swelled in CH₂Cl₂ for 40 min, and the drained resin was washed with THF (3 × 1 mL). To the resin was added TBAF/THF (1 M, 1.0 mL, 1.0 mmol, 20 equiv), and the reaction was allowed to proceed for 18 h. The drained resin was washed with THF (3 × 1 mL), and CH₂Cl₂ (3 × 1 mL), and air dried. After this alcohol resin was swelled in CH₂Cl₂ for 1 h and washed with DMF (3 × 1 mL), a pre-mixed solution of iodine (63 mg, 0.25 mmol, 5 equiv), PPh₃ (66 mg, 0.25 mmol, 5 equiv), and imidazole (17 mg, 0.25 mmol, 5 equiv) in DMF (1.0 mL) was added. After 18 h, the filtered resin was washed with DMF (3 × 1 mL), MeOH (3 × 1 mL), DMF (2 × 1 mL), and CH₂Cl₂ (3 × 1 mL).

Displacement of Iodo-Resin 27b with Amines and Subsequent Cleavage to Target Molecules 3: The air-dried resin 27b was swelled in CH₂Cl₂ for 40 min, and to the first 16 reaction vessels on one BillBoard were added a solution of 2-methoxyphenyl piperazine in anhydrous DMSO (1 M, 0.75 mL, 0.75 mmol, 15 equiv), while to the other 16 reaction vessels on the other BillBoard were added a solution of 3-chlorophenyl piperazine in anhydrous DMSO (1 M, 0.75 mL, 0.75 mmol, 15 equiv), and to the resin for quality control was added a tetrahydroisoquinoline solution in DMSO (1M, 0.75 mL, 0.75 mmol, 15 equiv). All reaction vessels were heated to 50 ◦C for 6 h with occasional agitation. The resulting resin product was drained, sequentially washed with DMF (2 × 1 mL), MeOH (2 × 1 mL), DMF (3 × 1 mL), CH₂Cl₂ (4 × 1 mL), and cleaved with 90% TFA/CH₂Cl₂ (1 mL) over 1.5 h, affording the target compound 3.

2-[(Cyclopropanecarbonyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-octanamide (3a): Product 3a was obtained from resin 23, 1-iodohexane, cyclopropanecarboxylic acid, and 2-methoxy-phenylpiperazine as an amorphous light yellow solid (10.5 mg, 46% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (92:8). Initial LC/MS purity 81%, t_R = 8.2 min; ¹H-NMR (CDCl₃): δ 0.71–0.78 (m, 2H), 0.86 (t, J = 6.9 Hz, 3H), 0.89–0.96 (m, 2H), 1.19–1.38 (m, 10H), 1.42–1.51 (m, 1H), 1.57–1.69 (m, 1H), 1.79–1.90 (m, 3H), 2.74–2.83 (m, 2H), 2.89–3.02 (m, 3H), 3.18–3.28 (m, 3H), 3.33–3.43 (m, 2H), 3.87 (s, 3H), 4.36 (dd, J = 7.8 Hz, J = 13.7 Hz, 1H), 6.60 (d, J = 7.1 Hz, 1H), 6.88 (d, J = 7.7 Hz, 1H), 6.91–6.97 (m, 2H), 7.01–7.09 (m, 2H), 7.52–7.65 (m, 1H). ¹³C-NMR (CDCl₃): δ 7.3, 7.4, 14.0, 14.6, 22.6, 24.4, 25.7, 29.0, 31.7, 32.8, 37.7, 49.3, 53.0, 53.8, 55.4, 55.9, 111.3, 118.6, 121.2, 123.8, 140.1, 152.2, 172.7, 173.8. HRMS calcd. for (M + H)⁺: C₂₆H₄₂N₄O₃ 459.3335, found 459.3313.

2-[(Cyclopropanecarbonyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(4-methoxy-phenyl)propanamide (3b): Product 3b was obtained from resin 23, 4-methoxybenzyl chloride, cyclopropanecarboxylic acid, and 2-methoxyphenylpiperazine as light yellow oil (12.2 mg, 49% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 78%, t_R = 6.1 min; ¹H-NMR (CDCl₃): δ 0.67–0.77 (m, 2H), 0.84–0.95 (m, 2H), 1.43–1.53 (m, 1H), 1.78–1.90 (m, 2H), 1.97–2.07 (m, 1H), 2.37 (t, J = 8.1 Hz, 1H), 2.68–2.79 (m, 2H), 2.84 (s, 1H), 2.98–3.05 (m, 2H), 3.20–3.29 (m, 4H), 3.33–3.42 (m, 2H), 3.76 (s, 3H), 3.86 (s, 3H), 4.50–4.79 (m, 1H), 6.81 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 6.91–6.95 (m, 2H), 7.03–7.08 (m, 1H), 7.14 (d, J = 8.5 Hz, 2H), 7.46 (brs, 1H). ¹³C-NMR (CDCl₃): δ 7.4, 7.5, 14.6, 17.7, 23.9, 29.6, 30.7, 37.6, 44.0, 48.7, 49.5, 52.8, 55.2, 55.5, 111.3, 113.9, 118.7, 121.2, 123.9, 128.8, 130.5, 139.7, 152.2, 158.5, 171.9, 174.0. HRMS calcd. for (M + H)⁺: C₂₈H₃₉N₄O₄ 495.2907, found 495.2914.

2-[(Cyclopropanecarbonyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(3-fluoro-phenyl)propanamide (3c): Product 3c was obtained from resin 23, 3-fluorobenzyl chloride, cyclopropanecarboxylic acid, and 2-methoxyphenylpiperazine as light yellow oil (4.6 mg, 19% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 65%, t_R = 6.5 min; ¹H-NMR (CDCl₃): δ 0.66–0.82 (m, 2H), 0.85–0.99 (m, 2H), 1.32–1.49 (m, 1H), 1.61–1.81 (m, 2H), 2.48–2.64 (m, 2H), 2.65–2.87 (m, 3H), 3.03–3.22 (m, 5H), 3.24–3.47 (m, 3H), 3.87 (s, 3H), 4.49–4.69 (m, 1H), 6.53 (brs, 1H), 6.87 (d, J = 8.1 Hz, 1H), 6.90–6.97 (m, 4H), 6.98–7.06 (m, 2H), 7.21–7.25 (m, 1H), 7.34 (brs, 1H). ¹³C-NMR (CDCl₃): δ 7.4, 7.5, 14.7, 24.4, 31.8, 38.7, 50.1, 53.2, 53.3, 54.9, 55.4, 77.6, 111.2 113.7, 116.4, 118.4, 121.1, 123.4, 125.1, 129.9, 139.4, 140.6, 152.2, 161.8, 163.8, 170.7, 173.5. HRMS calcd. for (M + H)⁺: C₂₇H₃₆FN₄O₃ 483.2771, found 483.2766.

