Diverse N-Substituted Azole-Containing Amino Acids as Building Blocks for Cyclopeptides

Abstract

The preparation of 16 oxazole- or thiazole-containing amino esters bearing a wide array of N-substitution is reported. These were accessed in 40–92% yield via an AgClO₄-promoted substitution reaction between a primary amine and a chloromethyl-functionalized thiazole or oxazole. These new synthetic building blocks will be useful for the preparation of new cyclopeptide analogues bearing heterocyclic backbone modifications. Four macrocyclic N-substituted oligoamides that include thiazole or oxazole heterocycles were obtained, following cyclooligomerization reactions of azole-modified N-substituted amino acids.

Introduction

A variety of both natural peptide-derived macrocycles and synthetic analogues thereof include heterocyclic units in the backbone.¹⁻⁴ Thiazole and oxazole are common in natural products such as dendroamide A, which exhibits multidrug resistance reversal activity⁵ (Figure 1). Researchers have sought synthetic routes to prepare azole-rich natural products and their analogues for development of bioactive agents and supramolecular receptors.⁶⁻⁸ In parallel, a number of groups aim to modulate cyclopeptide conformation and properties (e.g., proteolytic stability) by introducing varied heterocycles into the macrocycle backbone.⁹ New, diversely functionalized amino acid building blocks that include oxazole or thiazole will allow researchers to expand the suite of heterocycle-containing macrocycles for study and application.

Figure 1.

Structures of azole-rich natural product dendroamide A and of new N-substituted azole-containing amino acid building blocks explored in this work.

Introduction of N-substituted azole-containing units into cyclopeptide macrocycle backbones represents a promising but underexplored strategy to generate new molecules for structural and functional studies. N-substitution, including N-methylation, is known to influence macrocycle conformation and cell permeability of cyclic peptides.^10,11 However, only a single study details the preparation of N-methylated thiazole-based macrocycles.¹² Azoles have been incorporated into linear N-substituted oligoamides synthesized on solid support.^13,14 To access new azole-containing macrocycles that include N-substitution, diverse N-substituted azole-containing amino acids are needed (Figure 1).

Here, we introduce a method to prepare 16 diverse N-substituted, azole-containing amino ester building blocks via substitution reaction of a chloromethyl-functionalized azole with a primary amine. The compatibility of these building blocks with both solution phase and solid-phase approaches is an advantage for the synthesis of heterocycle-modified cyclopeptides. Additionally, we demonstrate a potential use of these azole-containing amino acids; four azole-rich macrocycles were prepared by their cyclooligomerization.

An attractive approach to prepare N-substituted azole-containing amino acid building blocks is a substitution reaction of the 2-chloromethyl-functionalized thiazole and oxazole esters, which are commercially available and synthetically accessible,^13,15 with a primary amine. A chief advantage of this strategy is that primary amines encompass a wide diversity of functionality which could translate to access to many analogues. An analogous substitution reaction has been employed using varied amine nucleophiles with resin-bound chloromethyl-functionalized azoles.^13,14 In solution, this substitution reaction is less common on similarly functionalized systems. The reaction of primary amines with chloromethyl-functionalized benzoxazoles and other 2-chloromethyl-functionalized azoles bearing aryl substituents at the 4- and/or 5-position has been reported.¹⁶⁻¹⁹ It is noteworthy that these more common substrates differ substantially from azoles used in the studies reported here. In the work described below, we report the reaction of azoles functionalized at the 4-position with an ester substituent; this group is susceptible to reaction with amine nucleophiles. Furthermore, azoles that include an electrophilic substituent at the 4-position present very different reactivity with amines than benzo-fused or aryl-substituted substrates. The reaction of similarly ester-functionalized 2-halomethyl azoles with secondary amines has been described,²⁰⁻²³ but their reaction with primary amines has been limited to a single example.²⁴ The scope of this reaction has not been previously explored. This article expands the examples of this important class of functionalized azoles by exploring the reaction of two azoles with a variety of primary amines.

