Methanesulfonamide Synthesis Essay

1. Introduction

Rigidin A (1), a pyrrolo[2,3-d]pyrimidine alkaloid, was first isolated by Kobayashi et al. from Okinawa marine tunicate Eudisromu cf. rigida in 1990 [1]. Later on, a series of its analogues, Rigidin B–E (25) (Figure 1), which were consequently isolated from the same marine species [2], were found to have strong inhibitory activity against calmodulin brain phosphodiesterase [3]. The core structure of rigidin A–E is a tetrasubstituted pyrrole fused to a pyrimidine, which is an important structural subunit in a variety of biologically active compounds. In the past decades, much attention have been drawn on the pyrrolo[2,3-d]pyrimidine analogues for biological and pharmaceutical applications [4,5,6,7,8,9,10,11].

Figure 1. Structures of marine alkaloids rigidin A–E.

Figure 1. Structures of marine alkaloids rigidin A–E.

Due to the fact that the content of rigidins in localized tunicate species is very low (0.0015% wet weight), only very limited amount of rigidins could be isolated for biological study. Therefore, total synthesis of rigidin was employed to provide sufficient samples for their biological assays.

Currently, there are four published synthetic routes for the total synthesis of rigidins. In 1993, Edstrom et al. presented the first report of total synthesis of rigidin A (1) with 1,3-dibenzyl protected 6-chlorouracil in 26% overall yield [12]. After a SN2 substitution of the 6-chloro group with N-benzylglycine, the pyrrolo[2,3-d]pyrimidine skeleton was formed by reflux in acetic anhydride. The two substitutes at 5- and 6-position were then attached on Stille cross-coupling and Friedel-Crafts acylation respectively.

Soon after, Sakamoto and his co-workers reported the second strategy for total synthesis of rigidin A (1) in 1994 [13,14]. With a multi-substituted bromopyrimidine as starting material, the pyrrolo[2,3-d]pyrimidine core was built by Stille cross-coupling reaction with vinylstannane and subsequent acidic hydrolysis. The two substitutes were then introduced by similar reactions reported by Edstrom [12]. Rigidin A (1) was obtained in less than 10% overall yield.

In 2006, Gupton et al. reported the third total synthesis strategy for rigidin A (1) and rigidin E (5), which used a symmetrical vinamidinium salt to construct 2,4-disubstituted pyrrole [15]. After C-6 substitute was introduced by Friedel-Crafts acylation, the pyrimidine moiety was constructed to accomplish the total synthesis of rigidin A (1) and rigidin E (5).

The first and second synthetic routes employed substituted pyrimidine as the starting material and subsequently constructed the pyrrole moiety. In contrast, the third route constructed multi-substituted pyrrole moiety before pyrimidine formation. All three routes suffered from harsh reaction conditions, expensive Palladium-based catalyst, lengthy route and lack of variability for diversity-oriented derivatives.

In 2011, Magedov reported that tetra- and pentasubstituted 2-aminopyrroles can be prepared via multi-component reactions of structurally diverse aldehydes and N-(aryl-, heteroaryl-, alkylsulfonamido)acetophenones with cyanoacetic acid derivatives, such as malononitrile and cyanoacetate [16]. Furthermore, this methodology was used successfully in total synthesis of rigidins A–D in moderate overall yield, which is the fourth total synthetic route [16]. So far, this is the most efficient total synthetic strategy. However, this protocol’s generality and reproducibility still needs to be proved. We describe herein an improved and practical synthetic route for the total synthesis of rigidin E.

2. Results and Discussion

Rigidin E contains many H-bond donor/acceptors, which may coordinate with heavy metal ions and form metal-rigidin complexes. The resulting metal contamination cannot usually be easily removed by a normal purification process. This promoted us to develop a metal-free strategy.

Our retrosynthetic analysis is showed in Figure 2. Principally, rigidin E may be synthesized from rigidin A through regioselective methylation at N-3. This reaction is very difficult to realize due to the three NH groups in rigidin E. Therefore, it is preferable to introduce the methyl group before constructing the tetrasubstituted 2-aminopyrrole.

Figure 2. Retrosynthetic analysis of rigidin E.

Figure 2. Retrosynthetic analysis of rigidin E.

Regioselective bromination of acetophenone 7 with phenyltrimethylammonium tribromide in anhydrous THF afforded 2-bromoacetophenone 8 in 80% yield [17]. 2-Amino-acetophenone 9 was synthesized using hexamethylenetetramine as an NH2 source in anhydrous chlorobenzene. Subsequent reaction with methanesulfonyl chloride gave methanesulfonamide 10 in 60% yield over two steps (Scheme 1).