2-[(Cyclopropanecarbonyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(4-fluoro-phenyl)propanamide (3d): Product 3d was obtained from resin 23, 4-fluorobenzyl bromide, cyclopropanecarboxylic acid, and 2-methoxyphenylpiperazine as light yellow oil (18.3 mg, 76% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 83%, t_R = 6.4 min; ¹H-NMR (CDCl₃): δ 0.66–0.77 (m, 2H), 0.78–0.94 (m, 2H), 1.42–1.55 (m, 1H), 1.81–1.99 (m, 2H), 2.79–2.93 (m, 2H), 3.02 (dd, J = 7.4 Hz, J = 13.8 Hz, 2H), 3.09 (dd, J = 6.5 Hz, J = 13.8 Hz, 2H), 3.22–3.43 (m, 6H), 3.87 (s, 3H), 4.68 (dd, J = 7.3 Hz, J = 14.6 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.91–7.02 (m, 5H), 7.04–7.09 (m, 1H), 7.16–7.24 (m, 2H), 7.62 (brs, 1H). ¹³C-NMR (CDCl₃): δ 7.4, 7.5, 14.7, 23.8, 36.5, 37.5, 48.1, 52.6, 54.8, 55.0, 55.4, 111.4, 115.2 115.4, 118.7, 121.2, 124.3, 130.9, 132.6, 139.2, 152.2, 160.9, 162.8, 172.1, 174.2. HRMS calcd. for (M + H)⁺: C₂₇H₃₆FN₄O₃ 483.2771, found 483.2755.

2-[(Cyclohexanecarbonyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}octanamide (3e): Product 3e was obtained from resin 23, 1-iodohexane, cyclohexanecarboxylic acid, and 2-methoxy-phenylpiperazine as light yellow oil (12.8 mg, 51% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 81%, t_R = 10.8 min; ¹H- NMR (CDCl₃): δ 0.85 (t, J = 6.8 Hz, 3H), 1.20–1.30 (m, 11H), 1.36–1.48 (m, 2H), 1.58–1.68 (m, 2H), 1.73–1.88 (m, 7H), 1.97–2.07 (m, 0.5H), 2.08–2.21 (m, 1H), 2.38 (t, J = 8.1 Hz, 0.5H), 2.65–2.79 (m, 2H), 2.82–2.96 (m, 3H), 3.13–3.26 (m, 3H), 3.29–3.48 (m, 3H), 3.88 (s, 3H), 4.35 (dd, J = 7.6 Hz, J = 13.9 Hz, 1H), 6.38 (brs, 1H), 6.87 (d, J = 8.1 Hz, 1H), 6.89–6.96 (m, 2H), 6.99–7.06 (m, 1H), 7.64 (brs, 1H). ¹³C-NMR (CDCl₃): δ 14.0, 17.7, 22.5, 24.6, 25.6, 25.7, 29.0, 29.3, 29.8, 30.7, 31.6, 32.8, 38.2, 45.3, 49.7, 53.2, 55.4, 111.3, 118.5, 121.1, 123.5, 140.4, 152.2, 172.5, 176.5. HRMS calcd. for (M + H)⁺: C₂₉H₄₉N₄O₃ 501.3805, found 501.3811.

2-[(Cyclohexanecarbonyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3(4-methoxy-phenyl)propanamide (3f): Product 3f was obtained from resin 23, 4-methoxybenzyl chloride, cyclo-hexanecarboxylic acid, and 2-methoxyphenylpiperazine as light yellow oil (9.9 mg, 37% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 65%, t_R = 8.7 min; ¹H-NMR (CDCl₃): δ 1.16–1.27 (m, 3H), 1.32–1.43 (m, 2H), 1.61–1.69 (m, 3H), 1.72–1.83 (m, 4H), 1.97–2.04 (m, 1H), 2.05–2.13 (m, 1H), 2.38 (t, J = 8.1 Hz, 1H), 2.45–2.52 (m, 2H), 2.62–2.66 (m, 1H), 2.68–2.73 (m, 1H), 2.84 (s, 1H), 2.97 (d, J = 7.1 Hz, 2H), 3.06–3.13 (m, 2H), 3.22–3.34 (m, 2H), 3.38 (t, J = 7.1 Hz, 1H), 3.76 (s, 3H), 3.86 (s, 3H), 4.50 (dd, J = 7.2 Hz, J = 14.6 Hz, 1H), 6.30 (d, J = 7.2 Hz, 1H), 6.80 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 7.9 Hz, 1H), 6.94 (d, J = 4.2 Hz, 2H), 6.99–7.05 (m, 1H), 7.11 (d, J = 8.6 Hz, 2H), 7.19 (brs, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 25.6, 25.7, 29.3, 29.7, 30.7, 38.1, 45.2, 49.5, 53.2, 54.7, 55.2, 55.4, 56.6, 111.2, 114.1, 118.4, 121.1, 123.3, 128.8, 130.4, 140.7, 152.2, 158.5, 171.1, 175.9. HRMS calcd. for (M + H)⁺: C₃₁H₄₅N₄O₄ 537.3441, found 537.3439.

2-[(Cyclohexanecarbonyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(3-fluoro-phenyl)propanamide (3g): Product 3g was obtained from resin 23, 3-fluorobenzyl chloride, cyclo-hexanecarboxylic acid, and 2-methoxyphenylpiperazine as yellow oil (13.9 mg, 53% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 65%, t_R = 9.1 min; ¹H-NMR (CDCl₃): δ 1.14–1.37 (m, 5H), 1.60–1.78 (m, 5H), 1.93–2.07 (m, 3H), 2.08-2.18 (m, 1H), 2.37 (t, J = 8.2 Hz, 1H), 2.84 (s, 1H), 2.94–3.05 (m, 3H), 3.16 (dd, J = 6.0 Hz, J = 13.9 Hz, 2H), 3.27–3.34 (m, 3H), 3.35–3.44 (m, 3H), 3.87 (s, 3H), 4.66 (dd, J = 7.9 Hz, J = 14.1 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 6.86–6.97 (m, 5H), 7.00 (d, J = 7.7 Hz, 1H), 7.05–7.11 (m, 1H), 7.21–7.25 (m, 1H), 7.60–7.70 (m, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 23.7, 25.7, 29.1, 29.7, 30.7, 37.7, 44.9, 47.7, 49.5, 52.5, 54.4, 55.5, 111.4, 113.7, 116.3, 118.9, 121.2, 124.5, 125.1, 130.0, 138.9, 139.5, 152.1, 161.8, 163.8, 172.3, 176.8. HRMS calcd. for (M + H)⁺: C₃₀H₄₂FN₄O₃ 525.3241, found 525.3222.