Results and Discussion

To identify optimal conditions for the substitution reaction, we monitored the reaction of methyl 2-(chloromethyl)oxazole-4-carboxylate (1) with isopropylamine or n-butylamine to generate secondary amines, 2 (Table 1). When the reaction was run with a large excess of amine in a range of solvents, amide byproduct 3 was observed (entries 1–4). This byproduct was especially problematic because it was challenging to separate 2 and 3 chromatographically; a substantial fraction of 2 co-eluted with 3, diminishing isolated yields of 2. Changing the reaction solvent to acetonitrile improved selectivity in favor of 2a (2a/3a = 33.3, entry 4). To promote the desired substitution reaction, the effects of adding non-nucleophilic amines (triethylamine, N,N-diisopropylethylamine, 1,8-diazabicyclo(5.4.0)undec-7-ene, and 4-dimethylaminopyridine) were evaluated, but reaction selectivity (ratio 2/3) was not improved (data not shown). AgClO₄ has been successfully used to mediate closely related substitution reactions,²⁵ but was not soluble in CH₂Cl₂. Its addition in a different solvent system (CH₂Cl₂/ethyl acetate, 4:1) improved reaction selectivity in favor of 2. Indeed, even in the presence of 25 equiv of isopropylamine, the product distribution changed dramatically in favor of 2a when 1.5 equiv of AgClO₄ was added (2a/3a = 81.8, entry 5). Unsurprisingly, reaction selectivity was lower when n-butylamine was used instead of the more sterically congested isopropylamine (2b/3b = 0.70, entry 6). Notably, amines did not react with the ethyl acetate solvent in these reactions (entries 5–7), suggesting that the adjacent azole makes the ester functional group more reactive toward acyl substitution reactions with amines. Decreasing the number of equivalents of n-butylamine to five equivalents improved selectivity for 2b over 3b when AgClO₄ was added (ratio 2/3 = 8.9, entry 7) more than when tetrabutylammonium iodide was added in an effort to activate the chloride in 1 for substitution (ratio 2/3 = 3.0, entry 8). Changing the solvent to acetonitrile in the presence of AgClO₄, only 2b was observed for the reaction of 1 with n-butylamine (entry 9). Heating the reaction mixture at 60 °C accelerated the consumption of 1 (5–6 h reaction times, entries 10 and 11). However, the reaction of the less sterically encumbered nucleophile n-butylamine gave a higher 2/3 ratio for the longer reaction at room temperature than when the reaction was heated over 5 h (>99 vs 12.2, entries 9 and 10). For all other nucleophiles, including more sterically demanding amines and/or weaker nucleophiles, heating the reaction produced higher yields of the desired products. Lastly, the addition of fewer equivalents of amine was evaluated (entry 12). When two equivalents of isopropylamine were used, the reaction time was considerably longer (52 h), but the yield and selectivity for 2 over 3 were not compromised. Overalkylation of the primary amine was not observed under any of the reaction conditions explored.

Table 1. Optimization of the Substitution Reaction between Chloromethyl-Functionalized Oxazole 1 and Primary Amines.

entry	R–NH2 (equiv)a	additive	solvent	Temp.	time (h)	ratio 2/3c	yield 2d
1	i-Pr–NH2 (30)		CH2Cl2	–10 °C to rt	24	7.0	48%
2	i-Pr–NH2 (30)		THF	–10 °C to rt	72	3.2	10%
3	i-Pr–NH2 (30)		Et2O	–10 °C to rt	96	1.5e	trace
4	i-Pr–NH2 (30)		CH3CN	–10 °C to rt	24	33.3	53%
5	i-Pr–NH2 (25)	AgClO4	CH2Cl2/EtOAc (4:1)	–10 °C to rt	24	81.8	56%
6b	n-Bu–NH2 (25)	AgClO4	CH2Cl2/EtOAc (4:1)	–10 °C to rt	6	0.70	trace
7	n-Bu–NH2 (5)	AgClO4	CH2Cl2/EtOAc (4:1)	–10 °C to rt	96	8.9	49%
8	n-Bu–NH2 (5)	(n-Bu)4NI	CH2Cl2	–10 °C to rt	72	3.0	12%
9	n-Bu–NH2 (5)	AgClO4	CH3CN	rt	24	>99f	70%
10	n-Bu–NH2 (5)	AgClO4	CH3CN	60 °C	5	12.2	58%
11	i-Pr–NH2 (5)	AgClO4	CH3CN	60 °C	6	>99f	66%
12	i-Pr–NH2 (2)	AgClO4	CH3CN	60 °C	52	>99f	61%

^aReaction conditions: methyl 2-(chloromethyl)oxazole-4-carboxylate (0.36 mmol, 1 equiv), amine (2–30 equiv), additive (1.5 equiv), solvent (5 mL).

^bUnder Ar.

^cRatios obtained by comparing peak integrations in the ¹H NMR spectrum of the crude reaction product.

^dIsolated yields of 2 following purification by column chromatography.

^eReaction did not proceed to completion.

^f3 was not observed in the crude ¹H NMR spectrum.

Applying the optimized conditions for the silver-mediated substitution reaction, sixteen N-substituted aminomethyl azole esters were prepared in 40–92% isolated yield (Table 2). Both the oxazole (1) and thiazole (4) derivatives were successfully reacted with primary amines bearing a range of functionality in the presence of AgClO₄ in acetonitrile at 60 °C. These examples highlighted the versatility of this reaction to generate diverse secondary amines including aliphatic (2a, 2b, 13, 14), aromatic and heteroaromatic (5, 6, 7, 9, 11, 12, 15), and aprotic or protected polar groups (8, 10, 16, 17, 18). In all reactions, no amide byproduct was observed in the crude ¹H NMR spectrum. Reaction times were generally longer for alkylation of 4 compared to the reaction of the corresponding oxazole 1 (see details in the Methods). Notably, both azole substrates reacted smoothly with the sterically encumbered l-(−)-α-methylbenzylamine to access 7 and 15 that bear chiral N-substituents. Predictably, the reactions of 1 with arylamine nucleophiles required longer reaction times (3–7 days). Nonetheless, N-aryl products 11 and 12 were isolated in useful yields. Taken together, this array of products demonstrated the flexibility of this approach to functionalize azoles with a diversity of secondary amines. Products from the reaction can be incorporated into traditional solution phase or appropriately modified for use in solid-phase peptide synthesis following N-protection and ester hydrolysis. These are thus useful precursors to a wide array of new peptidomimetics.

