Rapid Synthesis of Novel Pyrazino-Pyrido-Pyrimidinones Derived from Fumiquinazoline Alkaloids2,5-Diketopiperazines (2,5-DKPs) are valued in Medicinal Chemistry for their diverse properties and are considered privileged structures. The natural alkaloids class of fumiquinazolines, combining 2,5-DKP and quinazolinone cores within a pyrazino[1,2-b]quinazoline-3,6-dione framework, is known for its broad biological activity. Although few synthetic approaches to such structures have been explored, this study focuses on synthesizing novel fumiquinazolines analogs featuring a pyrazinopyrido[2,3-d]pyrimidine-5,7-dione core bearing various substituents on the pyrazine moiety (C-6 and C-9), classified into three series (A, B, and C). To investigate an efficient sequential one-pot method, we used a pyridine precursor and L-amino acids in two condensation and cyclization reactions, under controlled microwave irradiations at lower temperatures than previously reported. This approach generated protected bicyclic intermediates, particularly pyrido[2,3-d]pyrimidin-4(3H)-one derivatives, which were smoothly deprotected, allowing for final intramolecular cyclization to yield disubstituted fumiquinazoline analogs (series A) without observed epimerization. The optimized protocol revealed greater efficiency for monosubstituted analogs (series B and C), speeding up the tricyclic products synthesis without the need for intermediates isolation and deprotection. Overall, this study demonstrates the incorporation of diverse protected amino acids, featuring different electronic and structural side chains, via a rapid and effective sequential one-pot strategy, resulting in the synthesis of 19 novel fumiquinazoline analogs.
Keywords:
fumiquinazoline analogs; pyrazino-pyrido[2,3-d]pyrimidine-5,7-diones; 2,5-diketopiperazines; one-pot synthesis; green chemistry
Introduction
Natural products form a vast and intricate array of specialized metabolites originated from plants, animals, and microorganisms.1 Although these metabolites do not play a crucial role in the survival of the producing organism, they can confer significant adaptive advantages,2 such as defense mechanisms against predators3 and chemical signaling.4 Over the past decades, the investigation of natural compounds has been of utmost importance for the discovery of new drugs, including those with antimicrobial, anticancer, antiparasitic, and other actions.5, 6 2,5-Diketopiperazines (2,5-DKPs), also named piperazine-2,5-dione, are six-membered ring restrained dipeptides, found in numerous natural products isolated from bacteria, fungi, marine microorganisms, and even mammals. The primary biosynthetic pathway of 2,5-DKP involves the condensation of two naturally occurring α-amino acids, giving rise to a cis side chain arrangement whilst both amino acids have the same configurations (L or D), or trans if they are different (L and D). Additionally, these structures can harness the benefits of peptides while mitigating their drawbacks, which include resistance to proteolysis, conformational rigidity, controllable stereochemistry, capacity to keep low molecular weight (below 500 mol g−1) even when bearing substituent, and the ability to interact by both donor and acceptor hydrogen bonds, crucial for drug-like development.7, 8 Given the broad spectrum of activities exhibited by natural 2,5-DKPs related to anticancer, anti-oxidant, antiviral, antibacterial, and anti-inflammatory, in-depth exploration of their structure-activity relationships has driven the design of biologically active compounds, some of which had progressed to clinical trials, as depicted in Figure 1.8, 9, 10
The 2,5-DKP-fused quinazolinone nucleus is found in fumiquinazoline alkaloids and encompasses a pyrazino[1,2-b]quinazoline-3,6-dione tricyclic system, classified by structural complexity, Figure 2.11 The first discovered fumiquinazolines (fumiquinazoline A-G) were isolated from Aspergillus fumigatus found in the marine fish Pseudolabrus japonicus.12 Since then, diverse fumiquinazolines have been found in Penicillium, Aspergillus, Neosartorya, Acremonium and Scopulariopsis,11 with fumiquinazoline F being a major metabolite known for its antimicrobial activity against Micrococcus luteus (minimum inhibitory concentration, MIC 99 mg mL−1) and Staphylococcus aureus (MIC 137 mg mL−1).13 Notable antimicrobial activities against Candida albicans,14 methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), and aquatic bacteria Vibrio harveyi were also achieved, respectively, for cottoquinazoline D (MIC 22.6 µM), neofiscalin A (MIC 8 µg mL−1), and lapatin B (MIC 16 µg mL−1),15 while tumor cells growth inhibition was reported for fumiquinazoline J (half-maximal inhibitory concentration, IC50 values < 20 µM).16 In addition, studies of antiviral properties of glyantrypines revealed (−)-oxoglianthripin as H1N1 inhibitor with IC50 values comparable to ribavirin (19 μM) (Figure 2).17 Therefore, the significant therapeutic applications of these diketopiperazine-quinazolinone scaffolds reinforce their recognition as a privileged structure in Medicinal Chemistry.
Fumiquinazolines alkaloids containing the pyrazino[2,1-b]quinazoline-3,6-dione ring system, with the diketopiperazine-quinazolinone scaffold highlighted in pink.
The synthesis of pyrazino[2,1-b]quinazoline-3,6-dione cores is well-established through various methods, including: (i) an intramolecular aza-Wittig reaction involving the treatment of N-methyl L-phenylalanine with 2-azido benzoyl chloride;18 (ii) a four-step strategy comprising two consecutive condensations of L-tryptophan methyl ester, anthranilic acid, and Fmoc-D-alanine, followed by two intramolecular cyclizations;19 (iii) direct double cyclodehydration of linear tripeptides using solid support resin and zinc triflate;20 and (iv) a microwave-assisted, three-component, one-pot reaction utilizing selected N-Boc-amino acids and amino acid esters, designed to mimic the biosynthetic pathways of glyantrypine, fumiquinazoline F, and fiscalin B alkaloids.21 In general, the cLogP values of these tricyclic alkaloids indicate high lipid solubility. Introducing a nitrogen atom, for example by replacing the benzene ring of the quinazoline with a pyridine ring, could help balance solubility, as seen in pyrazino-pyrido-pyrimidinone analogues.
To the best of our knowledge, the synthesis of disubstituted pyrazino-pyrido-pyrimidinones has not been extensively investigated,22, 23 with only one reference noting the use of a microwave-assisted three-component one-pot reaction under harsh conditions, such as elevated temperature (220 °C, 1.5 min).22 Under these conditions, two products were isolated in low yields (9-12%) due to the steric hindrance caused by the amino acid side chains on the protected intermediates, resulting in high enantiomeric epimerization ratios, approaching 1:1.21, 22 To address these limitations, we investigated a multicomponent one-pot approach at a lower temperature (150 °C, 2.0 min) to synthesize disubstituted pyrazino-pyrido[2,3-d]pyrimidine-5,7-dione derivatives. However, pyrido[2,3-d]pyrimidin-4(3H)-one intermediates containing various amino acids side chains were isolated, and an additional step of N-Boc deprotection was included for spontaneously intramolecular cyclization. Thus, the target disubstituted pyrazinopyrido-pyrimidinones (20-39%) were obtained after classic chromatographic column using silica gel (CCC) and high-performance liquid chromatography (HPLC) purifications, without detectable epimerization. Our studies further revealed that monosubstituted products could be synthesized under the same conditions, without needing prior N-Boc deprotection (22-74%). Consequently, the substitution of the quinazolinone ring in fumiquinazoline with pyrido[2,3-d]pyrimidin-4(3H)-one,22 along with the introduction of substituents in the pyrazine (modified 2,5-DKP) core defined by the amino acid side chains used during synthesis, resulted in the generation of novel nineteen pyrazino-pyrido[2,3-d]pyrimidine-5,7-dione derivatives, which preserves the original configuration of the amino acids (series A-C, Scheme 1).
Chemical structures of 19 proposed fumiquinazoline analogs possessing the pyrazino-pyrido[2,3-d]pyrimidine-5,7-dione scaffold, classified as series A with the 2-ethyl-1H-indole substituent at C-6 (R2) highlighted in green and changes at C-9 (R1) in blue; series B with variations at C-6 (R2) in red and series C containing variations at C-9 (R2) in blue.
Results and Discussion
Driven by the need to synthesize pyrazino-pyrido[2,3-d] pyrimidine-5,7-dione core, we started from 2-amino nicotinic acid (20) and adapted the known method based on a sequential one-pot coupling of two L-amino acids.21 Despite the myriad possible combinations of protected amino acids for the first [N-Boc(R1)-aa] and second [methyl ester(R2)-aa] couplings, a selection was made to narrow the number of analogs and maintain diverse side chains displaying different physicochemical properties. Thus, seven L-amino acids were selected, glycine, valine, isoleucine, phenylalanine, tryptophan, tyrosine and methionine, for displaying their lateral chains at C-9 (R1), while preserving the L-tryptophan indole group at C-6 as observed in glyantrypine and fumiquinazoline F, G, and T (serie A, Scheme 2).
