Cycloaddition of 4-Acyl-1H-pyrrole-2,3-diones Fused at [e]-Side and Cyanamides: Divergent Approach to 4H-1,3-Oxazines

4-Acyl-1H-pyrrole-2,3-diones fused at [e]-side with a heterocyclic moiety are suitable platforms for the development of a hetero-Diels–Alder-reaction-based, diversity-oriented approaches to series of skeletally diverse heterocycles. These platforms are known to react as oxa-dienes with dienophiles to form angular 6/6/5/6-tetracyclic alkaloid-like heterocycles and are also prone to decarbonylation at high temperatures resulting in generation of acyl(imidoyl)ketenes, bidentate aza- and oxa-dienes, which can react with dienophiles to form skeletally diverse products (angular tricyclic products or heterocyclic ensembles). Based on these features, we have developed an approach to two series of skeletally diverse 4H-1,3-oxazines (tetracyclic alkaloid-like 4H-1,3-oxazines and 5-heteryl-4H-1,3-oxazines) via a hetero-Diels–Alder reaction of 4-acyl-1H-pyrrole-2,3-diones fused at [e]-side with cyanamides. The products of these transformations are of interest for drug discovery, since compounds bearing 4H-1,3-oxazine moiety are extensively studied for inhibitory activities against anticancer targets.


Introduction
Diversity-oriented synthesis (DOS) is a strategy to access structurally diverse libraries of small molecules from a single set of reagents [1,2]. This approach allows efficient exploration of the chemical space for the development of new drugs [3,4].
Initially, we tested the reaction of FPD 1a with cyanamide 2a in acetonitrile at room temperature (Table 1). According to UPLC-UV-MS data of the reaction mixture, the reaction proceeded very slowly. In a week, several unidentified side products were observed along with unreacted starting materials (conversion degree of FPD 1a of~20%). The UPLC-UV-MS yield of the desired product 3a was~10%. However, at elevating the reaction temperature up to 95 • C, the test reaction of FPD 1a with cyanamide 2a in acetonitrile proceeded smoothly and afforded the desired tetracyclic alkaloid-like 4H-1,3-oxazine 3a in an isolated yield of 85% (Table 1, Entry 3a). The reaction progress was monitored visually by the change of colour of the reaction mixture (FPD 1a has a deep violet colour, and product 3a is yellow). According to UPLC-UV-MS data of the reaction mixture, compound 3a was formed as a single product, and no side products were observed. Product 3a was isolated by a simple filtration directly from the reaction mixture. Since test results were satisfactory, we examined the substrate scope of this reaction by involving FPDs 1a-i, bearing various acyl substituents R 1 and heteroatoms X and cyanamides 2a-f, bearing various substituents at amino nitrogen atom (Table 1). Quinoxaline derivatives 3a-k were prepared using acetonitrile as the reaction solvent and isolated by a simple filtration directly from the reaction mixture. For the synthesis of 1,4-benzoxazine derivatives (X = O), toluene was used as a reaction solvent since compounds 3l,m were readily soluble in acetonitrile, and no precipitate was formed. In toluene compounds 3l,m formed precipitates after cooling of the reaction mixtures to room temperature, which eased their isolation.
It was found that the studied reaction proceeded well both with 5-oxa (X = O) and 5-aza (X = NH, NPh, NMe) FPDs 1. The reaction also worked well with various aryls and tert-butyl at acyl substituent R 1 of FPDs 1. Expectedly, the reaction of methoxy bearing FPD 1h did not result in cycloadduct 3k, since the methoxycarbonyl group COOMe is not electrophilic enough to participate in cycloaddition as a C=O part of the heterodiene system. The examined substituents in N,N-dialkylcyanamides 2a-d did not affect the reaction noticeably. However, our attempts to involve N-arylcyanamides 2e,f in HDA with FPDs 1a,l were not successful. In this case, the reaction proceeded with formation of insoluble hard-to-purify compounds, whose structure we did not succeed to identify. We assume that in this case other reaction course could occur instead of the formation of the desired compounds 3o-q, since N-arylcyanamides 2e,f has lower nucleophilicity at C≡N nitrogen than N,N-dialkylcyanamides 2a-d.
It is worthy of note that some of products 3 had a very low solubility in organic solvents all available to us. There were problems with acquisition on NMR spectra of such products, that's why in some cases, we had to record solid-state NMR (ssNMR) spectra.
It should be mentioned that in case of the reaction of 4-nitrophenyl substituted FPD 1d with 4-morpholinecarbonitrile 2b, the desired product 3n was observed only in trace amounts by UPLC-UV-MS of the reaction mixture. Prolongation of the reaction time (up to 14 days) and increasing the temperature (up to 120 • C) did not yield any positive results. We suppose that this phenomenon was caused by very low solubility of product 3n, which, possibly, under the examined conditions (FPDs 1 were used as suspensions in acetonitrile), formed a protective insoluble layer on the surfaces of solid particles of FPD 1d and, thus, prevented the reaction. It also should be mentioned that our attempts to perform the reaction of 4-nitrophenyl substituted FPD 1d with carbonitrile 2b in DMSO were also unsuccessful. This experiment was complicated by the fact that DMSO is a highly hygroscopic solvent and facilitated the hydrolysis reactions of the starting FPDs 1 and the products 3 (for hydrolysis studies of analogs of products 3, see [22]). In the case of compound 1d, NO 2 substituent makes FPD 1d very electrophilic and very reactive towards water.
Moreover, in the case of 1,4-benzoxazine products 3l,m (X = O), there were problems with monitoring them with UPLC-UV-MS and HPLC-UV (acetonitrile-water as eluents). Chromatograms of the reaction mixtures and individual compounds 3l,m (pure according to the NMR spectra) contained a lot of overlapped broad peaks, and the mass detector data showed signals of the desired products 3l,m only in trace amounts. Furthermore, such problems were never observed with quinoxaline products 3a-j (X = NH, NMe, NPh). We think that these could be explained by the occurrence of hydrolysis of compounds 3l,m on the LC column due to the presence of an ester moiety in their structures, which is a common feature of such compounds [22].
The study of melting in a capillary of compounds 3a-i,l,m revealed that under such conditions 5-heteryl-4H-1,3-oxazines 4a-i,l,m ( Table 2) were formed as sole products, and no regioisomeric pyrimidines G (Scheme 2) were observed (monitoring by UPLC-UV-MS). This transformation was then easily scaled up to 0.4 mmol (~200 mg) under solvent-free conditions. When scaling up, we found that an addition of small amounts (of about 0.1 equiv.) of the corresponding cyanamides 2a-d was required to increase the isolated yields of compounds 4a-i,l,m by reducing the side reactions leading to compounds H (monitoring by UPLC-UV-MS) (Scheme 2) characteristic of transformations involving in situ generation of acyl(imidoyl)ketenes C [24,32]. Compounds 4a-i,l,m were readily isolated by simple recrystallization of the crude reaction mixtures. No effect of the examined substituents on the formation of compounds 4a-i,l,m was observed. In the case of compound 3j (X = NH), no compound 4j was formed-instead of this compound, furoqinoxaline I was detected (monitoring by UPLC-UV-MS) (Scheme 2) [24,33].