Table 2. Amination of Chloromethyl-Substituted Thiazole or Oxazole via a Silver-Mediated Substitution Reaction (Isolated Yields in Parentheses).

^aReaction was run at room temperature.

To showcase the utility of these new building blocks for the preparation of novel azole-rich macrocycles, four of these were hydrolyzed and the resultant acids were subjected to cyclooligomerization reactions (Table 3). Cyclooligomerization reactions of azole-containing amino acids have been used in several instances to access complex azole-rich macrocycles with reasonable synthetic efficiency.^12,26 Upon cyclooligomerization mediated by pentafluorophenyl diphenylphosphinate (FDPP), acids derived from 2b produced the cyclic trimer, 19b as the major reaction product. In the earlier work from the Pattenden laboratory, cyclooligomerization of thiazole amino acids lacking N-substitution favored cyclic trimer products,²⁶ whereas N-methylated thiazole-containing amino acids yielded cyclic tetramers as the major product.¹² Cyclic tetramer 20b was a minor reaction product; other reaction products identified in the crude reaction mixture included small quantities of larger cyclic oligomers (pentamers, hexamers) and byproducts from FDPP (see the Supporting Information for chromatograms of the crude reaction products). Interestingly, linear oligomers were not observed. As shown in entries 1–3 in Table 3, changes to the number of equivalents of FDPP and to the reaction concentration changed the product distribution, consistent with the previous studies on cyclooligomerization reactions.²⁶

Table 3. Results from Hydrolysis and Cyclooligomerization Reactions.

entry	reactant	conditions for step 3	19/20a	yield of 19b
1	2b	3 equiv FDPP, 79 mM	2.0	5
2	2b	1.5 equiv FDPP, 79 mM	2.3	10
3	2b	1.5 equiv FDPP, 35 mM	4.1	12
4	2a	1.5 equiv FDPP, 35 mM	4.2c	9
5	5	1.5 equiv FDPP, 79 mM	4.5	13
6	14	1.5 equiv FDPP, 35 mM	3.1	11

^aRatios determined by comparison of HPLC chromatogram peak integrations (detection at 220 nm).

^bIsolated by semipreparative HPLC.

^cRatio of peak area for 19a/(20a + cyclic pentamer) because the cyclic pentamer co-elutes.

The hydrolysis–cyclooligomerization sequence likewise produced cyclic trimers as the major products for the reaction of 2a, 5, and 14. Table 3 reports conditions for the reaction conditions that produced highest selectivity for the respective cyclic trimer 19 (entries 4–6). Products obtained vary both azole and side-chain identities, demonstrating that a variety of structures can be accessed from these new azole synthons. Although isolated yields of reaction products following small-scale reaction and high-performance liquid chromatography (HPLC) purification were less than 13%, cyclooligomerization was a preparatively useful way to generate complex macrocycles in a short reaction sequence.

Analysis of oxazole-rich macrocycles 19a–c by ¹H and ¹³C NMR spectroscopy in CDCl₃ showed C₃-symmetrical structures, whereas spectra of thiazole-rich 19d showed evidence of multiple ring conformations. NMR spectral features of 19d in deuterated DMSO were sensitive to changes in temperature (see spectra in the Supporting Information). Macrocyclic peptoids (N-substituted glycine oligomers) of the same ring size (18 atoms, cyclic peptoid hexamers) similarly exhibit dynamic ring conformations.²⁷

In summary, an approach to the preparation of novel azole-containing N-substituted amino acid building blocks is reported. These building blocks are compatible with either traditional solid phase or solution phase peptide chemistry and a wide variety of structural features can be readily appended as N-substituents. N-Substituted heterocycle-rich cyclopeptide macrocycles, including cyclic trimers prepared in this work, will likely exhibit different conformation and physicochemical properties from the parent peptides. Their preparation and study is an exciting frontier and application of the work reported here.

Methods

All reagents and solvents were purchased from commercial sources and used without further purification. Infrared (IR) spectra were obtained using a Shimadzu IR Affinity-1S FT spectrometer. ¹H and ¹³C NMR spectra were recorded on a Bruker 400 MHz spectrometer using a 5 mm high-resolution direct-detection probe. Chemical shifts (δ) are reported in parts per million and are referenced to residual proton in the deuterated solvent, and spectra were acquired at room temperature for compound characterization. ¹H and ¹³C NMR spectra for all compounds are included in the Supporting Information. High-resolution mass spectral data were acquired using an Agilent 1260 Infinity II LC with a 6230 time of flight mass spectrometer (TOF MS) detector (electrospray ionization, positive ion mode) and were within 5 ppm of the expected values. Melting points were obtained using an MSRS DigiMelt MPA160 capillary melting point apparatus. Reaction monitoring was carried out using thin-layer chromatography (TLC) on silica gel (Merck KGaA, TLC Silica gel 60 F₂₅₄). Visualization of TLC results was effected with UV light and/or staining with aqueous KMnO₄ solution. Purification of the reaction products was carried out by flash column chromatography using silica gel 60 (215–400 mesh) purchased from Alfa Aesar. Esters 1 and 4 were prepared as described in the literature, and their spectral features identified were identical to those reported.¹³