Synthesis of pyrazino-pyrido[2,3-d]pyrimidine-5,7-diones, 1-6 (series A). Reaction conditions: (i) P(OPh)3, anhydrous Py, N-Boc-aa (R1), microwave irradiation: 100 °C, 10 min; (ii) anhydrous Py, L-tryptophan methyl ester 22, microwave irradiation: 150 °C, 2.0-3.0 min; (iii) TFA/DCM 1:1 v/v (2.0 mL), 2.0-3.0 h, room temperature (RT); (iv) 5% NaHCO3 solution until pH 7-8, RT.
In our hands, attempts to reproduce the sequential one-pot coupling of two L-amino acids under two microwave irradiation steps, respectively, at 150 °C for 10 min and 220 °C for 1.5 min, were unsuccessful to produce the pyrazino-pyrido[2,3-d]pyrimidine-5,7-diones.21, 22 For this reason, milder conditions were tested and revealed the formation of 4H-pyrido[2,3-d] [1,3]oxazin-4-one intermediate 21 (not isolated) under microwave irradiations at 100 °C, 10 min, as the product of the first coupling between 20 and N-Boc amino acid, followed by irradiation at 150 °C for 2 min for the second sequential reaction with tryptophan methyl-ester 22 to generate the final tricyclic products. However, after CCC purification, the 1H nuclear magnetic resonance (1H NMR) spectra revealed the presence of aromatic hydrogens (H-2, H-3 and H-4) and, unexpectedly, those corresponding to OMe and N-Boc protective groups around δ 3.83 (s, 3H) and 1.41 (s, 9H), respectively. In addition, major m/z consistent with the theoretical values of the protonated adducts [M + H]+ of pyrido[2,3-d]pyrimidin-4(3H)-one intermediates 23-28 (Scheme 2) were also detected at high-resolution electrospray ionization mass spectrometry (HRESI-MS). To address the lack of reactivity, primarily caused by steric hindrance from both amino acid side chains (C-6 and C-9), an additional deprotection step was performed to remove the amino-protecting group (N-Boc) under acidic conditions.8, 24 Following neutralization of the resulting ammonium salt derivatives (29-34), the free amino groups underwent an intramolecular nucleophilic attack on the carbonyl ester, resulting in the displacement of the methoxyl group and formation of the pyrazino-pyrido[2,3-d] pyrimidine-5,7-diones. Thus, HPLC purifications afforded six novel fumiquinazolines analogs (1-6) in 20-39% yields, which were identified by 1H NMR spectra with the characteristic signals of H-2, H-4 and H-3 protons around δ 8.99, 8.72 and 7.52, respectively, with no signal duplications. In addition, aromatic hydrogens related to indole moiety was observed ranging from δ 7.40-6.55 and H-6 and H-9 methine hydrogens between δ 5.65-5.43, and 4.85-2.50, respectively. The 13C spectra showed C-2, C-4, C-3 and C-6 signals preserved nearby δ 157.5, 138.1, 127.6 and 54.4, and C-9 varied approximately δ 56.2-53.4. Furthermore, HRESI-MS analyses displayed [M + H]+ adducts for all products. These data are comprehended in the Supplementary Information (SI) section.
After establishing the new protocol, we swiftly applied it to synthesizing both unsubstituted and monosubstituted derivatives, bypassing the need for the N-Boc deprotection step. Therefore, series B comprised the use of N-Boc-glycine as the first coupled amino acid to give R1 = H (C-9), and varied the second methyl ester-aa with R2 substituent (C-6), while in series C the first N-Boc-aa was changed to produce analogs with R1 substituent (C-9) and the methyl ester-glycine was fixed (R2 = H at C-6), Scheme 3. The coupling of 20 with the first and second protected-amino acids were tested under the same microwave irradiations conditions as previously described (100 °C, 10 min and 150 °C, 2 min). After slights changes just on the time of the second irradiation (2-3 min), the products 7-19 were formed via spontaneous loss of the N-Boc group, followed by an intramolecular cyclisation on the pyrido[2,3-d]pyrimidin-4(3H)-one systems 35, suitably substituted at C-6 (series B, Scheme 3a) or at C-9 (series C, Scheme 3b) depending on the precursors used. These results confirmed that steric hindrance significantly impacts the final intramolecular cyclization, particularly when both substituents R1 and R2 are present, making the process more challenging.
(a) General synthetic route to obtain series B (7-13) derivatives and respective yields. (b) General synthetic route to obtain series C (14-19) derivatives and respective yields. Reaction conditions: (i) P(OPh)3, anhydrous Py, N-Boc-glycine or N-Boc-aa (R1), microwave irradiation: 100 °C, 10 min; (ii) anhydrous Py, methyl ester glycine or metyl esther aa (R2), microwave irradiation: 150 °C, 2-3 min.
Attempts to purify derivative 7 (no substituents) by classical CCC in silica gel yielded an orange precipitate, rather than a white solid, after re-purification by HPLC. Although both samples showed identical 1H NMR signals, a contaminant was detected in the 31P NMR spectrum (δ 0.08) after purification just by CCC (SI section). This byproduct was further identified as metaphosphate ion (PO3−), the conjugate base of metaphosphoric acid (HPO3), originated from the triphenyl phosphite catalyst. Therefore, after HPLC purifications, the pyrazino-pyrido[2,3-d]pyrimidine-5,7-diones (7-19) were isolated without the metaphosphate ion with 22-74% yields, as shown in Scheme 3.
The pyrazino-pyrido-pyrimidinone core for all derivatives exhibited characteristic 1H NMR signals for the pyridine moiety, with H-2, H-4, and H-3 appearing as double doublets around δ 8.97, 8.51, and 7.55, respectively. In series B, the H-6 methine protons were assigned at δ 5.23-4.92, while H-9a and H-9b were noted over a broader range at δ 4.85-3.10. As for series C, H-9 remained at δ 4.6-4.8, and H-6a and H-6b were found within δ 4.71-3.02. Regarding the 13C NMR spectra, C-2, C-4, and C-3 consistently appeared near δ 156.7, 136.6, and 123.1, respectively, for both series. However, as for C-6 and C-9, their chemical shifts were influenced by amino acid lateral chains. When side chains were present at C-6 (series B), their signals appeared in the range of δ 59.9-55.2, while those at C-9 around δ 45.2-44.3. In addition, these shifts were reversed when a substituent was attached at C-9 (series C). The structures of the products were confirmed by HRESI-MS, which displayed mass-to-charge ratio (m/z) relations of protonated adducts [M + H]+ (SI section).
Conclusions
The synthesis of disubstituted pyrazino-pyrido[2,3-d] pyrimidine-5,7-diones, series A, proved difficult under harsh conditions as a result of the steric hindrance impose by amino acids side chains. Lower temperatures were employed during the two irradiation cycles to assist the sequential one-pot couplings of the first and second amino acids, specifically at 100 and 150 °C, respectively. However, these reaction conditions resulted in the formation of protected bicyclic intermediates related to pyrido[2,3-d] pyrimidin-4(3H)-one (23-28), where both the OMe and Boc groups remained intact. For this reason, the deprotection of N-Boc under gentler conditions, followed by neutralization of the corresponding ammonium salts, favored the intramolecular cyclization to give six novel disubstituted products preserving the pyrazino-pyrido-pyrimidinone core, named series A (1-6). Taking into account the additional step of deprotection, the yields were modest after both CCC and HPLC purifications but, most importantly, we did not observe any significant epimerization of the intermediates and products.
Subsequently, the optimized protocol was employed to obtain thirteen novel pyrazino-pyrido-pyrimidinone tricyclicfused analogs of glyantrypine, fumiquinazolines A, B and E, without the need for intermediate isolation and N-Boc deprotection steps. Then, rigorous purification processes (CCC and HPLC) were required to successfully obtain monosubstituted fumiquinazolines analogs as series B (7-13) and C (14-19). Altogether, the synthesis of functionalized analogs of fumiquinazolines was successfully achieved using the sequential one-pot strategy, which proved to be fast and convenient with the use of amino acid-based reagents and controlled reaction conditions.
