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

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 OPEN ACCESS 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 R 3 contains a basic amino group and n = 1 or 2. Compounds 3 have been shown to be selective binding agents for subclasses of 5hydroxytryptamine (5-HT, serotonin) receptors [8]. This paper reports a flexible solid-phase synthetic route to many variations of R 1 , R 2 , and R 3 on structures 2 and 3. It will be the basis for future synthetic laboratories compatible with the D3 process.  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 R 1 can be introduced during the course of the solid-phase synthesis utilizing the recently described chemistry outlined in Scheme 1 [6]. 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]. Path B [11] Path A [9] =

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 R 1 would possess both R and S configuration, not be restricted to naturally occurring amino acid side chains, and R 3 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 R 3 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 R 1 X: [R 1 = CH 2 Ph, CH 2 CH=CH 2 ; (CH 2 ) 7 CH 3 ; CH 2 Ph-4-CF 2 -P(O)(OEt) 2 ] (Scheme 3). The acylating agent (R 2 = 4-NC-Ph), nucleophile R 3 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. 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 R 3 ) in structure 2. The results with purified yields are shown in Table 2.

Conclusions
This procedure provides ready access to the fundamental peptide scaffold 1 with multiple substitutions at positions R 1 , R 2 , and R 3 . The average overall purified yield to combinatorially prepare the 32 compounds 3a-3af using this 11-step synthetic sequence was 46%. The R 3 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.
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 CH 2 Cl 2 -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 CH 2 Cl 2 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 F 254 , 0.25 mm pre-coated glass plates (EMD Chemical Inc., 5715-7). TLC plates were visualized using UV 254 . Column chromatography was performed on HyperSep SI® 3 mL cartridges (60108-315) preloaded 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. 1 H-NMR (500 MHz) and 13 C -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-d 1 (δ 7.26 ppm 1 H-NMR, 77.0 ppm 13 C-NMR) using TMS (0.00 ppm), or chloroform-d 1 mixed with methanol-d 4 (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 electrospray ionization. The LC/MS samples were analyzed as solutions in CH 3 CN, 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-C 8 column (PN835975-906; 4.6 × 50 mm, 3.5 μm) with linear gradients of 0.1% TFA in CH 3 CN 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.

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 CH 2 Cl 2 (3 × 1 mL). After the alcohol resin 26 was swelled in CH 2 Cl 2 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), H 2 O (1 × 1 mL), DMF (2 × 1 mL) and CH 2 Cl 2 (5 × 1 mL). After the air-dried resin was swelled in CH 2 Cl 2 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), H 2 O (2 × 1 mL), DMF (3 × 1 mL), CH 2 Cl 2 (4 × 1 mL), and air-dried. The resin product was then cleaved with 50% TFA/CH 2 Cl 2 (1 mL) over 1.5 h, and the filtrate of the reaction mixture was collected and combined with washes of CH 2 Cl 2 (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 CH 2 Cl 2 , or CH 2 Cl 2 with MeOH, if needed (total solution volume ≤0.5 mL), and purified using a pre-loaded silica gel cartridge with CH 2 Cl 2 -MeOH (95:5 or 93:7) to elute the purified product. Following solvent removal under N 2 flow, the purified product 2a-2d was normally obtained as an amorphous white solid or light yellow oil.

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 CH 2 Cl 2 for 1 h and washed with DMF (3 × 1 mL), a pre-mixed solution of iodine (63 mg, 0.25 mmol, 5 equiv), PPh 3 (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 CH 2 Cl 2 (3 × 1 mL). After mesylated resin 27a and iodinated resin 27b were swelled in CH 2 Cl 2 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), CH 2 Cl 2 (4 × 1 mL), and cleaved with 90% TFA/CH 2 Cl 2 (1 mL) over 1.5 h. The filtrate of the reaction mixture was collected and combined with washes of 50% TFA/CH 2 Cl 2 (1 × 1 mL) and CH 2 Cl 2 (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. 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 CH 2 Cl 2 for 1 h was evenly distributed to 33 of the reaction vessels in two separate 24-pack BillBoards via an isopycnic solution in CH 2 Cl 2 -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. 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), CH 2 Cl 2 (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/CH 2 Cl 2 (5 × 1 mL), CH 2 Cl 2 (2 × 1 mL). After the resin was swelled in CH 2 Cl 2 (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), CH 2 Cl 2 (3 × 1 mL), and dried in air.