In order to introduce the methyl group to N-3, 2-cyano-N-methylacetamide 11 was synthesized from ethyl cyanoacetate and methylamine in 90% yield [18,19]. Attempts to synthesize key intermediate 14 using the reported three-components reaction of compound 10, 11, and 12 proceeded in very low yield and was complicated by undesired byproducts (Scheme 2). As reported by Magedov [16], Knoevenagel adduct 13 may be the intermediate in the three-component reaction. We speculated that the intermediate 13 could not form efficiently because of the higher pKa of the methylene of N-methylacetamide compare to N-unsubstituted acetamide. Therefore, it is possible to solve this problem through synthesis of the intermediate 13 independently.

Scheme 1. Synthesis of methanesulfonamide 10. Reagents and conditions: (a) K2CO3, acetone, BnBr, 0 °C, 98%; (b) phenyltriethylammonium tribromide, THF, 80%; (c) hexamethylenetetramine, chlorobenzene, then conc. HCl; (d) MsCl, Et3N, acetone/H2O 2:1, 60% over two steps.

Scheme 1. Synthesis of methanesulfonamide 10. Reagents and conditions: (a) K2CO3, acetone, BnBr, 0 °C, 98%; (b) phenyltriethylammonium tribromide, THF, 80%; (c) hexamethylenetetramine, chlorobenzene, then conc. HCl; (d) MsCl, Et3N, acetone/H2O 2:1, 60% over two steps.

Scheme 2. Three-components reaction for synthesis the key intermediate 14 failed. Reagents and conditions: (a) K2CO3, EtOH, reflux.

Scheme 2. Three-components reaction for synthesis the key intermediate 14 failed. Reagents and conditions: (a) K2CO3, EtOH, reflux.

After Knoevenagel condensation of 2-cyano-N-methylacetamide 11 and 4-(benzyloxy)benzaldehyde 12 using piperidine as a catalyst, 3-(4-(benzyloxy)phenyl)-2-cyano-N-methylacrylamide 13 was obtained in 80% yield as a mixture of E/Z in about 1:1 ratio (Scheme 3) [20]. Then, a variation of the reported three-components reaction was conducted to synthesize the precursor 14.

Scheme 3. Synthesis of N-methylacrylamide 13. Reagents and conditions: (a) methylamine, H2O, 90%; (b) K2CO3, acetone, BnBr, 0 °C, 95%; (c) 11, piperidine, toluene, 80%.

Scheme 3. Synthesis of N-methylacrylamide 13. Reagents and conditions: (a) methylamine, H2O, 90%; (b) K2CO3, acetone, BnBr, 0 °C, 95%; (c) 11, piperidine, toluene, 80%.

After a careful screening of reaction conditions, the key intermediate 14 was successfully prepared by a cascade Michael addition/intermolecular cyclization between N-methylacrylamide 13 and methanesulfonamide 10 in one pot, with K2CO3 as base and ethanol as solvent under refluxing conditions. The present result proves that phenyl-2-cyanoacrylamide like Knoevenagel adduct was the intermediate for cyclization in the reported three-component reaction [21].

Carbonylation with oxalyl chloride in diglyme failed to give pyrimidinedione 15 [16]. Various reagents and conditions were tested, and triphosgene was found ideal to promote the I2-catalyzed cyclization in anhydrous THF to give pyrimidinedione 15 in 60% yield [22,23]. The final deprotection of benzyl groups using catalytic hydrogenation resulted in a complicated mixture. The reason might be the accompanied reduction of carbonyl at C-14. Finally, the protective groups were successfully removed by TMSI in situ prepared by TMSCl and NaI to afford rigidin E (5) in 88% yield (Scheme 4). All spectral data are consistent with those of the reported natural product [3].

Scheme 4. Total synthesis of rigidin E. Reagents and conditions: (a) K2CO3, EtOH, reflux, 50%; (b) triphosgene, I2, THF, 60%; (c) TMSCl, NaI, CH3CN, 88%.

Scheme 4. Total synthesis of rigidin E. Reagents and conditions: (a) K2CO3, EtOH, reflux, 50%; (b) triphosgene, I2, THF, 60%; (c) TMSCl, NaI, CH3CN, 88%.

3. Experimental Section

3.1. General

All reagents and catalysts were purchased from commercial sources (Acros or Sigma Aldrich) and used without purification. MeCN, chlorobenzene and DCM were dried with CaH2 and distilled prior to use. THF was dried with LiAlH4 and distilled prior to use. Thin layer chromatography was performed using silica gel GF-254 plates (Qing-Dao Chemical Company, China) with detection by UV (254 nm) or charting with 10% sulfuric acid in ethanol. Column chromatography was performed on silica gel (200–300 mesh, Qing-Dao Chemical Company, China). NMR spectra were recorded on a Bruker AV400 spectrometer, and chemical shifts (δ) are reported in ppm. 1H NMR and 13C NMR spectra were calibrated with TMS as internal standard, and coupling constants (J) are reported in Hz. The ESI-HRMS were obtained on a Bruker Dalton microTOFQ II spectrometer in positive ion mode. Melting points were measured on an electrothermal apparatus uncorrected.