Scheme 2. Plausible pathway of formation of compounds 4 and G.
We assume that the formation of compounds 4a-i,l,m proceeded through three stages (Scheme 2). First, compounds 3a-i,l,m underwent thermally initiated retro-HDA that afforded FPDs 1a-i and cyanamides 2a-d. Second, formed FPDs 1a-i decarbonylated (the evolution of carbon monoxide was indicated by a gas detector tube) to generate acyl(imidoyl)ketenes C. And finally, acyl(imidoyl)ketenes C reacted as oxa-dienes with cyanamides 2a-d to produce the desired 4H-1,3-oxazines 4a-i,l,m. We suppose that ketenes C reacted with cyanamides 2a-d exclusively as oxa-dienes, since this cycloaddition reaction proceeded via a charge-controlled polar transition state, as it was observed earlier in the reaction of ketenes C with carbodiimides [25].
To validate the proposed pathway of formation of compounds 4 (Scheme 2), we tested the one-pot solvent-free reaction of FPD 1a with cyanamide 2b. At heating of compound 1a with cyanamide 2b (reaction scale of 0.4 mmol, 1a:2b reagents ratio of 1:1.1) at 235-240 • C, we found that compound 4b was formed only in a yield of~45% (monitoring by UPLC-UV-MS), which was much lower than in the case of decomposition of compound 3b. We think that it was because of violation of heat and mass transfer processes during the solventfree reaction of compounds 1a and 2b. These violations promoted the thermolytical side reactions leading to compounds H [24,32] (monitored by UPLC-UV-MS) and decreased the yield of compound 4b. Thus, the development of a procedure to compounds 4 from the direct reaction of compounds 1 and 2 without isolation of compounds 3 is rather possible, but it requires additional optimization.
Then, to further validate the proposed pathway of formation of compounds 4 (Scheme 2), we performed the decomposition of compound 3b in the presence of FPD 1b at 240 • C and decomposition of compound 3a in the presence of cyanamide 2b at 240 • C and studied the obtained reaction mixtures by HPLC-UV. As a result, the decomposition of compound 3b (R 1 = Ph, R 2 = morpholino) in the presence of FPD 1b (R 1 = 4-ClC 6 H 4 ) at 240 • C afforded a mixture of compounds 4b (R 1 = Ph, R 2 = morpholino) and 4e (R 1 = 4-ClC 6 H 4 , R 2 = morpholino) along with a mixture of corresponding side products H. The decomposition of compound 3a (R 1 = Ph, R 2 = NEt 2 ) in the presence of cyanamide 2b (R 2 = morpholino) at 240 • C afforded a mixture of compounds 4a (R 1 = Ph, R 2 = NEt 2 ) and 4b (R 1 = Ph, R 2 = morpholino) along with the corresponding side product H. These crossover experiments indirectly confirm that the proposed pathway of formation of compounds 4 (Scheme 2) includes retro-HDA stage and formation of acyl(imidoyl)ketenes C.