General Procedure for the Synthesis of Methyl 2-(N-Alkylaminomethyl)azole-4-carboxylates (2a, 2b, 5–16)

A solution of 1 or 4 (0.72 mmol) and AgClO₄ (1.5 equiv, 1.08 mmol, 0.224 g) in 6 mL of acetonitrile was added to amine (3.6 mmol, 5.0 equiv) in 4 mL of acetonitrile in a round-bottom flask fitted with a reflux condenser and stirred at 60 °C for 6 h–7 days until TLC analysis indicated complete conversion of the reactant. A black precipitate formed over the course of the reaction. After cooling, the mixture was filtered through a Celite pad to remove the solid and the filtrate was then concentrated in vacuo. Saturated aqueous sodium bicarbonate (5 mL) was added to the filtrate, and the mixture was extracted with dichloromethane (5 × 5 mL). Organic extracts were washed with brine (5 mL) and water (5 mL). After drying over MgSO₄, the crude material was concentrated and purified using flash column chromatography (SiO₂, hexanes/ethyl acetate).

Methyl 2-((Isopropylamino)oxazole-4-carboxylate (2a)

The reaction was heated at 60 °C for 7 h. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 2a was isolated as a yellow oil (95.4 mg) in 66% yield. R_f = 0.2 (ethyl acetate/hexanes, 3:2). IR (thin film): 3275.0, 2964.5, 1741.2, 1710.5, 1583.5, 1438.9, 1321.24, 1205.5, 1085.9, 1004.9, 952.8, 804.3, and 769.6 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.13 (s, 1H), 3.92 (s, 2H), 3.85 (s, 3H), 2.82–2.76 (m, 1H), 1.98 (br s, 1H), and 1.03 (d, J = 8 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 163.3, 160.5, 143.0, 132.1, 51.1, 47.0, 42.6, and 21.5; HRMS (ESI-TOF) m/z: calcd for C₉H₁₄N₂O₃ ([M + H⁺]), 199.1077; found, 199.1059.

Methyl 2-((Butylamino)methyl)oxazole-4-carboxylate (2b)

The reaction was stirred at room temperature for 24 h. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 2b was isolated as a yellow oil (108.5 mg) in 70% yield. R_f = 0.34 (ethyl acetate/hexanes, 1:1). IR (thin film): 3505.0, 3216.3, 2960.1, 2928.6, 1745.1, 1586.9, 1499.1, 1473.1, 1343.9, 1265.8, 1085.9, 989.9, and 824.5 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.14 (s, 1H), 3.89 (s, 2H), 3.85 (s, 3H), 2.55 (t, J = 8 Hz, 2H), 1.54 (br s, 1H), 1.44–1.37 (m, 2H), 1.31–1.22 (m, 2H), and 0.83 (t, J = 8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.2, 161.5, 144.0, 133.1, 52.1, 48.9, 45.9, 31.8, 20.2, and 13.8; HRMS (ESI-TOF) m/z: calcd for C₁₀H₁₆N₂O₃ ([M + H⁺]), 213.1234; found, 213.1210.

Methyl 2-((Benzylamino)methyl)oxazole-4-carboxylate (5)

The reaction was heated at 60 °C for 7 h. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 5 was isolated as a yellow oil (137.8 mg) in 77% yield. R_f = 0.14 (ethyl acetate/hexanes, 1:1). IR (thin film): 3519.0, 2864.2, 1730.1, 1539.3, 1454.3, 1438.9, 1354.0, 1321.2, 1201.6, 1085.0, 999.1, 808.1, 748.3, 698.2, and 623.0 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.15 (s, 1H), 7.25–7.18 (m, 5H), 3.88 (s, 2H), 3.84 (s, 3H), 3.75 (s, 2H), and 2.00 (sb, 1H); ¹³C NMR (100 MHz, CDCl₃)): δ (ppm) 164.0, 161.4, 144.1, 139.0, 133.0, 128.3, 128.1, 127.1, 52.9, 52.0, and 45.0; HRMS (ESI-TOF) m/z: calcd for C₁₃H₁₄N₂O₃ ([M + H⁺]), 247.1077; found, 247.1049.