Experimental
All reagents, chemicals, and solvents were purchased from commercial suppliers and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) using commercially available precoated plates and visualized with ultraviolet (UV) light at 254 nm. Chromatographic purifications on the column were performed using flash silica gel (40-63 μm). Microwave chemical reactor model Discover®, serial number DU 8608 (CEM, USA) was used. The 1H NMR and 13C NMR spectra were recorded on Bruker Avance DRX 300 (300 MHz), DRX 400 (400 MHz) or DPX 500 (500 MHz) spectrometers (Bruker, Switzerland) at the University of São Paulo, Ribeirão Preto, Brazil. The chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS), with the multiplicity (s: singlet, d: doublet, t: triplet, dd: double doublet, m: multiplet), coupling constant (J), given in hertz (Hz), and the number of hydrogens deduced from the relative integral in parentheses. The assignments were made with the aid of two-dimensional analyses gCOSY (gradient-selected correlation spectroscopy), gHMQC (gradient-selected heteronuclear multiple quantum coherence), and gHMBC (gradient-selected heteronuclear multiple bond coherence). Mass spectrometry analyses by ESI-MS were performed using positive and negative ionization mode on a Bruker Daltonics UltrOTOF-Q-ESI-TOF instrument (Bruker, Switzerland). Shimadzu HPLC system with a Shim-PaK C-18 reverse-phase column, Shim-PacK CLC-ODS (M) (250 × 10.0 mm or 4.6 mm, Shimadzu, Japan), were applied to further purification under different gradient conditions (A: H2O, B: MeCN) and flow rates, with UV detection (190 and 250 nm).
General protocol for obtaining intermediates belonging to series A (23-28)
In different flasks, 2-amino nicotinic acid (20) (30 mg, 0.22 mmol, 1.0 eq.) was added, along with each respective N-Boc-protected amino acid: N-Boc-Ala (41.6 mg), N-Boc-Iso (50.9 mg), N-Boc-Phe (58.3 mg), N-Boc-Tyr (61.9 mg), N-Boc-Trp (66.9 mg), and N-Boc-Met (54.8 mg). In each flask was added anhydrous pyridine (1.5 mL) and triphenylphosphite (72 μL, 0.24 mmol, 1.1 eq.) and then irradiated in a microwave reactor (M.W.) for 10 min at 100 °C. After cooling to room temperature, to each mixture was then added tryptophan methyl ester (66 mg, 0.22 mmol, 1.0 eq.) and irradiated again in the M.W. at 150 °C for 2.0-3.0 min. After purification by flash chromatography column with silica gel (hexane 100% → AcOEt 100%), the protected intermediates with the groups -N-Boc and-OMe of series A (23-28) were obtained as slightly yellowish oils with yields between 20-35%. Their structures were confirmed by spectroscopic analyses (1H NMR) and by HRESI-MS.
Compound 23
27.2 mg, 0.048 mmol, 22%; microwave irradiation time in the second step: 2.5 min; 1H NMR (300 MHz, CDCl3) δ 8.92 (dd, 1H, J2,3 4.5 Hz, J2,4 1.9 Hz, H-2), 8.62 (d, 1H, J3,4 7.8 Hz, H-4), 8.27 (s, 1H, NH-17), 7.45-7.41 (m, 2H, H-3, H-12), 7.25 (d, 1H, J14,15 8.1 Hz, H-15), 7.09 (apparent t, 1H, J13,14 = J14,15 7.3 Hz, H-14), 6.94 (apparent t, 1H, J12,13 = J13,14 7.3 Hz, H-13), 6.81 (d, 1H, J 2.0 Hz, H-10), 5.77 (s, 1H, NH), 4.88 (dd, 1H, J6,8a 9.8 Hz, H-6), 4.48-4.40 (m, 1H, H-18), 3.82-3.77 (m, 2H, H-18a, H-18b), 3.74 (s, 3H, OMe), 1.36 (s, 9H, NHBoc), 0.55 (d, 3H, J18,24 5.5 Hz, CH3-24); HRMS (ESI) m/z, calcd. for C26H30N5O5+ [M + H]+: 492.2241, found: 492.2230.
Compound 24
29.5 mg, 0.045 mmol, 20%; microwave irradiation time in the second step: 2.0 min; 1H NMR (300 MHz, CDCl3) δ 8.92 (dd, 1H, J2,3 4.5 Hz, J2,4 2.2 Hz, H-2), 8.63 (dd, 1H, J3,4 8.0 Hz, J2,4 2.2 Hz, H-4), 8.14 (d, 1H J 5.4 Hz, NH-17), 7.61 (dd, 1H, J2,3 4.5 Hz, J3,4 8.0 Hz, H-3), 7.37 (d, 1H, J 5.5 Hz, arom-H), 7.16 (dd, 2H, J 15.6 Hz, J 6.5 Hz, arom-H), 6.88-6.84 (m, 1H, arom-H), 6.68-6.60 (m, 1H, arom-H), 5.79 (d, 1H, J 8.7 Hz, NH), 5.05 (dd, 1H, J6,8a 11.5 Hz, J6,8b 5.0 Hz, H-6), 3.91-3.86 (m, 1H, H-8a), 3.78 (s, 3H, OMe), 3.65 (d, 1H, J18,25 8.7 Hz, H-18), 3.57 (dd, 1H, J8a,8b 15.1 Hz, J6,8b 5.0 Hz, H-8b), 1.80-1.69 (m, 1H, H-25a), 1.67-1.57 (m, 1H, H-25b), 1.41 (s, 9H, NHBoc), 0.95 (d, 3H, J27,24 7.2 Hz, CH3-27), 0.70 (t, 3H, J25,26a = J25,26b 7.4 Hz, CH3-26); HRMS (ESI) m/z, calcd. for C29H36N5O5+ [M + NH4+]+: 552.2876, found: 552.2810.
Compound 25
37.1 mg, 0.054 mmol, 29%; microwave irradiation time in the second step: 3.0 min; 1H NMR (300 MHz, CDCl3) δ 8.89 (dd, 1H, J2,3 4.5 Hz, J2,4 2.0 Hz, H-2), 8.62 (dd, 1H, J3,4 7.9 Hz, J2,4 2.0 Hz, H-4), 7.98 (s, 1H, NH-17), 7.44 (dd, 1H, J2,3 4.5 Hz, J3,4 7.9 Hz, H-3), 7.19-7.12 (m, 6H, arom-H), 7.02 (d, 1H, J12,13 2.3 Hz, H-10), 7.00-6.96 (m, 2H, arom-H), 6.74 (apparent t, 1H, J 7.1 Hz, arom-H), 5.63 (d, 1H, J 8.1 Hz, NH), 5.02-4.90 (m, 1H, H-6), 4.70 (dd, 1H, J18,24a 5.5 Hz, J18,24b 12.4 Hz, H-18), 4.14-4.07 (m, 1H, H-8a), 3.82 (s, 3H, OMe), 3.73-3.67 (m, 1H, H-8b), 3.13 (dd, 1H, J24a,18 5.0 Hz, J24a,24b 13.7 Hz, H-24a), 2.85 (dd, 1H, J24b,18 6.5 Hz, J24a,24b 13.7 Hz, H-24b), 1.36 (s, 9H, NHBoc); HRMS (ESI) m/z, calcd. for C32H34N5O5+ [M + H]+: 568.2554, found: 568.2534.
Compound 26
34.5 mg, 0.047 mmol, 22%; microwave irradiation time in the second step: 3.0 min. In the spectrum of this derivative, the occurrence of rotamers in a 1:0.75 ratio was observed due to the duplication of some signals. 1H NMR (300 MHz, CDCl3) δ 8.93 (dd, 1H, J2,3 4.6 Hz, J2,4 2.0 Hz, H-2), 8.57 (dd, 1H, J3,4 7.9 Hz, J2,4 2.0 Hz, H-4), 8.07 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 8.02 (s, 1H, NH-17), 7.44 (apparent t, 3H, J 6.3 Hz, arom-H), 7.33 (dd, 2H, J 7.6 Hz, J 4.7 Hz, arom-H), 7.23-7.15 (m, 3H, arom-H), 7.13-7.05 (m, 2H, arom-H), 6.93 (apparent t, 3H, J 7.5 Hz, arom-H), 6.82-6.66 (m, 3H, arom-H), 6.45-6.37 (m, 2H, arm-H), 5.66 (d, 1H, J 8.1 Hz, NH), 5.04-4.94 (m, 1H, H-6), 4.85-4.72 (m, 1H, H-18), 3.75 (s, 3H, OMe), 3.29 (dd, 1H, J8a,8b 14.5 Hz, J6,8a 6.1 Hz, H-24b), 3.18-3.08 (m, 2H, H-24a, H-8b), 2.49 (dd, 1H, J24a,24b 14.8 Hz, J18,24b 5.1 Hz, H-24b), 1.28 (s, 9H, NHBoc); HRMS (ESI) m/z, calcd. for C34H35N6O5+ [M + H]+: 607.2663, found: 607.2665.