2-[(Cyclohexanecarbonyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(4-fluoro-phenyl)propanamide (3h): Product 3h was obtained from resin 23, 4-fluorobenzyl bromide, cyclo-hexanecarboxylic acid, and 2-methoxyphenylpiperazine as an amorphous light yellow solid (16.3 mg, 62% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 65%, t_R = 9.0 min; ¹H-NMR (CDCl₃): δ 1.13-1.27 (m, 3H), 1.28-1.41 (m, 2H), 1.59-1.66 (m, 1H), 1.67–1.82 (m, 6H), 1.98–2.05 (m, 0.4H), 2.05–2.14 (m, 1H), 2.37 (t, J = 8.2 Hz, 0.4H), 2.54–2.66 (m, 2H), 2.75–2.86 (m, 3H), 2.93–3.00 (m, 1H), 3.02–3.08 (m, 1H), 3.09–3.20 (m, 3H), 3.22–3.41 (m, 3H), 3.86 (s, 3H), 4.58 (dd, J = 7.3 Hz, J = 14.8 Hz, 1H), 6.48 (brs, 1H), 6.87 (d, J = 8.1 Hz, 1H), 6.91–6.99 (m, 4H), 7.00–7.07 (m, 1H), 7.12–7.20 (m, 2H), 7.49 (brs, 1H). ¹³C-NMR (CDCl₃): δ 17.6, 24.4, 25.7, 29.2, 29.7, 30.7, 37.9, 45.1, 49.5, 53.1, 54.5, 55.4, 56.2, 111.3, 115.4, 118.5, 121.1, 123.6, 130.9, 132.5, 140.3, 152.2, 160.9, 162.8, 171.3, 176.3. HRMS calcd. for (M + H)⁺: C₃₀H₄₂FN₄O₃ 525.3241, found 525.3244.

2-[(Benzoyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}octanamide (3i): Product 3i was obtained from resin 23, 1-iodohexane, benzoic acid, and 2-methoxyphenylpiperazine as light yellow oil (17.4 mg, 70% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 83%, t_R = 10.1 min; ¹H-NMR (CDCl₃): δ 0.81–0.89 (m, 3H), 1.21–1.41 (m, 8H), 1.71–1.83 (m, 1H), 1.84–1.98 (m, 3H), 1.99–2.06 (m, 0.4H), 2.35 (t, J = 8.2 Hz, 0.5H), 2.83 (s, 1H), 2.90–2.99 (m, 2H), 3.02–3.13 (m, 2H), 3.19–3.29 (m, 3H), 3.31–3.49 (m, 3H), 3.85 (s, 3H), 4.57 (dd, J = 7.7 Hz, J = 13.7 Hz, 1H), 6.82–6.97 (m, 3H), 7.01–7.10 (m, 1H), 7.29–7.36 (m, 1H), 7.40 (t, J = 7.5 Hz, 2H), 7.48 (t, J = 7.4 Hz, 1H), 7.86 (d, J = 7.3 Hz, 2H), 7.94 (brs, 1H). ¹³C-NMR (CDCl₃): δ 14.0, 17.7, 22.5, 23.9, 25.8, 29.0, 30.7, 31.6, 32.6, 37.0, 48.4, 49.5, 52.8, 54.5, 55.4, 111.3, 118.7, 121.2, 124.1, 127.3, 128.6, 131.8, 133.6, 139.5, 152.2, 167.6, 173.1. HRMS calcd. for (M + H)⁺: C₂₉H₄₃N₄O₃ 495.3335, found 495.3337.

2-[(Benzoyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3(4-methoxyphenyl)-propan-amide (3j): Product 3j was obtained from resin 23, 4-methoxybenzyl chloride, benzoic acid, and 2-methoxyphenylpiperazine as an amorphous light yellow solid (10.3 mg, 39% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 71%, t_R = 7.9 min; ¹H-NMR (CDCl₃): δ 1.72-1.87 (m, 2H), 1.96–2.09 (m, 1H), 2.37 (t, J = 8.2 Hz, 1H), 2.63–2.73 (m, 2H), 2.79–2.91 (m, 3H), 3.10–3.20 (m, 5H), 3.31–3.39 (m, 2H), 3.76 (s, 3H), 3.86 (s, 3H), 4.74 (dd, J = 7.2 Hz, J = 14.4 Hz, 1H), 6.83 (d, J = 8.6 Hz, 2H), 6.86–6.89 (m, 1H), 6.89–6.97 (m, 2H), 7.02–7.09 (m, 2H), 7.18 (d, J = 8.6 Hz, 2H), 7.37–7.45 (m, 3H), 7.46–7.51 (m, 1H), 7.74–7.79 (m, 2H). ¹³C-NMR (CDCl₃): δ 17.7, 24.2, 29.6, 30.7, 37.9, 49.2, 49.4, 52.9, 55.2, 55.4, 55.6, 111.3, 114.1, 118.6, 121.2, 123.7, 127.1, 128.6, 128.7, 130.4, 131.7, 133.8, 152.2, 158.6, 167.1, 171.4. HRMS calcd. for (M + H)⁺: C₃₁H₂₉N₄O₄ 531.2971, found 531.2951.

2-[(Benzoyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(3-fluorophenyl)-propanamide (3k): Product 3k was obtained from resin 23, 3-fluorobenzyl chloride, benzoic acid, and 2-methoxyphenylpiperazine as yellow oil (12.6 mg, 49% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 75%, t_R = 8.4 min; ¹H- NMR (CDCl₃): δ 1.76–1.93 (m, 2H), 1.96–2.08 (m, 1H), 2.37 (t, J = 8.2 Hz, 1H), 2.71–2.82 (m, 2H), 2.83–2.86 (m, 1H), 2.87–2.97 (m, 2H), 3.14–3.25 (m, 5H), 3.28–3.34 (m, 1H), 3.36–3.40 (m, 1H), 3.86 (s, 3H), 4.81 (dd, J = 7.2 Hz, J = 14.6 Hz, 1H), 6.86–6.89 (m, 1H), 6.89–6.96 (m, 3H), 6.97–7.01 (m, 1H), 7.02–7.08 (m, 2H), 7.16–7.22 (m, 1H), 7.23–7.26 (m, 1H), 7.37–7.43 (m, 2H), 7.45–7.51 (m, 1H), 7.59–7.70 (m, 1H), 7.73–7.80 (m, 2H). ¹³C-NMR (CDCl₃): δ 17.7, 24.0, 29.6, 30.7, 38.3, 48.9, 49.4, 52.9, 55.3, 55.4, 111.3, 113.8, 113.9, 118.7, 121.2, 123.9, 125.1, 127.2, 128.6, 130.1, 131.8, 133.6, 139.4, 139.5, 152.2, 161.9, 163.8, 167.2, 171.3. HRMS calcd. for (M + H)⁺: C₃₀H₃₆FN₄O₃ 519.2771, found 519.2782.