Methyl 2-(((Pyridine-3-ylmethyl)amino)methyl)oxazole-4-carboxylate (6)

The reaction was heated at 60 °C for 6 h. Following purification by flash column chromatography with 50:1 CH₂Cl₂/CH₃OH, 6 was isolated as a yellow oil (52.6 mg) in 40% yield. R_f = 0.38 (CH₂Cl₂/CH₃OH, 9:1). IR (thin film): 3240.5, 1739.7, 1579.7, 1479.4, 1425.4, 1321.2, 1199.7, 1143.7, 1085.0, 1001.0, 804.3, 769.6, and 713.6 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.48–8.42 (m, 2H), 8.15 (s, H), 7.62 (d, J = 8.0 Hz, 1H),7.18 (q, J = 4.0 Hz, 1H), 3.90 (s, 2H), 3.84 (s, 3H), 3.78 (s, 2H), and 2.35 (sb, 1H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 163.7, 161.4, 149.6, 148.6, 144.1, 135.9, 134.5, 133.2, 123.4, 52.1, 50.2, and 45.1; HRMS (ESI-TOF) m/z: calcd for C₁₂H₁₃N₃O₃ ([M + H⁺]), 248.1030; found, 248.1012.

Methyl (S)-2-(((1-Phenylethyl)amino)methyl)oxazole-4-carboxylate (7)

The reaction was heated at 60 °C for 24 h. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 7 was isolated as a yellow oil (150.0 mg) in 79% yield. R_f = 0.23 (ethyl acetate/hexanes, 1:1). IR (thin film): 3280.0, 2970.3, 1741.7, 1583.5, 1450.4, 1436.9, 1321.2, 1197.7, 1141.8, 1091.7, 1002.9, 806.25, 761.8, and 700.1 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.12 (s, 1H), 7.27–7.17 (m, 5H), 3.85 (s, 3H), 3.80–3.69 (m, 3H), 2.04 (sb, 1H), and 1.33 (d, J = 8.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)): δ (ppm) 164.2, 161.5, 144.1, 143.9, 133.1, 128.5, 127.2, 126.6, 57.6, 52.0, 43.9, and 24.1; HRMS (ESI-TOF) m/z: calcd for C₁₄H₁₆N₂O₃ ([M + H⁺]), 261.1234; found, 261.1218.

Methyl 2-(((2-tert-Butoxycarbonyl)amino)methyl)oxazole-4-carboxylate (8)

The reaction was heated at 60 °C for 20 h. Following purification by flash column chromatography with 1:4 hexanes/ethyl acetate, 8 was isolated as a brown oil (150.0 mg) in 68% yield. R_f = 0.11 (ethyl acetate/hexanes, 2:1). IR (thin film): 3299.0, 2981.9, 1734.0, 1670.3, 1490.5, 1438.9, 1373.3, 1239.9, 1166.9, 1145.7, 1103.2, and 1098.4 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.16 (s, 1H), 3.97 (s, 2H), 3.83 (s, 3H), 3.21–3.18 (m, 2H), 2.79–2.76 (m, 2H), and 1.35 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.2, 161.3, 156.2, 144.2, 132.5, 52.0, 48.8, 45.5, 36.4, 31.2, and 28.2; HRMS (ESI-TOF) m/z: calcd for C₁₃H₂₁N₃O₅ ([M + H⁺]), 300.1554; found, 300.1546.

Methyl 2-(((3-(1H-Imidazole-1-yl)propyl)amino)methyl)oxazole-4-carboxylate (9)

The reaction was heated at 60 °C for 6 h. Following purification by flash column chromatography with 1:5 hexanes/ethyl acetate, 9 was isolated as a yellow oil (120.2 mg) in 63% yield. R_f = 0.29 (CH₂Cl₂/CH₃OH, 9:1). IR (thin film): 3360.0, 2951.0, 1735.9, 1583.5, 1508.3, 1436.9, 1321.2, 1228.6, 1201.6, 1145.7, 1119.5, 1001.0, 916.1, 806.2, 765.7, and 665.4 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.10 (s, 1H), 7.34 (s, 1H), 6.88 (s, 1H), 6.79 (s, 1H), 3.92 (t, J = 8 Hz, 2H), 3.80 (s, 2H), 3.77 (s, 3H), 2.47 (t, J = 8.0 Hz, 2H), 2.36 (sb, 1H), and 1.83–1.76 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 163.8, 161.4, 144.1, 137.1, 133.0, 129.1, 118.8, 52.0, 45.7, 45.3, 44.2, and 30.9; HRMS (ESI-TOF) m/z: calcd for C₁₂H₁₆N₄O₃ ([M + H⁺]), 265.1295; found, 265.1280.

Methyl 2-(((2-Methoxyethyl)amino)methyl)oxazole-4-carboxylate (10)

The reaction was heated at 60 °C for 6 h. Following purification by flash column chromatography with 1:5 hexanes/ethyl acetate, 10 was isolated as a brown oil (128.6 mg) in 82% yield. R_f = 0.16 (ethyl acetate/hexanes, 8:1). IR (thin film): 3285.0, 2889.3, 1741.7, 1726.2, 1583.5, 1436.9, 1319.3, 1197.7, and 1085.0 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.18 (s, 1H), 3.92 (s, 2H), 3.88 (s, 3H), 3.41 (t, J = 8.0 Hz, 2H), 3.26 (s, 3H), 2.75 (t, J = 8.0 Hz, 2H), and 2.25 (sb, 1H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 163.8, 161.3, 144.1, 132.9, 71.5, 58.5, 51.9, 48.3, and 45.8; HRMS (ESI-TOF) m/z: calcd for C₉H₁₄N₂O₄ ([M + H⁺]), 215.1026; found, 215.1015.