Compound 27
30.5 mg, 0.052 mmol, 24%; microwave irradiation time in the second step: 3.0 min; 1H NMR (300 MHz, CD3OD) δ 8.76 (dd, 1H, J23 4.7 Hz, J24 1.9 Hz, H-2), 8.62 (dd, 1H, J34 7.9 Hz, J24 1.9 Hz, H-4), 7.53 (dd, 1H, J23 4.7 Hz, J3,4 7.9 Hz, H-3), 7.13-7.05 (m, 2H, H-15, H-12), 6.93 (s, 1H, H-10), 6.86-6.81 (m, 1H, H-14), 6.65 (d, 2H, J 8.3 Hz, H-27, H-29), 6.55 (t, 1H, J12,13 = J13,14 7.8 Hz, H-13), 6.42 (d, 2H, J 8.3 Hz, H-26, H-30), 6.08 (d, 1H, J 7.0 Hz, NH), 5.43-5.38 (m, 1H, H-18), 4.68-4.65 (m, 1H, H-6), 3.81 (s, 3H, OMe), 3.71-3.67 (m, 2H, H-8a, H-8b), 3.00 (dd, 1H, J24a,18 4.8 Hz, J24a,24b 13.8 Hz, H-24a), 2.85 (dd, 1H, J24b,18 4.8 Hz, J24a,24b 13.8 Hz, H-24b), 1.39 (s, 9H, NHBoc); HRMS (ESI) m/z, calcd. for C32H34N5O6+ [M + H]+: 584.2504, found: 584.2489.
Compound 28
42.5 mg, 0.077 mmol, 35%; microwave irradiation time in the second step: 2.5 min; 1H NMR (300 MHz, CDCl3) δ 8.91 (dd, 1H, J2,3 4.6 Hz, J2,4 1.9 Hz, H-2), 8.63 (dd, 1H, J3,4 7.9 Hz, J2,4 1.9 Hz, H-4), 8.14 (s, 1H, NH-17), 7.44 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 7.18-7.13 (m, 3H, arom-H), 6.94 (apparent t, 1H, J13,14 = J14,15 7.6 Hz, H-14), 6.62 (t, 1H, J12,13 = J13,14 7.3 Hz, H-13), 5.85 (d, 1H, J 8.0 Hz, NH), 4.34-4.29 (m, 1H, H-6), 4.71 (t, 1H, J18,9a = J18,9b 7.7 Hz, H-18), 3.83 (s, 3H, OMe), 3.84-3.71 (m, 2H, H-8a, H-8b), 2.61-2.43 (m, 3H, H-19b, H-20a, H-20b), 2.02 (s, 3H, S-CH3), 1.62-1.57 (m, 1H, H-19a), 1.41 (s, 9H, NHBoc); HRMS (ESI) m/z, calcd. for calculated for C28H34N5O5+ [M + H]+: 552.2275, found: 552.2272.
General protocol for synthesis of final products of series A (1-6)
Each protected intermediate (23-28) was treated with a mixture of trifluoroacetic acid/dichloromethane (TFA/DCM) in a 1:1 v/v ratio (2.0 mL) and stirred at room temperature for 2 h. Subsequently, the solvents were removed under reduced pressure, and the crude reaction mixtures were neutralized with a 5% NaHCO3 solution until pH ca. 7.0 (ca. 1.0 mL). Finally, DCM was added, and extraction was performed using a separation funnel. The organic phase was separated, dried, and purified by chromatography column using flash silica gel (hexane 100% → AcOEt 100%), followed by purification using Shimadzu Shim-PaK HPLC system under gradient conditions (A: H2O, B: MeCN, 0-80% B) and flow rate of 1.0 mL min−1. In this manner, the final products of the indole series A (1-6) were obtained as slightly yellow oils with yields ranging from 20-39%. These compounds had their structures confirmed and signals assigned by a set of spectroscopic analyses (1H NMR, 13C NMR, gCOSY, gHMQC, gHMBC) and by HRESI-MS.
6-((1H-Indol-2-yl)methyl)-9-methyl-dihydro-5H-pyrazino [1,2-a]pyrido[2,3-d]pyrimidine-5,7-(6H)-dione (1)
6.6 mg, 0.018 mmol, 33%; 1H NMR (400 MHz, CDCl3) δ 8.94 (dd, 1H, J2,3 4.6 Hz, J2,4 1.9 Hz, H-2), 8.69 (dd, 1H, J2,4 1.9 Hz, J3,4 7.9 Hz, H-4), 7.69 (dd, 1H, JNH,9b 5.7 Hz, JNH,9a 3.3 Hz, NH-8), 7.48 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 7.23 (d, 1H, J20,21 8.2 Hz, H-21), 7.16 (d, 1H, J18,19 8.1 Hz, H-18), 7.01 (apparent t, 1H, J18,19 = J19,20 7.6 Hz, H-19), 6.75 (apparent t, 1H, J19,20 = J20,21 7.5 Hz, H-20), 6.71 (s, 1H, H-22), 5.45 (apparent dd, 1H, J6,15a 4.4 Hz, J6,15b 3.7 Hz, H-6), 3.69 (q, 1H, J9,25 7.0 Hz, H-9), 3.69 (dd, 2H, J6,15a 4.4 Hz, J15a,15b 9.3 Hz, H-15a, H-15b), 0.95 (d, 3H, J9,25 7.0 Hz, CH3-25); 13C NMR (100 MHz, CDCl3) δ 171.8 (C-7), 162.7 (C-5), 156.3 (C-2), 154.8 (C-11), 138.5 (C-14), 138.1 (C-4), 132.4 (C-16), 131.8 (C-23), 127.2 (C-24), 124.1 (C-20), 120.9 (C-19), 119.6 (C-21), 116.5 (C-3), 112.7 (C-18), 110.6 (C-12), 109.5 (C-22), 54.1 (C-6), 53.6 (C-9), 25.7 (C-15), 19.6 (C-25); HRMS (ESI) m/z, calcd. for C20H18N5O2+ [M + H]+: 360.1455, found: 360.1463. Under the reported chromatographic conditions, the retention time (tR) of the compound was 11.53 min.
6-((1H-Indol-2-yl)methyl)-9-(sec-butyl)-dihydro-5H-pyrazino [1,2-a]pyrido[2,3-d]pyrimidine-5,7(6H)-dione (2)
7.0 mg, 0.017 mmol, 29%; 1H NMR (400 MHz, CDCl3) δ 9.01 (dd, 1H, J2,3 4.6 Hz, J2,4 2.0 Hz, H-2), 8.74 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 8.09 (broad s, 1H, NH-8), 7.53 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 7.50 (d, 1H, J20,21 8.2 Hz, H-21), 7.29 (d, 1H, J18,19 8.1 Hz, H-18), 7.14 (ddd, 1H, J18,19 8.1 Hz, J19,20 7.1 Hz, J19,21 1.0 Hz, H-19), 6.99 (ddd, 1H, J19,20 7.1 Hz, J20,21 8.2 Hz, J18,20 0.9 Hz, H-20), 6.54 (d, 1H, J22,NH 2.1 Hz, H-22), 5.65 (dd, 1H, J6,15a 2.7 Hz, J6,15b 5.3 Hz, H-6), 3.79 (dd, 1H, J6,15a 2.7 Hz, J15a,M5b 15.1 Hz, H-15a), 3.59 (dd, 1H, J6,15b 5.4 Hz, J15a,15b 15.1 Hz, H-15b), 2.57-2.51 (m, 1H, H-9), 1.18-1.10 (m, 2H, H-26a, H-26b), 0.79-0.71 (m, 1H, H-25), 0.64 (d, 3H, J25,28 6.7 Hz, CH3-28), 0.41 (t, 3H, = J26b,27 7.4 Hz, CH3-27); 13C NMR (100 MHz, CDCl3) δ 169.0 (C-7), 161.2 (C-5), 157.1 (C-11), 156.4 (C-2), 155.2 (C-14), 137.2 (C-4), 136.3 (C-23), 127.2 (C-24), 123.7 (C-22), 123.2 (C-3), 122.8 (C-19), 120.6 (C-20), 118.9 (C-18), 115.6 (C-12), 111.4 (C-21), 109.2 (C-16), 72.0 (C-6), 56.0 (C-9), 32.5 (C-25), 26.1 (C-26), 13.6 (C-28), 11.1 (C-27); HRMS (ESI) m/z calcd. for C23H24N5O2+ [M + H]+: 402.1925, found: 402.1907. Under the reported chromatographic conditions, the retention time of the compound was 13.19 min.