2-[(Benzoyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(4-fluorophenyl)-propanamide (3l): Product 3l was obtained from resin 23, 4-fluorobenzyl bromide, benzoic acid, and 2-methoxy-phenylpiperazine as brownish oil (19.7 mg, 76% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 86%, t_R = 8.3 min; ¹H-NMR (CDCl₃): δ 1.78–1.93 (m, 2H), 1.95–2.06 (m, 0.5H), 2.36 (t, J = 8.2 Hz, 0.5H), 2.75–2.88 (m, 3H), 2.93–3.04 (m, 2H), 3.07–3.16 (m, 2H), 3.17–3.32 (m, 5H), 3.34–3.41 (m, 1H), 3.85 (s, 3H), 4.71–4.92 (m, 1H), 6.85–6.91 (m, 2H), 7.01–7.09 (m, 1H), 7.19–7.25 (m, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.46 (t, J = 7.4 Hz, 1H), 7.77 (d, J = 7.4 Hz, 2H), 7.85 (brs, 1H). ¹³C-NMR (CDCl₃): δ 17.6, 23.8, 29.6, 30.7, 37.5, 48.5, 49.5, 52.7, 55.4, 55.5, 111.3, 115.5, 118.7, 121.2, 124.1, 127.2, 128.5, 130.9, 131.9, 132.5, 133.4, 139.5, 152.2, 160.9, 162.8, 167.5, 171.8. HRMS calcd. for (M + H)⁺: C₃₀H₃₆FN₄O₃ 519.2771, found 519.2745.

2-[(Quinolin-2-oyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}octanamide (3m): Product 3m was obtained from resin 23, 1-iodohexane, quinaldic acid, and 2-methoxyphenylpiperazine as brownish oil (18.4 mg, 67% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (95:5). Initial LC/MS purity 80%, t_R = 12.1 min; ¹H-NMR (CDCl₃): δ 0.85 (t, J = 6.9 Hz, 3H), 1.24–1.30 (m, 4H), 1.32–1.49 (m, 4H), 1.78–1.92 (m, 1H), 1.94–2.10 (m, 3.5H), 2.37 (t, J = 8.1 Hz, 0.5H), 2.83 (s, 1H), 2.96–3.10 (m, 3H), 3.19–3.32 (m, 4H), 3.34–3.42 (m, 2H), 3.43–3.53 (m, 1H), 3.84 (s, 3H), 4.59 (dd, J = 7.3 Hz, J = 13.7 Hz, 1H), 6.82–6.96 (m, 3H), 6.99–7.09 (m, 1H), 7.57–7.71 (m, 2H), 7.76 (t, J = 8.2 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 8.21–8.34 (m, 2H), 8.61–8.82 (m, 1H). ¹³C-NMR (CDCl₃): δ 14.0, 17.6, 22.5, 25.8, 28.9, 30.7, 31.6, 32.3, 48.2, 49.5, 52.7, 54.2, 55.4, 111.3, 118.8, 121.2, 124.1, 127.7, 128.1, 129.4, 129.9, 130.2, 137.6, 139.4, 146.5, 148.9, 152.1, 164.9, 172.4. HRMS calcd. for (M + H)⁺: C₃₂H₄₄N₅O₃ 546.3444, found 546.3419.

2-[(Quinolin-2-oyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(4-methoxyphenyl)-propanamide (3n): Product 3n was obtained from resin 23, 4-methoxybenzyl chloride, quinaldic acid, and 2-methoxyphenylpiperazine as brownish oil (10.5 mg, 36% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 66%, t_R = 9.9 min; ¹H-NMR (CDCl₃): δ 1.61–1.87 (m, 2H), 1.95–2.08 (m, 1H), 2.37 (t, J = 8.1 Hz, 1H), 2.56–2.67 (m, 2H), 2.77–2.88 (m, 3H), 3.08–3.15 (m, 2H), 3.16–3.28 (m, 3H), 3.32–3.35 (m, 1H), 3.36–3.39 (m, 1H), 3.77 (s, 3H), 3.85 (s, 3H), 4.76 (dd, J = 7.7 Hz, J = 14.4 Hz, 1H), 6.81–6.88 (m, 4H), 6.89–6.93 (m, 1H), 6.98–7.05 (m, 1H), 7.15–7.22 (m, 1H), 7.23–7.26 (m, 2H), 7.57–7.66 (m, 1H), 7.71–7.80 (m, 1H), 7.86 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 8.27 (d, J = 8.5 Hz, 1H), 8.78 (d, J = 8.1 Hz, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 24.4, 29.6, 30.7, 37.7, 49.4, 52.9, 55.2, 55.4, 55.5, 56.1, 111.2, 114.0, 118.7, 121.1, 123.5, 127.7, 128.1, 128.8, 129.4, 129.9, 130.2, 130.5, 137.5, 146.5, 149.1, 152.2, 158.6, 164.5, 170.9. HRMS calcd. for (M + H)⁺: C₃₄H₄₀N₅O₄ 582.3080, found 582.3058.

2-[(Quinolin-2-oyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(3-fluorophenyl)-propanamide (3o): Product 3o was obtained from resin 23, 3-fluorobenzyl chloride, quinaldic acid, and 2-methoxyphenylpiperazine as brownish oil (15.3 mg, 54% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 77%, t_R = 10.3 min; ¹H- NMR (CDCl₃): δ 1.88–2.12 (m, 3H), 2.37 (t, J = 8.1 Hz, 1H), 2.84 (s, 1H), 2.93–3.04 (m, 2H), 3.19–3.44 (m, 9H), 3.86 (s, 3H), 4.84 (dd, J = 7.0 Hz, J = 14.5 Hz, 1H), 6.85–6.98 (m, 4H), 7.02–7.10 (m, 2H), 7.13 (d, J = 7.7 Hz, 1H), 7.27-7.33 (m, 1H), 7.40–7.49 (m, 1H), 7.62 (t, J = 7.1 Hz, 1H), 7.73–7.81 (m, 1H), 7.85 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.76 (d, J = 7.8 Hz, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 23.7, 29.6, 30.7, 37.9, 47.8, 49.4, 52.5, 54.9, 55.4, 111.3, 114.1, 116.3, 118.5, 118.8, 121.2, 124.3, 125.2, 127.7, 128.1, 129.4, 129.9, 130.2, 137.5, 139.3, 146.5, 148.8, 152.1, 161.9, 162.7, 163.8, 164.8, 171.3. HRMS calcd. for (M + H)⁺: C₃₃H₃₇FN₅O₃ 570.2880, found 570.2853.