Methyl 2-((Phenylamino)methyl)oxazole-4-carboxylate (11)

The reaction was heated at 60 °C for 7 days. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 11 was isolated as a brown oil (67.2 mg) in 40% yield. R_f = 0.20 (ethyl acetate/hexanes, 1:1). IR (thin film): 3280.0, 2860.0, 2840.0, 1708.9, 1600.0, 1488.8, 1440.8, 1315.4, 1240.2, 1201.6, 1103.2, 1006.8, 752.2, and 692.4 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.11 (s, 1H), 7.14–7.10 (m, 2H), 6.70 (t, 2H, J = 8.0 Hz, J’ = 8.0 Hz), 6.63 (d, 2H, J = 8.0 Hz), 4.43 (s, 2H), 4.33 (br s, 1H), and 3.84 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 163.1, 161.4, 146.5, 144.2, 133.3, 129.4, 118.7, 113.1, 52.2, and 41.2; HRMS (ESI-TOF) m/z: calcd for C₁₂H₁₂N₂O₃ ([M + H⁺]), 233.0926; found, 233.0938.

Methyl 2-(((4-Methoxyphenyl)amino)methyl)oxazole-4-carboxylate (12)

The reaction was heated at 60 °C for 3 days. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 12 was isolated as a brown oil (163.1 mg) in 85% yield. R_f = 0.38 (ethyl acetate/hexanes, 1:1). IR (thin film): 3271.0, 2833.4, 1678.0, 1519.5, 1463.9, 1440.8, 1238.3, 1201.6, 1180.4, 1033.8, 825.5, and 721.3 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.16 (s, 1H), 6.74 (d, 2H, J = 8.0 Hz), 6.65 (d, 2H, J = 8.0 Hz), 4.42 (s, 2H), 3.89 (s, 3H), 3.73 (s, 3H), and 3.56 (br s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 163.5, 161.5, 152.7, 140.8, 139.9, 133.0, 114.8, 114.5, 55.6, 52.1, and 42.0; HRMS (ESI-TOF) m/z: calcd for C₁₃H₁₄N₂O₄ ([M + H⁺]), 263.1032; found, 263.1054.

Methyl 2-((Isopropylamino)methyl)thiazole-4-carboxylate (13)

The reaction was heated at 60 °C for 24 h. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 13 was isolated as a yellow solid (51.0 mg) in 66% yield. mp = 46.5–48.8 °C; R_f = 0.17 (ethyl acetate/hexanes, 2:1). IR (thin film): 3245.0, 3103.4, 2945.3, 2922.1, 1703.1, 1494.8, 1435.0, 1319.3, 1305.8, 1232.5, 1170.7, 1126.4, 1085.9, 987.5, 910.4, 860.2, and 771.0 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.05 (s, 1H), 4.06 (s, 2H), 3.87 (s, 3H), 2.86–2.80 (m, 1H), 1.89 (br s, 1H), and 1.03 (d, J = 8.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.0, 161.9, 146.5, 127.7, 52.3, 48.7, 48.4, and 22.9; HRMS (ESI-TOF) m/z: calcd for C₉H₁₄N₂O₂S ([M + H⁺]), 215.0849; found, 215.0832.

Methyl 2-((Butylamino)methyl)thiazole-4-carboxylate (14)

The reaction was heated at 60 °C for 20 h. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 14 was isolated as a yellow solid (111.8 mg) in 68% yield. mp = 52.0–53.6 °C; R_f = 0.22 (ethyl acetate/hexanes, 2:1). IR (thin film): 3305.9, 3116.9, 2951.0, 2927.9, 1707.0, 1548.8, 1490.9, 1458.1, 1435.0, 1321.2, 1305.8, 1234.4, 1085.9, 977.9, and 801.3 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.11 (s, 1H), 4.11 (s, 2H), 3.91 (s, 3H), 2.67 (t, J = 8.0 Hz, 2H), 2.00 (br s, 1), 1.50–1.43 (m, 2H), 1.38–1.30 (m, 2H), and 0.88 (t, J = 8.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 174.5, 161.9, 146.5, 127.8, 52.3, 49.3, 32.1, 20.2, and 13.9; HRMS (ESI-TOF) m/z: calcd for C₁₀H₁₆N₂O₂S ([M + H⁺]), 229.1005; found, 229.0991.

Methyl (S)-2-(((1-Phenylethyl)amino)methyl)thiazole-4-carboxylate (15)

The reaction was heated at 60 °C for 6 h. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 15 was isolated as a yellow solid (182.1 mg) in 92% yield. mp = 97.3–98.5 °C; R_f = 0.45 (ethyl acetate/hexanes, 1:1). IR (thin film): 3327.2, 3089.9, 2922.1, 1712.7, 1492.9, 1452.4, 1334.7, 1300.0, 1217.0, 1099.4, 1176.5, 981.7, 856.3, 779.2, and 759.9 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.99 (s, 1H), 7.23–7.11 (m, 5H), 3.93–3.75 (m, 6H), 2.08 (br s, 1H), and 1.28 (d, J = 8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 174.10, 161.86, 146.52, 144.47, 128.54, 127.74, 127.22, 126.54, 57.79, 52.22, 48.58, and 24.02; HRMS (ESI-TOF) m/z: calcd for C₁₄H₁₆N₂O₂S ([M + H⁺]), 277.1005; found, 277.0982.