6-((1H-Indol-2-yl)methyl)-9-benzyl-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d]pyrimidine-5,7(6H)-dione (3)
3.0 mg, 0.0069 mmol, 23%; 1H NMR (400 MHz, CDCl3) δ 9.03 (dd, 1H, J2,4 2.0 Hz, J2,3 4.5 Hz, H-2), 8.73 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 8.12 (broad s, 1H, NH-8), 7.54 (dd, 1H, J2,3 4.5 Hz, J3,4 7.9 Hz, H-3), 7.46-7.38 (m, 4H, H-18, H-20, H-21, H-29), 7.23-7.16 (m, 5H, H-19, H-27, H-28, H-30, H-31), 6.94 (apparent t, 1H, J19,20 = J20,21 7.1 Hz, H-20), 6.61 (d, 1H, J22,NH 2.3 Hz, H-22), 5.61 (dd, 1H, J6,15a 2.6 Hz, J6,15b 5.4 Hz, H-6), 3.81-3.72 (m, 2H, H-9, H-15a), 3.61 (dd, 1H, J6,15b 5.4 Hz, J15a,15b 15.2 Hz, H-15b), 3.01 (dd, 1H, J9,25a 2.7 Hz, J25a,25b 11.3 Hz, H-25a), 2.57 (dd, 1H, J25a,25b 11.3 Hz , J9,25b 15.1 Hz, H-15a); 13C NMR (100 MHz, CDCl3) δ 168.9 (C-7), 161.1 (C-5), 156.7 (C-2), 155.3 (C-11), 137.1 (C-14), 137.0 (C-4), 136.4 (C-23), 134.9 (C-16), 131.2 (C-26), 129.4 (C-3), 128.7 (C-30), 128.6 (C-28), 127.6 (C-31), 127.1 (C-27), 124.0 (C-24), 123.9 (C-22), 123.2 (C-29), 123.1 (C-19), 120.9 (C-20), 119.0 (C-21), 115.8 (C-12), 111.4 (C-18), 57.9 (C-6), 53.4 (C-9), 38.1 (C-25), 29.9 (C-15); HRMS (ESI) m/z, calcd. for C26H22N5O2+ [M + H]+: 436.1768, found: 436.1759. Under the reported chromatographic conditions, the retention time of the compound was 13.67 min.
6,9-bis((1H-Indol-2-yl)methyl)-dihydro-5H-pyrazino[1,2-a] pyrido[2,3-d]pyrimidine-5,7(6H)-dione (4)
10.6 mg, 0.022 mmol, 39%; 1H NMR (400 MHz, CDCl3) δ 9.00 (dd, 1H, J2,3 4.6 Hz, J2,4 2.0 Hz, H-2), 8.72 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 7.52 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 7.47 (d, 1H, J20,21 8.2 Hz, H-21), 7.42 (d, 1H, J18,19 8.1 Hz, H-18), 7.33 (d, 1H, J30,31 7.9 Hz, H-31), 7.24 (d, 1H, J28,29 8.2 Hz, H-28), 7.14-7.09 (m, 1H, H-30), 7.08-7.04 (m, 1H, H-29), 7.02-6.99 (m, 1H, H-19), 6.98-6.94 (m, 1H, H-20), 6.56 (s, 1H, H-22), 5.71 (s, 1H, H-27), 5.43 (apparent dd, 1H, J6,15b 4.7 Hz, J6,15a 3.1 Hz, H-6), 4.51 (apparent dd, 1H, J9,25a 2.8 Hz, J9,25b 11.5 Hz, H-9), 3.76 (dd, 1H, J6,15a 3.1 Hz, J15a,15b 15.2 Hz, H-15a), 3.66 (dd, 1H, J6,15b 4.7 Hz, J15a,15b 15.2 Hz, H-15b), 3.20 (dd, 1H, J9,25a 2.8 Hz, J25a,25b 13.9 Hz, H-25a); 13C NMR (100 MHz, CDCl3) δ 166.4 (C-7), 161.4 (C-5), 157.3 (C-2), 156.8 (C-11), 155.0 (C-14), 137.1 (C-4), 136.4 (C-23), 136.1 (C-34), 132.5 (C-16), 131.2 (C-26), 128.1 (C-3), 126.3 (C-24), 124.4 (C-33), 123.9 (C-22), 123.8 (C-27), 122.7 (C-30), 122.4 (C-19), 120.3 (C-28), 119.8 (C-21), 119.3 (C-31), 118.7 (C-18), 115.5 (C-12), 111.9 (C-29), 111.5 (C-20), 57.3 (C-6), 56.2 (C-9), 33.2 (C-25), 29.9 (C-15); HRMS (ESI) m/z, calcd. for C28H23N6O2+ [M + H]+: 475.1877, found: 475.1873. Under the reported chromatographic conditions, the retention time of the compound was 13.70 min.
6-((1H-Indol-2-yl)methyl)-9-(4-hydroxybenzyl)-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d]pyrimidine-5,7(6H)-dione (5)
5.4 mg, 0.012 mmol, 32%; 1H NMR (400 MHz, CDCl3) δ 8.99-8.98 (m, 1H, H-2), 8.72 (dd, 1H, J2,4 1.8 Hz, J3,4 7.9 Hz, H-4), 7.73 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 7.37 (d, 1H, J20,21 8.1 Hz, H-21), 7.34 (d, 1H, J18,19 8.0 Hz, H-18), 7.17 (t, 1H, J18,19 = J19,20 7.5 Hz, H-19), 6.87 (t, 1H, J19,20 = J20,21 7.6 Hz, H-20), 6.61 (d, 2H, J27,18 = J30,31 8.3 Hz, H-27, H-31), 6.58 (s, 1H, H-22), 6.36 (d, 2H, J27,28 = J30,31 8.3 Hz, H-28, H-30), 5.56 (dd, 1H, J6,15a 5.3 Hz, J6,15b 2.6 Hz, H-6), 3.73 (dd, 1H, J6,15a 2.4 Hz, J15a,15b 15.0 Hz, H-15a), 3.60-3.51 (m, 2H, H-9, H-15b), 2.90 (dd, 1H, J9,25a 3.6 Hz, J25a,25b 10.9 Hz, H-25a), 2.90 (dd, 1H, J9,25b 14.6 Hz, J25a,25b 10.9 Hz, H-25b); 13C NMR (100 MHz, CDCl3) δ 168.0 (C-7), 162.6 (C-5), 156.5 (C-2), 156.0 (C-11), 147.3 (C-19), 142.1 (C-4), 137.2 (C-14), 136.3 (C-23), 132.5 (C-16), 131.1 (C-26), 129.8 (C-3), 129.0 (C-30), 125.7 (C-28), 125.2 (C-31), 124.0 (C-27), 122.9 (C-24), 120.5 (C-22), 118.7 (C-29), 116.1 (C-19), 115.8 (C-20), 111.5 (C-12), 108.8 (C-18), 57.9 (C-6), 53.5 (C-9), 37.3 (C-25), 29.9 (C-15); HRMS (ESI) m/z, calcd. for C26H22N5O3+ [M + H]+: 452.1717, found: 452.1706. Under the reported chromatographic conditions, the retention time of the compound was 13.69 min.
6-((1H-Indol-2-yl)methyl)-9-(2-(methylthio)ethyl)-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d]pyrimidine-5,7(6H)-dione (6)
3.5 mg, 0.008 mmol, 20%; 1H NMR (400 MHz, CDCl3) δ 8.99 (d, 1H, J2,3 4.1 Hz, H-2), 8.70 (d, 1H, J3,4 7.9 Hz, H-4), 8.39 (broad s, 1H, NH-8), 7.50 (dd, 1H, J2,3 4.1 Hz, J3,4 7.9 Hz, H-3), 7.35 (d, 1H, J20,21 8.1 Hz, H-21), 7.29 (d, 1H, J18,19 8.1 Hz, H-18), 7.10 (t, 1H, J18,19 = J19,20 7.4 Hz, H-19), 6.90 (t, 1H, J19,20 = J20,21 7.4 Hz, H-20), 6.99 (broad s, 1H, H-22), 5.64-5.61 (m, 1H, H-6), 3.72 (d, 1H, J15a,15b 15.2 Hz, H-15a), 3.18-3.14 (m, 1H, H-9), 2.44-2.36 (m, 2H, H-26a, H-26b), 2.34-2.26 (m, 1H, H-25), 1.91 (s, 3H, SCH3-28); 13C NMR (100 MHz, CDCl3) δ 168.9 (C-7), 161.1 (C-5), 157.1 (C-2), 156.7 (C-11), 155.3 (C-14), 137.0 (C-4), 136.4 (C-16), 134.9 (C-23), 129.4 (C-24), 127.6 (C-21), 123.2 (C-22), 120.9 (C-19), 119.0 (C-3), 115.8 (C-12), 111.4 (C-18), 57.9 (C-6), 53.4 (C-9), 29.9 (C-26), 28.0 (C-15), 27.4 (C-25), 19.4 (C-28); HRMS (ESI) m/z, calcd. for C22H22N5O2S+ [M + H]+: 420.1489, found: 420.1494. Under the reported chromatographic conditions, the retention time of the compound was 11.22 min.