2-[(Quinolin-2-oyl)amino]-N-{[4-(2-methoxyphenyl)-piperazin-1-yl]propyl}-3-(4-fluorophenyl)-propanamide (3p): Product 3p was obtained from resin 23, 4-fluorobenzyl bromide, quinaldic acid, and 2-methoxyphenylpiperazine as brownish oil (22.1 mg, 78% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 85%, t_R = 10.2 min; ¹H-NMR (CDCl₃): δ 1.92–2.08 (m, 2.5H), 2.32–2.42 (m, 0.5H), 2.83 (s, 1H), 2.93–3.04 (m, 2H), 3.18–3.44 (m, 10H), 3.85 (s, 3H), 4.83 (dd, J = 7.3 Hz, J = 14.7 Hz, 1H), 6.84–6.95 (m, 3H), 6.96–7.02 (m, 2H), 7.03–7.09 (m, 1H), 7.28–7.36 (m, 2H), 7.47–7.56 (m, 1H), 7.61 (t, J = 7.4 Hz, 1H), 7.76 (t, J = 7.1 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.26 (d, J = 8.5 Hz, 1H), 8.74 (d, J = 7.8 Hz, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 23.6, 29.6, 30.7, 36.5, 37.5, 47.7, 49.4, 52.5, 54.6, 55.2, 55.4, 111.3, 115.4, 118.6, 118.9, 121.2, 124.3, 127.7, 128.1, 129.4, 129.9, 130.2, 131.0, 132.5, 139.1, 146.5, 148.9, 152.1, 160.9, 162.4, 162.9, 164.7, 171.5. HRMS calcd. for (M + H)⁺: C₃₃H₃₇FN₅O₃ 570.2880, found 570.2870.

2-[(Cyclopropanecarbonyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}octanamide (3q): Product 3q was obtained from resin 23, 1-iodohexane, cyclopropanecarboxylic acid, and 3-chloro-phenylpiperazine as brownish oil (11.2 mg, 48% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 68%, t_R = 9.7 min; ¹H- NMR (CDCl₃): δ 0.66–0.79 (m, 2H), 0.81–0.97 (m, 5H), 1.18–1.37 (m, 8.6H), 1.38–1.52 (m, 1H), 1.56–1.70 (m, 1H), 1.76–1.84 (m, 1H), 1.84–1.93 (m, 1.6H), 1.96–2.08 (m, 0.4H), 2.37 (t, J = 8.1 Hz, 0.4H), 2.72–2.83 (m, 2H), 2.84 (s, 1H), 2.87–3.01 (m, 3H), 3.27–3.44 (m, 6H), 4.34 (dd, J = 7.5 Hz, J = 14.1 Hz, 1H), 6.62 (brs, 1H), 6.75–6.81 (m, 1H), 6.83–6.90 (m, 2H), 7.15–7.22 (m, 1H), 7.39–7.57 (m, 1H). ¹³C-NMR (CDCl₃): δ 7.4, 7.5, 14.0, 14.6, 17.7, 22.5, 24.5, 25.6, 29.0, 31.6, 32.6, 47.7, 49.5, 52.4, 53.9, 55.7, 114.4, 116.5, 120.5, 130.3, 135.1, 151.4, 172.7, 174.1. HRMS calcd. for (M + H)⁺: C₂₅H₄₀ClN₄O₂ 463.2840, found 463.2841.

2-[(Cyclopropanecarbonyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3(4-methoxy-phenyl)propanamide (3r): Product 3r was obtained from resin 23, 4-methoxybenzyl chloride, cyclopropanecarboxylic acid, and 3-chlorophenyl piperazine as yellow oil (10.2 mg, 41% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (92:8). Initial LC/MS purity 60%, t_R = 7.7 min; ¹H-NMR (CDCl₃): δ 0.67–0.79 (m, 2H), 0.85–0.94 (m, 2H), 1.39–1.44 (m, 1H), 1.71–1.83 (m, 2H), 1.97–2.08 (m, 1H), 2.37 (t, J = 8.2 Hz, 1H), 2.57–2.64 (m, 2H), 2.75–2.82 (m, 2H), 2.83–2.85 (m, 1H), 2.97–3.01 (m, 1H), 3.24–3.30 (m, 5H), 3.36–3.41 (m, 1H), 3.77 (s, 3H), 4.45–4.68 (m, 1H), 6.64 (brs, 1H), 6.77 (d, J = 7.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 7.0 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 7.18 (t, J = 8.3 Hz, 1H). ¹³C-NMR (CDCl₃): δ 7.4, 7.5, 13.5, 14.6, 17.7, 29.6, 30.7, 37.7, 47.7, 49.5, 52.3, 55.3, 113.9, 114.4, 116.4, 120.4, 128.8, 130.3, 130.4, 135.1, 151.4, 158.5, 171.6, 173.8. HRMS calcd. for (M + H)⁺: C₂₇H₃₆ClN₄O₃ 499.2476, found 499.2467.

2-[(Cyclopropanecarbonyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3-(3-fluorophenyl)-propanamide (3s): Product 3s was obtained from resin 23, 3-fluorobenzyl chloride, cyclo-propanecarboxylic acid, and 3-chlorophenylpiperazine as light yellow oil (8.0 mg, 33% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 57%, t_R = 8.2 min; ¹H-NMR (CDCl₃): δ 0.67–0.81 (m, 2H), 0.86–0.96 (m, 2H), 1.37–1.47 (m, 1H), 1.77 (brs, 2H), 2.57–2.89 (m, 5H), 3.04–3.12 (m, 2H), 3.22–3.41 (m, 7H), 4.47–4.72 (m, 1H), 6.55 (brs, 1H), 6.74–6.81 (m, 1H), 6.85–6.89 (m, 2H), 6.91–6.96 (m, 2H), 7.00 (d, J = 7.7 Hz, 1H), 7.19 (t, J = 8.4 Hz, 2H), 7.23–7.26 (m, 1H). ¹³C-NMR (CDCl₃): δ 7.5, 7.6, 14.6, 24.4, 38.3, 47.9, 52.5, 54.9, 56.0, 113.7, 114.3, 116.2, 116.4, 120.3, 125.2, 130.0, 130.2, 135.1, 139.5, 151.5, 161.8, 163.8, 171.4, 173.8. HRMS calcd. for (M + H)⁺: C₂₆H₃₃ClFN₄O₂ 487.2276, found 487.2299.

2-[(Cyclopropanecarbonyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3-(4-fluorophenyl)-propanamide (3t): Product 3t was obtained from resin 23, 4-fluorobenzyl bromide, cyclo-propanecarboxylic acid, and 3-chlorophenylpiperazine as brown oil (9.9 mg, 41% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 73%, t_R = 8.1 min; ¹H-NMR (CDCl₃): δ 0.67–0.78 (m, 2H), 0.83–0.94 (m, 2H), 1.37–1.47 (m, 1H), 1.69–1.85 (m, 2H), 2.58–2.73 (m, 2H), 2.75–2.94 (m, 4H), 2.98–3.07 (m, 2H), 3.21–3.39 (m, 6H), 4.58 (dd, J = 7.3 Hz, J = 14.7 Hz, 1H), 6.63 (brs, 1H), 6.74–6.79 (m, 1H), 6.84–6.91 (m, 2H), 6.94–7.01 (m, 2H), 7.10–7.26 (m, 4H). ¹³C-NMR (CDCl₃): δ 7.4, 7.6, 14.6, 24.3, 37.7, 47.6, 52.3, 55.1, 55.6, 114.5, 115.4, 116.5, 120.6, 130.3, 130.9, 132.6, 135.1, 151.3, 160.9, 162.8, 171.4, 173.9. HRMS calcd. for (M + H)⁺: C₂₆H₃₃ClFN₄O₂ 487.2276, found 487.2292.