General Information
1 H and 13 C NMR spectra (Supplementary Materials) were acquired on a Bruker Avance III 400 HD spectrometer (Switzerland) (at 400 and 100 MHz, respectively) at 313 K in CDCl 3 (stab. with Ag) or DMSO-d 6 using the TMS or HMDS signal (in 1 H NMR) or solvent residual signals (in 13 C NMR, 77.00 for CDCl 3 , 39.51 for DMSO-d 6 ; in 1 H NMR, 7.26 for CDCl 3 , 2.50 for DMSO-d 6 ) as internal standards. 13 C ssNMR spectra were acquired on a Bruker Avance III 400 WB NMR spectrometer (Switzerland) (at 100 MHz). Melting points were measured on a Mettler Toledo MP70 apparatus (Switzerland). Elemental analyses were carried out on a Vario MICRO Cube analyzer (Germany). The reaction conditions were optimized using UPLC-UV-MS (Waters ACQUITY UPLC I-Class system (USA); Acquity UPLC BEH C18 column, grain size of 1.7 µm; acetonitrile-water (water containing 0.1% formic acid) as eluents; flow rate of 0.6 mL/min; ACQUITY UPLC PDA eλ Detector (wavelength range of 230-780 nm); Xevo TQD mass detector; electrospray ionization (ESI); positive and negative ion detection; ion source temperature of 150 • C; capillary voltage of 3500-4000 V; cone voltage of 20-70 V; vaporizer temperature of 200 • C) and HPLC-UV (Hitachi Chromaster Japan); NUCLEODUR C18 Gravity column (particle size 3 µm; eluent acetonitrile-water, flow rate 1.5 mL/min); Hitachi Chromaster 5430 diode array detector (λ 210-750 nm)). CO was indicated by gas detector tubes Gazoopredelitel GH-4 (USSR) (specifications 12.43.20-76). The single crystal X-ray analyses of compounds 3a, 3i, 4b, 4f, 4g, and 4i were performed on an Xcalibur Ruby diffractometer (Agilent Technologies, UK). The empirical absorption correction was introduced by multi-scan method using SCALE3 AB-SPACK algorithm [34]. Using OLEX2 [35], the structures were solved with the SHELXS [36] program and refined by the full-matrix least-squares minimization in the anisotropic approximation for all non-hydrogen atoms with the SHELXL [37] program. Hydrogen atoms were positioned geometrically and refined using a riding model. Thin-layer chromatography (TLC) was performed on Merck silica gel 60 F 254 plates using EtOAc/toluene, 1:5 v/v, toluene, EtOAc as eluents. Starting compounds 1a-j were obtained according to reported procedures [25,33,38,39]. Toluene for procedures involving compounds 1 was dried over Na before the use. Acetonitrile for procedures involving compounds 1 was dried over molecular sieves 4Å before the use. All other solvents and reagents were purchased from commercial vendors and used as received. Procedures involving compounds 1, 3 were carried out in oven-dried glassware.

Synthetic Methods and Analytic Data of Compounds
3.2.1. General Procedure to Compounds 3a-j,l,m A suspension of the corresponding FPD 1 (0.76 mmol) [25,33,38,39] and the corresponding cyanamide 2 (0.84 mmol) in 4 mL of a solvent (anhydrous acetonitrile (for 1a-h) or anhydrous toluene (for 1i)) was stirred and heated at 95 • C for 16 h (until the disappearance of the dark violet color of the compound 1) in an oven-dried capped vial. Then the reaction mixture was cooled to room temperature, and the resulting precipitate was filtered off to afford the desired compound 3. Compound 3 was pure enough and was used further without additional purification.   trione (3i). After cooling the reaction mixture, no precipitate was formed. As such, the reaction solvent (acetonitrile) was removed on a rotary evaporator. The resulting solid was dissolved in toluene (2 mL). Then, petroleum ether (bp 70-100 • C) (6 mL) was added to the toluene solution, and the resulting precipitate was filtered off to afford compound 3i.

General Procedure to Compounds 4a-i,l,m
A mixture of the corresponding compound 3 (0.4 mmol) and the corresponding cyanamide 2 (0.04 mmol) was put into an oven-dried tube, pressed slightly, and then heated in a metal bath at 190-245 • C (the temperature for each compound is given in Table 2; caution: CO evolves during the reaction) for 3 min. The reaction mixture was cooled to room temperature and recrystallized from about 3 mL of a solvent (acetonitrile (for 3a-h) or toluene (for 3l,m)) to give the appropriate compound 4. In the case of compound 3i, the reaction mixture was cooled to room temperature, dissolved in 1 mL of ethyl acetate. Then, 5 mL of n-hexane were added to it, and the resulting precipitate was filtered off to afford compound 4i.   (2)