General protocol for obtaining products from series B (7-13)
In separate round-bottom flasks, 2-amino nicotinic acid (20) (60 mg, 0.43 mmol, 1.0 eq.), N-Boc-glycine (75.3 mg, 0.43 mmol, 1.0 eq.), anhydrous pyridine (1.5 mL), and triphenyl phosphite (124 μL, 0.47 mmol, 1.1 eq.) were added. Each flask was then irradiated in microwave reactor for 10 min at 100 °C. After cooling the mixture to room temperature, each respective amino acid protected at the carboxyl group with a -OMe group was added separately (0.43 mmol, 1.0 eq.): methyl ester glycine (54.0 mg), methyl ester alanine (60.0 mg), methyl ester isoleucine (78.1 mg), methyl ester phenylalanine (92.8 mg), methyl ester tryptophan (109.5 mg), methyl ester tyrosine (99.6 mg), and methyl ester methionine (85.9 mg). Each mixture was irradiated again in microwave at 150 °C for 2.0-3.0 min. After preliminary purification by CCC with flash silica gel (AcOEt 100% → AcOEt/MeOH 8:2 v/v), the products were then purified using a Shimadzu HPLC system under gradient conditions (A: H2O, B: MeCN, 0-40% B) and flow rate of 3.0 mL min−1. In this way, the final products of series B (7-13) were obtained as slightly yellow solids with yields ranging from 22-59%. These compounds had their structures confirmed and signals assigned by a set of spectroscopic analyses (1H NMR, 13C NMR, gCOSY, gHMQC, gHMBC) and HRESI-MS.
8,9-Dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d]pyrimidine-5,7-(6H)-dione (7)
33.7 mg, 0.15 mmol, 36%; microwave irradiation time in the second step: 2.0 min; 1H NMR (300 MHz, DMSO-d6) δ 8.95 (dd, 1H, J2,3 4.5 Hz, J2,4 2.0 Hz, H-2), 8.65 (broad s, 1H, NH-8), 8.51 (dd, 1H, J4,3 7.9 Hz, J4,2 2.0 Hz, H-4), 7.55 (dd, 1H, J2,3 4.5 Hz, J4,3 7.9 Hz, H-3), 4.50-4.48 (m, 4H, CH2-6, CH2-9); 13C NMR (75 MHz, DMSO-d6) δ 166.1 (C7=O), 160.9 (C5=O), 157.5 (C-11), 156.6 (C-2), 153.9 (C-14), 136.4 (C-4), 122.9 (C-3), 115.4 (C-12), 45.3 (C-6/C-9), 45.3 (C-6/C-9); HRMS (ESI) m/z, calcd. for C10H9N4O2+ [M + H]+: 217.0720, found: 217.0714. Under the reported chromatographic conditions, the retention time of the compound was 9.51 min.
6-Methyl-8,9-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d] pyrimidine-5,7-(6H)-dione (8)
48.0 mg, 0.21 mmol, 49%; microwave irradiation time in the second step: 2.0 min; 1H NMR (300 MHz, DMSO-d6) δ 8.93 (dd, 1H, J2,3 4.5 Hz, J2,4 1.9 Hz, H-2), 8.67 (d, 1H, JNH,9b 4.6 Hz, NH-8), 8.48 (dd, 1H, J2,4 1.9 Hz, J3,4 7.9 Hz, H-3), 7.53 (dd, 1H, J2,3 4.5 Hz, J3,4 7.9 Hz, H-4), 4.94 (q, 1H, J6,15 7.2 Hz, H-6), 4.83 (d, 1H, J9a,9b 17.4 Hz, H-9a), 4.32 (dd, 1H, JNH,9b 4.6 Hz, J9a,9b 17.4 Hz, H-9b), 1.50 (d, 3H, J 7.1 Hz, CH3-15); 13C NMR (75 MHz, DMSO-d6) δ 168.5 (C7=O), 160.7 (C5=O), 157.4 (C-11), 156.6 (C-2), 153.20 (C-14), 136.4 (C-4), 122.9 (C-3), 115.6 (C-12), 52.2 (C-6), 44.6 (C-9), 16.4 (C-15); HRMS (ESI) m/z, calcd. for
C11H11N4O2+ [M + H]+: 281.0877, found: 281.0874. Under the reported chromatographic conditions, the retention time of the compound was 11.26 min.
6-(sec-Butyl)-8,9-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d] pyrimidine-5,7-(6H)-dione (9)
44.0 mg, 0.16 mmol, 38%; microwave irradiation time in the second step: 2.5 min; 1H NMR (300 MHz, DMSO-d6) δ 8.99 (dd, 1H, J2,3 4.5 Hz, J2,4 2.0 Hz, H-2), 8.67 (d, 1H, JNH,9b 5.0 Hz, NH-8), 8.55 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 7.58 (dd, 1H, J2,3 4.5 Hz, J3,4 7.9 Hz, H-3), 4.92 (d, 1H, J6,15 6.8 Hz, H-6), 4.84 (d, 1H, J9a,9b 18.1 Hz, H-9a), 4.31 (dd, 1H, JNH,9b 5.0 Hz, J9a,9b 18.1 Hz, H-9b), 2.09-2.00 (m, 1H, H-15), 1.46-1.39 (m, 1H, H-16a), 1.25-1.15 (m, 1H, H-16b), 0.99 (d, 3H, J15,18 6.8 Hz, CH3-18), 0.89 (t, 3H, J16,17 7.3 Hz, CH3-17); 13C NMR (75 MHz, DMSO-d6) δ 166.8 (C7=O), 161.3 (C5=O), 157.3 (C-11), 156.7 (C-2), 153.7 (C-14), 136.7 (C-4), 123.0 (C-3), 115.5 (C-12), 59.9 (C-6), 45.2 (C-9), 37.1 (C-15), 26.4 (C-16), 15.7 (C-18), 11.6 (C-17); HRMS (ESI) m/z, calcd. for C14H17N4O2+ [M + H]+: 273.1346, found: 273.1339. Under the reported chromatographic conditions, the retention time of the compound was 19.82 min.
6-Benzyl-8,9-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d] pyrimidine-5,7-(6H)-dione (10)
56.0 mg, 0.18 mmol, 43%; microwave irradiation time in the second step: 2.5 min; 1H NMR (300 MHz, DMSO-d6) δ 8.95 (dd, 1H, J2,3 4.6 Hz, J2,4 2.0 Hz, H-2), 8.58 (d, 1H, JNH,9a 4.4 Hz, NH-8), 8.52 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 7.56 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 7.26-7.18 (m, 3H, H-18, H-19, H-20), 7.00-6.97 (m, 2H, H-17, H-21), 5.21 (t, 1H, J6,15a/15b 5.3 Hz, H-6), 4.02 (dd, 1H, JNH,9a 4.4 Hz, J9a,9b 17.7 Hz, H-9a), 3.34 (d, 1H, J9a,9b 17.7 Hz, H-9b), 3.25 (d, 2H, J6,15a/15b 5.3 Hz, CH2-15); 13C NMR (75 MHz, DMSO-d6) δ 167.0 (C7=O), 160.8 (C5=O), 157.2 (C-11), 156.8 (C-2), 153.2 (C-14), 136.6 (C-4), 135.6 (C-16), 130.0 (C-17/ C-21), 129.0 (C-17/ C-21), 127.9 (C-19), 123.1 (C-3), 115.5 (C-12), 57.1 (C-6), 44.3 (C-9), 36.7 (C-15); HRMS (ESI) m/z, calcd. for C17H15N4O2+ [M + H]+: 307.1190, found: 307.1182. Under the reported chromatographic conditions, the retention time of the compound was 21.18 min.