2-[(Cyclohexanecarbonyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}octanamide (3u): Product 3u was obtained from resin 23, 1-iodohexane, cyclohexanecarboxylic acid, and 3-chlorophenyl piperazine as brown oil (10.9 mg, 43% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 66%, t_R = 12.2 min; ¹H-NMR (CDCl₃): δ 0.76–0.92 (m, 3H), 1.16–1.33 (m, 11H), 1.35–1.47 (m, 2H), 1.55–1.69 (m, 2H), 1.71-1.95 (m, 7H), 1.97–2.07 (m, 0.5H), 2.09–2.22 (m, 1H), 2.38 (t, J = 8.0 Hz, 0.5H), 2.78–2.86 (m, 2H), 2.89–3.04 (m, 3H), 3.25–3.45 (m, 6H), 4.15–4.44 (m, 1H), 6.38 (brs, 1H), 6.78 (d, J = 8.1 Hz, 1H), 6.82–6.93 (m, 2H), 7.18 (t, J = 8.3 Hz, 1H), 7.53 (brs, 1H). ¹³C-NMR (CDCl₃): δ 14.0, 17.7, 22.5, 25.5, 25.7, 28.9, 29.3, 29.8, 31.6, 32.4, 37.3, 45.2, 47.6, 49.5, 52.4, 53.5, 55.5, 114.5, 116.6, 120.6, 130.3, 135.1, 151.3, 172.8, 176.7. HRMS calcd. for (M + H)⁺: C₂₈H₄₆ClN₄O₂ 505.3309, found 505.3318.

2-[(Cyclohexanecarbonyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3(4-methoxyphenyl)-propanamide (3v): Product 3v was obtained from resin 23, 4-methoxybenzyl chloride, cyclo-hexanecarboxylic acid, and 3-chlorophenylpiperazine as an amorphous off-white solid (6.9 mg, 26% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 55%, t_R = 10.1 min; ¹H-NMR (CDCl₃): δ 1.17–1.26 (m, 3H), 1.31–1.41 (m, 2H), 1.61–1.67 (m, 1H), 1.71–1.84 (m, 7H), 1.98–2.05 (m, 0.6H), 2.07–2.15 (m, 1H), 2.37 (t, J = 8.2 Hz, 0.6H), 2.59–2.71 (m, 3H), 2.84 (s, 1H), 2.98 (d, J = 6.7 Hz, 2H), 3.24–3.35 (m, 6H), 3.78 (s, 3H), 4.51 (dd, J = 7.1 Hz, J = 14.3 Hz, 1H), 6.22 (brs, 1H), 6.75–6.79 (m, 1H), 6.82 (d, J = 8.5 Hz, 2H), 6.86–6.89 (m, 2H), 6.96–7.07 (m, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.16–7.22 (m, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 24.4, 25.5, 25.7, 29.3, 29.7, 30.7, 37.7, 45.1, 49.4, 52.4, 54.8, 55.3, 113.9, 114.4, 116.4, 120.4, 128.8, 130.2, 130.4, 135.1, 151.4, 158.5, 171.5, 176.2. HRMS calcd. for (M + H)⁺: C₃₀H₄₂ClN₄O₃ 541.2945, found 541.2943.

2-[(Cyclohexanecarbonyl)amino]-N-{[4-(3-chloroyphenyl)-piperazin-1-yl]propyl}-3-(3-fluorophenyl)-propanamide (3w): Product 3w was obtained from resin 23, 3-fluorobenzyl chloride, cyclo-hexanecarboxylic acid, and 3-chlorophenylpiperazine as an amorphous light yellow solid (8.5 mg, 32% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 54%, t_R = 10.6 min; ¹H-NMR (CDCl₃): δ 1.17–1.28 (m, 3H), 1.29–1.41 (m, 2H), 1.62–1.81 (m, 7H), 1.99–2.14 (m, 2H), 2.37 (t, J = 8.0 Hz, 1H), 2.49–2.59 (m, 2H), 2.62–2.73 (m, 3H), 2.84 (s, 1H), 2.96–3.09 (m, 2H), 3.18–3.25 (m, 3H), 3.27–3.31 (m, 1H), 3.38 (t, J = 7.0 Hz, 1H), 4.48–4.64 (m, 1H), 6.32 (brs, 1H), 6.77 (d, J = 8.2 Hz, 1H), 6.83–6.89 (m, 2H), 6.90–6.96 (m, 2H), 6.98 (d, J = 7.6 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.21–7.26 (m, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 24.7, 25.5, 25.6, 29.3, 29.7, 30.7, 38.3, 45.1, 48.1, 49.5, 52.6, 54.4, 56.2, 113.8, 113.9, 114.2, 116.2, 116.3, 120.0, 125.1, 130.2, 139.3, 151.7, 161.8, 163.8, 170.9, 176.2. HRMS calcd. for (M + H)⁺: C₂₉H₃₉ClFN₄O₂ 529.2746, found 529.2736.

2-[(Cyclohexanecarbonyl)amino]-N-{[4-(3-chloroyphenyl)-piperazin-1-yl]propyl}-3-(4-fluorophenyl)-propanamide (3x): Product 3x was obtained from resin 23, 4-fluorobenzyl bromide, cyclo-hexanecarboxylic acid, and 3-chlorophenylpiperazine as an amorphous yellow solid (11.7 mg, 44% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 72%, t_R = 10.5 min; ¹H-NMR (CDCl₃): δ 1.11–1.28 (m, 3H), 1.29–1.41 (m, 2H), 1.60–1.67 (m, 1H), 1.69–1.82 (m, 5H), 1.97–2.04 (m, 0.5H), 2.05–2.14 (m, 1H), 2.37 (t, J = 8.2 Hz, 0.5H), 2.59–2.73 (m, 2H), 2.75–2.92 (m, 4H), 2.97 (dd, J = 7.1 Hz, J = 13.7 Hz, 1H), 3.06 (dd, J = 7.2 Hz, J = 13.6 Hz, 1H), 3.23–3.35 (m, 5H), 3.35–3.40 (m, 1H), 4.55 (dd, J = 7.1 Hz, J = 14.4 Hz, 1H), 6.36 (d, J = 6.0 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 6.83–6.90 (m, 2H), 6.96 (t, J = 8.5 Hz, 2H), 7.11–7.22 (m, 3H), 7.24–7.26 (m, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 24.4, 25.7, 29.2, 29.7, 30.7, 37.7, 45.1, 47.7, 49.5, 52.4, 54.6, 55.6, 114.4, 115.3, 116.5, 120.5, 130.3, 130.9, 132.6, 135.1, 151.3, 160.9, 162.8, 171.4, 176.4. HRMS calcd. for (M + H)⁺: C₂₉H₃₉ClFN₄O₂ 529.2746, found 529.2741.