Methyl 2-(((2-tert-Butoxycarbonyl)amino)ethyl)amino)methyl)thiazole-4-carboxylate (16)

The reaction was heated at 60 °C for 24 h. Following purification by flash column chromatography with 1:4 hexanes/ethyl acetate, 16 was isolated as a yellow oil (200.0 mg) in 88% yield. R_f = 0.17 (ethyl acetate/hexanes, 4:1). IR (thin film): 3360.0, 2980.0, 1693.5, 1510.0, 1454.3, 1367.5, 1236.3, 1165.0, 1091.7, and 765.0 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.07 (s, 1H), 4.10 (s, 2H), 3.84 (s, 3H), 3.23–3.17 (m, 2H), 2.76 (m, 2H), 1.95 (s, 1H), and 1.33 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 173.5, 162.1, 156.4, 146.0, 128.1, 60.3, 52.4, 49.9, 48.7, 35.1, and 28.3; HRMS (ESI-TOF) m/z: calcd for C₉H₁₄N₂O₂S ([M + H⁺]), 316.1326; found, 316.1299.

Methyl 2-(((2-Methoxyethyl)amino)methyl)thiazole-4-carboxylate (17)

The reaction was heated at 60 °C for 24 h. Following purification by flash column chromatography with 1:5 hexanes/ethyl acetate, 17 was isolated as a brown oil (95.3 mg) in 58% yield. R_f = 0.14 (ethyl acetate/hexanes, 10:1). IR (thin film): 3445.0, 3080.0, 2889.3, 1724.3, 1492.9, 1454.3, 1226.7, and 1085.0 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.08 (s, 1H), 4.17 (s, 2H), 3.88 (s, 3H), 3.47 (t, J = 8.0 Hz, 2H), 3.30 (s, 3H), 3.03 (sb, 1H), and 2.88 (t, J = 8.0 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 172.56, 162.96, 146.56, 128.13, 71.19, 58.87, 52.49, 50.38, and 48.73; HRMS (ESI-TOF) m/z: calcd for C₉H₁₄N₂O₃S ([M + H⁺]), 231.0798; found, 231.0769.

Methyl 2-(((2-((Triisopropylsilyl)ethyl)amino)methyl)thiazole-4-carboxylate (18)

The reaction was heated at 60 °C for 24 h. Following purification by flash column chromatography with 1:1 hexanes/ethyl acetate, 18 was isolated as a brown oil (164.8 mg) in 80% yield. R_f = 0.36 (ethyl acetate/hexanes, 1:1). IR (thin film): 3360.0, 2941.4, 2889.3, 2864.2, 1722.4, 1462.0, 1319.3, 1238.3, 1209.3, 1085.0, 1068.5, 993.3, and 881.4 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.07 (s, 1H), 4.11 (s, 2H), 3.86 (s, 3H), 3.75 (t, J = 4.0 Hz, 2H), 2.75 (t, J = 4.0 Hz, 2H), and 0.99–0.97 (m, 21H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 174.7, 161.9, 146.4, 127.8, 62.3, 52.2, 51.3, 50.5, 17.9, and 11.8; HRMS (ESI-TOF) m/z: calcd for C₁₇H₃₂N₂O₃SSi ([M + H⁺]), 373.1976; found, 373.1958.

General Procedure for the Synthesis of 19a–d by Cyclooligomerization of 2a, 2b, 5, 14

To a solution of 2a, 2b, 5, or 14 (0.317 mmol) in 3 mL methanol in a 20 mL scintillation vial was added a 1 M aqueous solution of sodium hydroxide (0.33 mL, 0.38 mmol, 1.2 equiv). The mixture was stirred with heating in a 70 °C sand bath for 24 h. After completion of the reaction, the mixture was cooled and quenched with sodium bicarbonate (0.133 g, 1.585 mmol, 5.0 equiv). The mixture was then concentrated in vacuo at 25 °C, and the solid residue was suspended in 3 mL water/acetonitrile (1:1), frozen, and lyophilized overnight. Then, 4 mL of dry dimethylformamide (DMF) and FDPP (0.153 g, 0.3975 mmol, 1.5 equiv) were added to the vial, and the reaction mixture was stirred at room temperature for 3 days. DMF was removed by toluene azeotrope distillation. The residue was partitioned between 1 M aqueous sodium hydroxide (5 mL) and ethyl acetate (5 mL), and the aqueous layer was extracted with ethyl acetate (4 × 5 mL). The combined organic layers were washed with 5 mL water and dried over MgSO₄, then concentrated by rotary evaporation.