6-((1H-Indol-2-yl)methyl)-8,9-dihydro-5H-pyrazino[1,2-a] pyrido[2,3-d]pyrimidine-5,7-(6H)-dione (11)
88.2 mg, 0.26 mmol, 59%; microwave irradiation time in the second step: 3.0 min; 1H NMR (300 MHz, DMSO-d6) δ 10.98 (broad s, 1H, NH-17), 8.95 (dd, 1H, J2,3 4.6 Hz, J2,4 2.0 Hz, H-2), 8.58 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 8.39 (d, 1H, JNH,9a 4.3 Hz, NH-8), 7.58 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 7.32 (d, 1H, J20,21 8.1 Hz, H-21), 7.26 (d, 1H, J18,19 7.9 Hz, H-18), 7.01 (apparent t, 1H, J18,19 = J19,20 7.5 Hz, H-19), 6.92 (d, 1H, J22,NH 2.2 Hz, H-22), 6.77 (apparent t, 1H, J19,20 = J20,21 7.5 Hz, H-20), 5.23 (t, 1H, J6,15a/15b 4.8 Hz, H-6), 3.88 (dd, 1H, JNH,9a 4.3 Hz, J9a,9b 17.5 Hz, H-9a), 3.43 (d, 2H, J6,15a/15b 4.8 Hz, H-15a, H-15b), 3.10 (d, 1H, J9a,9b 17.5 Hz, H-9b); 13C NMR (75 MHz, DMSO-d6) δ 167.8 (C7=O), 161.1 (C5=O), 157.3 (C-11), 156.7 (C-2), 153.2 (C-14), 136.5 (C-4), 136.5 (C-23), 127.6 (C-24), 125.1 (C-22), 122.9 (C-3), 121.7 (C-19), 119.1 (C-20), 118.1 (C-18), 115.6 (C-12), 111.9 (C-21), 108.1 (C-16), 57.1 (C-6), 44.5 (C-9), 26.9 (C-15); HRMS (ESI) m/z, calcd. for C19H16N5O2+ [M + H]+: 346.1299, found: 346.1292. Under the reported chromatographic conditions, the retention time of the compound was 21.74 min.
6-(p-Hydroxybenzyl)-8,9-dihydro-5H-pyrazino[1,2-a] pyrido[2,3-d]pyrimidine-5,7-(6H)-dione (12)
31.1 mg, 0.096 mmol, 22%; microwave irradiation time in the second step: 2.5 min; 1H NMR (300 MHz, DMSO-d6) δ 9.45 (s, 1H, OH-22), 8.96 (dd, 1H, J2,3 4.6 Hz, J2,4 2.0 Hz, H-2), 8.55 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 8.51 (d, 1H, JNH,9a 4.4 Hz, NH-8), 7.57 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 6.73 (d, 2H, J16,17 = J18,19 8.4 Hz, H-16, H-19), 6.59 (d, 2H, J16,17 = J18,19 8.4 Hz, H-17, H-18), 5.15 (t, 1H, J6,15a = J6,15b 4.8 Hz, H-6), 3.98 (dd, 1H, 4.4 Hz, J9a,9b 17.6 Hz, H-9a), 3.19-3.14 (m, 3H, H-9b, H-15a, H-15b); 13C NMR (75 MHz, DMSO-d6) δ 167.2 (C7=O), 160.8 (C5=O), 157.3 (C-11), 157.3 (C-21), 156.8 (C-2), 153.2 (C-14), 136.6 (C-4), 131.0 (C-16/ C-19), 125.4 (C-20), 123.0 (C-3), 115.8 (C-17/C-18), 115.5 (C-12), 57.3 (C-6), 44.4 (C-9), 35.9 (C-15); HRMS (ESI) m/z, calcd. for C17H15N4O3+ [M + H]+: 323.1139, found: 323.1134. Under the reported chromatographic conditions, the retention time of the compound was 16.58 min.
6-(2-(Methylthio)ethyl)-8,9-dihydro-5H-pyrazino[1,2-a] pyrido[2,3-d]pyrimidine-5,7-(6H)-dione (13)
37.6 mg, 0.13 mmol, 30%; microwave irradiation time in the second step: 2.0 min; 1H NMR (300 MHz, DMSO-d6) δ 8.98 (dd, 1H, J2,3 4.5 Hz, J 2,4 1.9 Hz, H-2), 8.71 (d, 1H, JNH,9a 4.7 Hz, NH-8), 8.54 (dd, 1H, J2,4 1.9 Hz, J3,4 7.9 Hz, H-4), 7.57 (dd, 1H, J2,3 4.5 Hz, J3,4 7.9 Hz, H-3), 5.14 (t, 1H, J6,5a/15b 7.2 Hz, H-6), 4.85 (dd, 1H, J9a,9b 17.5 Hz, H-9a), 4.29 (dd, 1H, J9b,NH 4.7 Hz, J9a,9b 17.5 Hz, H-9b), 2.62-2.57 (m, 2H, CH2-16), 2.22-2.12 (m, 2H, CH2-15), 2.05 (s, 3H, CH3-17); 13C NMR (75 MHz, DMSO-d6) δ 167.4 (C7=O), 161.1 (C5=O), 157.4 (C-11), 156.6 (C-2), 153.4 (C-14), 136.6 (C-4), 123.0 (C-3), 115.8 (C-12), 55.2 (C-6), 44.7 (C-9), 34.8 (C-16), 30.5 (C-15), 14.9 (C-17); HRMS (ESI) m/z, calcd. for C13H14N4O2S+ [M + H]+: 291.0910, found: 291.0905. Under the reported chromatographic conditions, the retention time of the compound was 16.99 min.
General protocol for obtaining products from series C (14-19)
In different round-bottom flasks, 2-amino nicotinic acid (20) (60 mg, 0.43 mmol, 1.0 eq.), anhydrous pyridine (1.5 mL), and triphenyl phosphite (124 µL, 0.47 mmol, 1.1 eq.) were added and, respectively, in each mixture a different amino acid protected with the-N-Boc group (0.43 mmol, 1.0 eq.) was added, namely: N-Boc-alanine (81.4 mg), N-Boc-isoleucine (99.5 mg), N-Boc-phenylalanine (131.6 mg), N-Boc-tryptophan (148.4 mg), N-Boc-tyrosine (120.8 mg), and N-Bocmethionine (106.8 mg). Each flask was then irradiated in microwave for 10 min at 100 °C. After cooling to room temperature, methyl ester glycine (54.0 mg, 0.43 mmol, 1.0 eq.) was added in each flask, and each mixture was irradiated again in microwave for 2.0-3.0 min at 150 °C. After purification by flash chromatography column (EtOAc 100% → EtOAc/MeOH 8:2 v/v), the products were purified on a Shimadzu Shim-PaK HPLC system under gradient conditions (A: H2O, B: MeCN, 0-40% B) and flow rate of 3.0 mL min−1. Thus, the final products of series C (14-19) were obtained as slightly yellowish solids with yields between 31-74%. These compounds had their structures confirmed and signals assigned by a set of spectroscopic analyses (1H NMR, 13C NMR, gCOSY, gHMQC, gHMBC) and by HRESI-MS.
9-Methyl-8,9-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d] pyrimidine-5,7-(6H)-dione (14)
47.6 mg, 0.21 mmol, 48%; microwave irradiation time in the second step: 2.0 min; 1H NMR (300 MHz, DMSO-d6) δ 8.95 (dd, 1H, J2,3 4.6 Hz, J2,4 2.0 Hz, H-2), 8.74 (broad s, 1H, NH-8), 8.51 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 7.55 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 4.71-4.61 (m, 2H, H-6a, H-9), 4.48 (d, 1H, J6a,6b 17.4 Hz, H-6b), 1.56 (d, 3H, J9,15 6.8 Hz, CH3-15); 13C NMR (75 MHz, DMSO-d6) δ 165.8 (C7=O), 160.9 (C5=O), 157.4 (C-11), 156.7 (C-14), 156.6 (C-2), 136.3 (C-4), 123.1 (C-3), 115.3 (C-12), 50.9 (C-9), 45.2 (C-6), 19.6 (C-15); HRMS (ESI) m/z, calcd. for C11H11N4O2+ [M + H]+: 281.0877, found: 281.0874. Under the reported chromatographic conditions, the retention time of the compound was 11.13 min.
9-(sec-Butyl)-8,9-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d] pyrimidine-5,7-(6H)-dione (15)
54.8 mg, 0.20 mmol, 47%; microwave irradiation time in the second step: 2.5 min. It is worth noting that rotamers were detected during its assignment by spectroscopic techniques, which is why some signals appeared duplicated. 1H NMR (300 MHz, DMSO-d6) δ 8.97 (dd, 1H, J2,3 4.5 Hz, J2,4 1.9 Hz, H-2), 8.73 and 8.66 (broad s, 1H, NH-8), 8.53 (dd, 1H, J2,4 1.9 Hz, J3,4 7.9 Hz, H-4), 7.56 (dd, 1H, J2,3 4.5 Hz, J3,4 7.9 Hz, H-3), 4.64 and 4.61 (d, 1H, J6a,6b 18.1 Hz, H-6a), 4.42 (d, 1H, J6a,6b 18.1 Hz, H-6b), 4.40-4.39 and 4.28 (dd, 1H, JNH-8,9b 4.0 Hz, J9,15 6.2 Hz, H-9), 2.21-2.09 (m, 1H, H-15), 1.63-1.51 (m, 1H, H-16a), 1.32-1.21 (m, 1H, H-16b), 0.97-0.87 (m, 6H, CH3-17, CH3-18); 13C NMR (75 MHz, DMSO-d6) δ 165.42 and 165.40 (C7=O), 161.1 (C5=O), 156.7 (C-11), 155.4 (C-2), 154.9 (C-14), 136.3 (C-4), 123.07 and 123.02 (C-3), 115.3 and 115.2 (C-12), 60.56 and 59.59 (C-9), 45.2 (C-6), 40.8 (C-15), 25.9 and 24.6 (C-16), 15.8 and 14.5 (C-18), 11.9 and 11.5 (C-17); HRMS (ESI) m/z, calcd. for C14H17N4O2+ [M + H]+: 273.1346, found: 273.1340. Under the reported chromatographic conditions, the retention time of the compound was 11.24 min.