2-[(Benzoyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}octanamide (3y): Product 3y was obtained from resin 23, 1-iodohexane, benzoic acid, and 3-chlorophenylpiperazine as brown oil (10.3 mg, 41% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 68%, t_R = 11.5 min; ¹H-NMR (CDCl₃): δ 0.78–0.89 (m, 3H), 1.10–1.41 (m, 9H), 1.68–1.81 (m, 1H), 1.84–1.94 (m, 2H), 1.97–2.08 (m, 0.4H), 2.37 (t, J = 8.1 Hz, 0.4H), 2.70–2.80 (m, 2H), 2.81–2.93 (m, 3H), 3.23–3.46 (m, 6H), 4.38–4.69 (m, 1H), 6.76 (d, J = 8.6 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H), 7.05 (brs, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.42 (t, J = 7.5 Hz, 2H), 7.49 (t, J = 7.3 Hz, 1H), 7.59 (brs, 1H), 7.83 (d, J = 7.4 Hz, 2H). ¹³C-NMR (CDCl₃): δ 14.0, 17.7, 22.5,, 29.0, 31.6, 32.7, 37.8, 47.7, 52.5, 54.2, 55.9, 114.4, 116.4, 120.4, 127.2, 128.6, 130.2, 131.8, 133.7, 135.1, 151.4, 167.4, 172.4. HRMS calcd. for (M + H)⁺: C₂₈H₄₀ClN₄O₂ 499.2840, found 499.2840.

2-[(Benzoyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3(4-methoxyphenyl)-propanamide (3z): Product 3z was obtained from resin 23, 4-methoxybenzyl chloride, benzoic acid, and 3-chlorophenyl piperazine as an amorphous white solid (5.2 mg, 19% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 59%, t_R = 9.4 min; ¹H -MR (CDCl₃): δ 1.64–1.75 (m, 2H), 2.43–2.73 (m, 6H), 3.05–3.21 (m, 6H), 3.26–3.39 (m, 2H), 3.78 (s, 3H), 4.69 (dd, J = 7.5 Hz, J = 13.7 Hz, 1H), 6.74 (d, J = 7.4 Hz, 1H), 6.79–6.87 (m, 4H), 6.92 (d, J = 6.0 Hz, 1H), 7.08 (brs, 1H), 7.14–7.21 (m, 3H), 7.39 (t, J = 7.6 Hz, 2H), 7.48 (t, J = 7.4 Hz, 1H), 7.74 (d, J = 7.3 Hz, 2H). ¹³C-NMR (CDCl₃): δ 24.6, 37.9, 38.5, 48.2, 52.7, 55.3, 55.6, 56.6, 114.1, 116.1, 119.9, 127.1, 128.6, 128.7, 130.1, 130.4, 131.8, 133.7, 135.0, 151.7, 158.7, 167.0, 170.9. HRMS calcd. for (M + H)⁺: C₃₀H₃₆ClN₄O₃ 535.2476, found 535.2470.

2-[(Benzoyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3-(3-fluorophenyl)-propanamide (3aa): Product 3aa was obtained from resin 23, 3-fluorobenzyl chloride, benzoic acid, and 3-chlorophenylpiperazine as light yellow oil (9.2 mg, 35% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 61%, t_R = 9.9 min; ¹H- NMR (CDCl₃): δ 1.61–1.77 (m, 2H), 1.96–2.07 (m, 1H), 2.37 (t, J = 8.1 Hz, 1H), 2.49–2.54 (m, 1H), 2.55–2.63 (m, 2H), 2.64–2.69 (m, 1H), 2.84 (s, 1H), 3.14–3.19 (m, 4H), 3.25–3.40 (m, 3H), 4.64–4.85 (m, 1H), 6.75 (d, J = 7.6 Hz, 1H), 6.85 (d, J = 7.4 Hz, 2H), 6.91–7.01 (m, 2H), 7.02–7.09 (m, 2H)), 7.14–7.21 (m, 1H), 7.22–7.26 (m, 1H), 7.29–7.36 (m, 1H), 7.39 (t, J = 7.6 Hz, 2H), 7.46–7.52 (m, 1H), 7.75 (d, J = 7.8 Hz, 2H). ¹³C-NMR (CDCl₃): δ 17.7, 14.5, 19.6, 30.7, 38.4, 48.1, 49.5, 52.6, 55.3, 56.4, 113.9, 114.0, 114.2, 116.2, 116.4, 119.9, 125.1, 127.1, 128.6, 130.1, 131.9, 133.5, 135.0, 139.4, 151.7, 161.9, 163.8, 167.2, 170.8. HRMS calcd. for (M + H)⁺: C₂₉H₃₃ClFN₄O₂ 523.2276, found 523.2283.

2-[(Benzoyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3-(4-fluorophenyl)-propanamide (3ab): Product 3ab was obtained from resin 23, 4-fluorobenzyl bromide, benzoic acid, and 3-chloro-phenylpiperazine as an amorphous light yellow solid (14.2 mg, 54% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 73%, t_R = 9.8 min; ¹H-NMR (CD₃OD/CDCl₃): δ 1.74–1.85 (m, 2H), 1.97–2.07 (m, 1H), 2.37 (t, J = 8.1 Hz, 1H), 2.63–2.76 (m, 2H), 2.82–2.92 (m, 3H), 3.09 (dd, J = 7.2 Hz, J = 13.5 Hz, 1H), 3.16 (dd, J = 7.4 Hz, J = 13.7 Hz, 1H), 3.23–3.34 (m, 5H), 4.63–4.84 (m, 1H), 6.72–6.81 (m, 1H), 6.82–6.92 (m, 2H), 6.97 (t, J = 8.6 Hz, 2H), 7.09–7.26 (m, 3H), 7.41 (t, J = 7.6 Hz, 2H), 7.47–7.53 (m, 1H), 7.76 (d, J = 7.8 Hz, 2H). ¹³C-NMR (CD₃OD/CDCl₃): δ 17.6, 24.3, 29.6, 30.7, 37.5, 47.5, 52.3, 55.4, 114.5, 115.4, 115.5, 116.6, 120.6, 127.2, 128.6, 130.3, 130.8, 130.9, 132.0, 132.4, 133.3, 135.1, 151.2, 160.9, 162.9, 167.6, 168.8. HRMS calcd. for (M + H)⁺: C₂₉H₃₃ClFN₄O₂ 523.2276, found 523.2252.