Analysis of the cyclooligomerization product ratios was effected by comparing peak areas obtained by analytical RP-HPLC. Crude reaction mixtures were eluted from an AAPPTec Spirit Peptide C18 column (5 μM, 0.46 cm × 15 cm) using a 60–90% linear gradient of methanol (solvent B) in 0.1% aqueous trifluoroacetic acid (TFA; solvent A) at 0.75 mL/min. Peaks eluted were detected by absorbance at 220 and 254 nm, and chromatograms were visualized and peaks from the 220 nm chromatogram were integrated with EZChrom software. Analytical chromatograms of both crude and purified compounds are shown in the Supporting Information.

Cyclic products were purified by semipreparative RP-HPLC. Compounds were dissolved in 1.5:1:0.5 ethyl acetate/CH₃OH/0.1% aqueous TFA, then eluted from an AAPPTec Spirit Peptide C18 column (5 μM, 10.0 mm × 25 cm) using a linear gradient of methanol (solvent B) in 0.1% aqueous TFA (solvent A) at 3 mL/min flow rate. Peaks eluted were detected by absorbance at 220 and 254 nm, and data were visualized with EZChrom software. Purified compounds were isolated as powders following lyophilization.

Cyclic Trimer 19a

A white powder (9.5 mg) was isolated in 9% yield. IR (thin film): 2910.5, 2880.0, 1635.7, 1581.1, 1444.6, 1415.7, 1180.4, 1111.0, 1076.2, and 729.0 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.96 (s, 3H), 4.93–4.82 (m, 9H), and 1.14 (d, J = 4.0 Hz, 18H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 162.0, 160.7, 143.3, 137.0, 46.3, 40.2, 29.7, and 19.8; HRMS (ESI-TOF) m/z: calcd for C₂₄H₃₀N₆O₆ ([M + H⁺]), 499.2300; found, 499.2330.

Cyclic Trimer 19b

A white powder (17.2 mg) was isolated in 12% yield. IR (thin film): 2956.8, 2931.8, 2872.0, 1637.5, 1581.6, 1463.9, 1419.6, 1315.4, 1172.7, 1105.2, 962.4, and 752.2 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.02 (s, 3H), 4.99 (br s, 6H), 3.56–3.55 (m, 6H), 1.58–1.54 (m, 6H), 1.35–1.29 (m, 6H), and 0.88 (t, J = 8.0 Hz, 9H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.9, 160.1, 144.1, 136.5, 48.7, 45.8, 29.4, 20.0, 52.22, and 13.8; HRMS (ESI-TOF) m/z: calcd for C₂₇H₃₆N₆O₆ ([M + H⁺]), 541.2769; found, 541.2795.

Cyclic Trimer 19c

A pale pink powder (26.5 mg) was isolated in 13% yield. IR (thin film): 2961.0, 2924.0, 2852.7, 1638.9, 1581.6, 1494.8, 1452.4, 1419.6, 1357.8, 1319.3, 1201.6, 1165.0, 1111.0, 1080.1, 1029.9, 970.1, 758.2, and 700.1 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.09 (s, 3H), 7.30–7.19 (m, 15H), and 4.88–4.82 (m, 12H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 162.0, 160.1, 144.4, 136.4, 136.0, 128.8, 128.5, 127.9, 50.8, and 44.3; HRMS (ESI-TOF) m/z: calcd for C₃₆H₃₀N₆O₆ ([M + H⁺]), 643.2300; found, 643.2325.

Cyclic Trimer 19d

A white powder (72.3 mg) was isolated in 11% yield. IR (thin film): 2956.8, 2926.0, 2872.0, 1639.5, 1485.1, 1408.0, 1317.3, 1226.7, 933.5, and 744.5 cm^–1. Owing to the presence of multiple rotameric states as detailed in the text, both ¹H and ¹³C NMR spectra show poor signal dispersion, and peaks often appear as complex multiplets. ¹³C NMR spectra exhibit multiple peaks for carbons attributed to different conformers. See spectra shown in the Supporting Information for more details, including spectra acquired at elevated temperatures. ¹H NMR (400 MHz, DMSO-d₆, room temperature): δ (ppm) 8.35–7.54 (m, 3H), 5.73–4.20 (m, 6H), 3.88–3.07 (m, 6H, overlaps with HOD peak), and 1.75–0.67 (m, 21H); ¹³C NMR (100 MHz, CDCl₃, room temperature): δ (ppm) 169.9, 168.8, 166.3, 165.4, 165.2, 163.4, 163.3, 161.9, 150.2, 149.9, 149.4, 149.0, 51.1, 50.8, 49.9, 49.3, 48.8, 47.2, 47.0, 33.7, 31.9, 31.1, 30.8, 30.2, 30.0, 29.7, 29.4, 28.9, 26.7, 22.7, 20.3, 20.2, 19.8, 14.2, 14.1, 13.9, 13.8, and 13.7; HRMS (ESI-TOF) m/z: calcd for C₂₇H₃₆N₆O₃S₃ ([M + H⁺]), 589.2084; found, 589.2145.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b03682.

• ¹H and ¹³C NMR spectra of new compounds and analytical HPLC chromatograms for 19a–d (PDF)

The authors declare no competing financial interest.