9-Benzyl-8,9-dihydro-5H-pyrazino[1,2-a]pyrido[2,3-d] pyrimidine-5,7-(6H)-dione (16)
67.2 mg, 0.22 mmol, 51%; microwave irradiation time in the second step: 2.5 min; 1H NMR (300 MHz, DMSO-d6) δ 9.00 (dd, 1H, J2,3 4.5 Hz, J2,4 2.0 Hz, H-2), 8.76 (d, 1H, J 3.6 Hz, NH-8), 8.51 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 7.58 (dd, 1H, J2,3 4.5 Hz, J3,4 7.9 Hz, H-3), 7.26-7.23 (m, 3H, H-18, H-19, H-20), 7.07 (dd, 2H, J 3.0 Hz, J 6.4 Hz, H-17, H-21), 4.85 (dd, 1H, J9,15a 4.9 Hz, J9,15b 9.8 Hz, H-9), 4.35 (d, 1H, J6a,6b 17.9 Hz, H-6a), 3.32 (d, 1H, J6a,6b 17.9 Hz, H-6b), 3.26-3.16 (m, 2H, CH2-15); 13C NMR (75 MHz, DMSO-d6) δ 164.9 (C7=O), 160.8 (C5=O), 157.4 (C-11), 156.9 (C-2), 155.0 (C-14), 136.4 (C-4), 136.0 (C-16), 130.3 (C-17/C-21), 128.9 (C-18/C-20), 127.6 (C-19), 123.2 (C-3), 115.1 (C-12), 57.0 (C-9), 44.7 (C-6), 41.8 (C-15); HRMS (ESI) m/z, calcd. for C17H15N4O2+ [M + H]+: 307.1190, found: 307.1183. Under the reported chromatographic conditions, the retention time of the compound was 20.19 min.
9-((1H-Indol-2-yl)methyl)-8,9-dihydro-5H-pyrazino[1,2-a] pyrido[2,3-d]pyrimidine-5,7-(6H)-dione (17)
109.7 mg, 0.32 mmol, 74%; microwave irradiation time in the second step: 3.0 min; 1H NMR (300 MHz, DMSO-d6) δ 11.02 (broad s, 1H, NH-17), 9.02 (dd, 1H, J2,3 4.3 Hz, J2,4 1.4 Hz, H-2), 8.72 (d, 1H, JNH-8,9 3.3 Hz, NH-8), 8.41 (dd, 1H, J2,4 1.4 Hz, J3,4 7.8 Hz, H-4), 7.56 (dd, 1H, J2,3 4.3 Hz, J3,4 7.8 Hz, H-3), 7.31 (d, 1H, J18,19 8.4 Hz, H-18), 7.05 (d, 1H, J22,NH-17 1.8 Hz, H-22), 6.94 (d, 2H, J 7.6 Hz, H-20, H-21), 6.57 (t, 1H, J18,19 = J19,20 7.5 Hz, H-19), 4.85 (apparent q, 1H, J9,15a = J9,15b 4.5 Hz, H-9), 4.15 (dd, 1H, J6a,6b 17.9 Hz, H-6a), 3.45 (dd, 1H, J9,15a 5.1 Hz, J15a,15b 14.4 Hz, H-15a), 3.27 (dd, 1H, J9,15b 4.3 Hz, J15a,15b 14.4 Hz, H-15b), 2.81 (d, 1H, J6a,6b 17.9 Hz, H-6b); 13C NMR (75 MHz, DMSO-d6) δ 164.8 (C7=O), 160.6 (C5=O), 157.5 (C-11), 156.8 (C-2), 155.6 (C-14), 136.4 (C-4), 136.2 (C-23), 127.4 (C-16), 125.7 (C-22), 123.0 (C-3), 121.6 (C-20), 117.9 (C-21), 118.9 (C-19), 115.1 (C-12), 112.0 (C-18), 107.8 (C-24), 56.9 (C-9), 44.6 (C-6), 32.8 (C-15); HRMS (ESI) m/z, calcd. for C19H16N5O2+ [M + H]+: 346.1299, found: 346.1293. Under the reported chromatographic conditions, the retention time of the compound was 20.23 min.
9-(p-Hydroxybenzyl)-8,9-dihydro-5H-pyrazino[1,2-a] pyrido[2,3-d]pyrimidine-5,7-(6H)-dione (18)
43.0 mg, 0.13 mmol, 31%; microwave irradiation time in the second step: 2.5 min; 1H NMR (300 MHz, DMSO-d6) δ 9.00 (dd, 1H, J2,3 4.6 Hz, J2,4 2.0 Hz, H-2), 8.72 (d, 1H, JNH,9a 4.2 Hz, NH), 8.51 (dd, 1H, J2,4 2.0 Hz, J3,4 7.9 Hz, H-4), 7.58 (dd, 1H, J2,3 4.6 Hz, J3,4 7.9 Hz, H-3), 6.78 (d, 2H, J16,17 = J18,19 8.4 Hz, H-16, H-19), 6.60 (d, 2H, J16,17 = J18,19 8.5 Hz, H-17, H-18), 4.78 (dd, 1H, J9,15a 4.2 Hz, J9,15b 8.7 Hz, H-9), 4.29 (d, 1H, J6a,6b 17.9 Hz, H-6a), 3.19-3.02 (m, 3H, H-6b, H-15a, H-15b); 13C NMR (75 MHz, DMSO-d6) δ 167.8 (C7=O), 160.8 (C5=O), 157.4 (C-11), 157.0 (C-2), 156.9 (C-14), 155.1 (C-21), 136.4 (C-4), 131.3 (C-20), 123.5 (C-16/C-19), 123.2 (C-3), 115.8 (C-17, C-18), 115.0 (C-12), 57.3 (C-9), 44.6 (C-6), 41.5 (C-15); HRMS (ESI) m/z, calcd. for C17H15N4O3+ [M + H]+: 323.1139, found: 323.1135. Under the reported chromatographic conditions, the retention time of the compound was 14.86 min.
9-(2-(Methylthio)ethyl)-8,9-dihydro-5H-pyrazino[1,2-a] pyrido[2,3-d]pyrimidine-5,7-(6H)-dione (19)
46.3 mg, 0.16 mmol, 37%; microwave irradiation time in the second step: 2.5 min; 1H NMR (300 MHz, DMSO-d6) δ 8.95 (dd, 1H, J2,3 4.5 Hz, J2,4 1.9 Hz, H-2), 8.89 (d, 1H, JNH,9 3.0 Hz, NH-8), 8.50 (dd, 1H, J2,4 1.9 Hz, J3,4 7.9 Hz, H-4), 7.55 (dd, 1H, J2,3 4.5 Hz, J3,4 7.9 Hz, H-3), 4.66-4.61 (m, 1H, H-9), 4.55 (d, 2H, J6a,6b 3.13 Hz, H-6a, H-6b), 2.70-2.59 (m, 2H, CH2-16), 2.23-2.15 (m, 2H, CH2-15), 2.06 (s, 3H, CH3-17); 13C NMR (75 MHz, DMSO-d6) δ 165.6 (C7=O), 161.0 (C5=O), 157.4 (C-11), 156.6 (C-2), 155.5 (C-14), 136.3 (C-4), 123.1 (C-3), 115.4 (C-12), 54.5 (C-9), 45.0 (C-6), 33.8 (C-15), 29.3 (C-16), 14.9 (C-17); HRMS (ESI) m/z, calcd. for C13H14N4O2S+ [M + H]+: 291.0837, found: 291.0906. Under the reported chromatographic conditions, the retention time of the compound was 15.70 min.
Supplementary Information
Supplementary information (detailed NMR spectra, HRMS (ESI) analysis and chromatographic data) is available free of charge at http://jbcs.sbq.org.br as aPDF file.
Acknowledgments
This investigation has received financial assistance from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant number 2018/13518-1, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) award number 420395/2018-0 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) finance code 001.
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