2-[(Quinolin-2-oyl)amino]-N-{[4-(3-chloroyphenyl)-piperazin-1-yl]propyl}octanamide (3ac): Product 3ac was obtained from resin 23, 1-iodohexane, quinaldic acid, and 3-chlorophenylpiperazine as brown oil (12.4 mg, 45% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (95:5). Initial LC/MS purity 72%, t_R = 13.3 min; ¹H-NMR (CDCl₃): δ 0.86 (t, J = 6.9 Hz, 3H), 1.24–1.32 (m, 5H), 1.34–1.51 (m, 4H), 1.79–1.93 (m, 1H), 1.96–2.09 (m, 3H), 2.84 (s, 1H), 2.92–3.19 (m, 4H), 3.33–3.48 (m, 6H), 4.53 (dd, J = 7.6 Hz, 13.7 Hz, 1H), 6.72 (dd, J = 2.1 Hz, J = 8.3 Hz, 1H), 6.78–6.84 (m, 1H), 6.89 (d, J = 7.9 Hz, 1H), 7.17 (t, J = 8.1 Hz, 1H), 7.39 (brs, 1H), 7.58–7.66 (m, 1H), 7.72–7.81 (m, 1H), 7.86 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.28 (d, J = 8.5 Hz, 1H), 8.63 (d, J = 7.5 Hz, 1H). ¹³C-NMR (CDCl₃): δ 14.0, 17.7, 22.5, 25.9, 28.9, 31.6, 32.1, 36.9, 46.9, 52.1, 54.3, 55.2, 114.6, 116.7, 118.6, 120.9, 127.7, 128.2, 129.4, 129.9, 130.3, 135.1, 137.6, 146.5, 148.9, 150.9, 164.9, 172.4. HRMS calcd. for (M + H)⁺: C₃₁H₄₁ClN₅O₂ 550.2949, found 550.2936.

2-[(Quinolin-2-oyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3-(4-methoxyphenyl)-propanamide (3ad): Product 3ad was obtained from resin 23, 4-methoxybenzyl chloride, quinaldic acid, and 3-chlorophenylpiperazine as brown oil (8.0 mg, 27% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (94:6). Initial LC/MS purity 58%, t_R = 11.3 min; ¹H-NMR (CDCl₃): δ 1.62–1.73 (m, 2H), 1.97–2.06 (m, 1H), 2.37 (t, J = 8.2 Hz, 1H), 2.42–2.47 (m, 1H), 2.48–2.59 (m, 3H), 2.84 (s, 1H), 2.97–3.07 (m, 3H), 3.17 (dd, J = 7.6 Hz, J = 13.7 Hz, 1H), 3.26 (dd, J = 5.8 Hz, J = 13.8 Hz, 1H), 3.32–3.39 (m, 2H), 3.78 (s, 3H), 4.76 (dd, J = 7.5 Hz, J = 14.0 Hz, 1H), 6.62 (d, J = 8.3 Hz, 1H), 6.69 (brs, 1H), 6.79 (d, J = 7.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H), 7.04–7.14 (m, 2H), 7.22–7.26 (m, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.69–7.77 (m, 1H), 7.84 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 8.16–8.28 (m, 2H), 8.75 (d, J = 7.9 Hz, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 24.7, 29.6, 30.7, 37.5, 47.9, 49.5, 52.8, 55.4, 113.8, 114.1, 115.7, 118.7, 127.7, 128.2, 128.8, 129.4, 129.8, 130.0, 130.2, 130.6, 134.9, 137.6, 146.4, 148.9, 156.6, 158.7, 164.6, 170.7. HRMS calcd. for (M + H)⁺: C₃₃H₃₇ClN₅O₃ 586.2585, found 586.2600.

2-[(Quinolin-2-oyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3-(3-fluorophenyl)-propanamide (3ae): Product 3ae was obtained from resin 23, 3-fluorobenzyl chloride, quinaldic acid, and 3-chlorophenylpiperazine as brown oil (9.4 mg, 33% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (95:5). Initial LC/MS purity 59%, t_R = 11.6 min; ¹H- NMR (CDCl₃): δ 1.58–1.70 (m, 2H), 1.95–2.08 (m, 1H), 2.35–2.42 (m, 2H), 2.43–2.50 (m, 3H), 2.84 (s, 1H), 2.94–3.03 (m, 3H), 3.21–3.27 (m, 1H), 3.29–3.40 (m, 3H), 4.81 (dd, J = 7.9 Hz, J = 14.0 Hz, 1H), 6.92–7.00 (m, 1H), 7.04–7.13 (m, 3H), 7.17–7.22 (m, 1H), 7.27–7.32 (m, 1H), 7.57–7.64 (m, 1H), 7.71–7.77 (m, 1H), 7.84 (d, J = 8.2 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 8.15–8.28 (m, 2H), 8.73 (d, J = 8.4 Hz, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 24.7, 29.6, 30.7, 38.1, 39.2, 48.1, 52.9, 54.9, 57.0, 113.6, 113.9, 114.0, 115.5, 116.4, 116.6, 118.6, 119.3, 125.4, 127.7, 128.2, 129.4, 129.8, 129.9, 130.3, 134.9, 137.6, 139.4, 146.4, 148.8, 151.7, 161.9, 163.9, 164.6, 170.2. HRMS calcd. for (M + H)⁺: C₃₂H₃₄ClFN₅O₂ 574.2385, found 574.2402.

2-[(Quinolin-2-oyl)amino]-N-{[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-3-(4-fluorophenyl)-propanamide (3af). Product 3af was obtained from resin 23, 4-fluorobenzyl bromide, quinaldic acid, and 3-chlorophenylpiperazine as brown oil (15.5 mg, 54% isolated yield) following chromatographic purification over silica gel with CH₂Cl₂–MeOH (93:7). Initial LC/MS purity 72%, t_R = 11.5 min; ¹H- NMR (CDCl₃): δ 1.84–1.98 (m, 2H), 1.98–2.05 (m, 0.5H), 2.37 (t, J = 8.1 Hz, 0.5H), 2.79–3.17 (m, 5H), 3.20–3.44 (m, 8H), 4.79 (dd, J = 7.2 Hz, J = 14.7 Hz, 1H), 6.71 (dd, J = 2.1 Hz, J = 8.3 Hz, 1H), 6.78–6.84 (m, 1H), 6.89 (dd, J = 1.1 Hz, J = 7.9 Hz, 1H), 6.96–7.04 (m, 2H), 7.17 (t, J = 8.1 Hz, 1H), 7.27–7.40 (m, 3H), 7.57–7.66 (m, 1H), 7.72–7.81 (m, 1H), 7.85 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 8.16 (d, J = 8.5 Hz, 1H), 8.26 (d, J = 8.5 Hz, 1H), 8.72 (d, J = 7.8 Hz, 1H). ¹³C-NMR (CDCl₃): δ 17.7, 23.9, 29.6, 30.7, 37.1, 37.4, 46.9, 49.4, 52.0, 55.2, 114.5, 115.4, 115.6, 116.7, 118.5, 120.9, 127.7, 128.2, 129.4, 129.8, 130.3, 131.0, 132.4, 135.1, 137.6, 146.5, 148.8, 150.8, 160.9, 162.9, 164.8, 171.2. HRMS calcd. for (M + H)⁺: C₃₂H₃₄ClFN₅O₂ 574.2385, found 574.2376.