3.2. 1-(4-(Benzyloxy)phenyl)-2-bromoethanone 8

To a solution of 1-(4-(benzyloxy)phenyl)ethanone 7 (2.26 g, 10 mmol) in anhydrous THF (20 mL) was added phenyltrimethylammonium tribromide (3.76 g, 10 mmol). The resulting solution was stirred at room temperature. A white precipitate was formed and the solution became yellowish over several minutes. After 30 min, the mixture was poured into ice water (50 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was recrystallized from ethanol (15 mL) to afford the title product 8 (2.43 g, 80% yield).

MP: 67–69 °C; Rf = 0.30 (PE–EtOAc, 50:1). 1H NMR (400 MHz, CDCl3): δ = 4.40 (s, 2H, CH2Br), 5.14 (s, 2H, C6H5CH2), 7.03 (d, J = 8.8 Hz, 2H, C6H4, H-3,5), 7.35–7.44 (m, 5H, C6H5), 7.96 (d, J = 8.8 Hz, 2H, C6H4, H-2,4). 13C NMR (100 MHz, CDCl3): δ = 30.7, 70.2, 114.9, 127.5, 128.3, 128.7, 130.6, 131.4, 135.9, 163.3, 190.0. MS (ESI): m/z = 304.8 [M + H]+.

3.3. N-(2-(4-(Benzyloxy)phenyl)-2-oxoethyl)methanesulfonamide 10

1-(4-(Benzyloxy)phenyl)-2-bromoethanone 8 (3.04 g, 10 mmol) was dissolved in anhydrous chlorobenzene (8.5 mL). The solution was added dropwise to a solution of hexamethylenetetramine (1.15 g, 11 mmol) in anhydrous chlorobenzene (10 mL) at 30 °C. The reaction mixture was stirred at 30 °C for 4 h and filtered. The filter cake was washed with ethanol (10 mL) and dried in vacuo. The filter cake was redissolved in a mixture of concentrated hydrochloric acid (10 mL) and ethanol (20 mL) with vigorous stirring. The resulting reaction mixture was stirred occasionally under N2 atmosphere for 48 h. The reaction mixture was cooled to 0 °C and filtered to give compound 9. It was redissolved in H2O/acetone (1:2, 600 mL). Then methanesulfonyl chloride (1.71 g, 15 mmol) was added to the mixture. The mixture was placed in an ice bath and triethylamine (2.53 g, 25 mmol) was added dropwise over 30 min. Then acetone (14 mL) was added to the above mixture. The reaction mixture was stirred at room temperature for 10 h, and then the volatiles were evaporated under reduced pressure. The solid started to precipitate. To the obtained slurry were added ethyl acetate (20 mL) and saturated NH4Cl (20 mL) aqueous solution. The organic layer was separated and washed with saturated NaHCO3 (20 mL) aqueous solution and brine (20 mL), dried with anhydrous MgSO4, and concentrated to afford the title product 10 (1.91 g, 60% yield).

Mp: 138–140 °C; Rf = 0.43 (CH3OH–CH2Cl2, 1:50). 1H NMR (400 MHz, CDCl3): δ = 2.99 (s, 3H, CH3), 4.61 (s, 2H, CH2NH), 5.15 (s, 2H, C6H5CH2), 5.32–5.49 (b, 1H, NH), 7.04 (d, J = 8.8 Hz, 2H, C6H4, H-3,5), 7.25–7.42 (m, 5H, C6H5), 7.91 (d, J = 8.8 Hz, 2H, C6H4, H-2,4). 13C NMR (100 MHz, CDCl3): δ = 40.7, 48.8, 70.3, 115.1, 126.9, 127.5, 128.4, 128.7, 130.3, 135.8, 163.7, 191.6. MS (ESI): m/z = 342.5 [M + Na]

1. Introduction

Khellactone coumarins, which constitute a small branch of the coumarin family, are notable because of their extensive bioactivities, including anti-HIV [1], anti-platelet aggregation [2], calcium antagonist activity [3], P-glycoprotein inhibitory ability, etc. [4,5]. Particullarly, the famous compound DCK [3'R,4'R-di-O-(camphanoyl-(+)-cis-khellactone] in this class, along with its derivatives, have received increasing attention due to their potent anti-HIV activity [6]. As shown in Figure 1, khellactone coumarins contain two chiral carbons, C-3' and C-4', and most of the reported compounds possess 3'R,4'R configuration. Lots of literature has shown that the rigid stereochemistry of 3'R and 4'R-configured khellactone derivatives is crucial for anti-HIV activity [7,8,9].

Figure 1. Structures of cis-khellactone with (a) 3'R,4'R or (b) 3'S,4'S configuration.

Figure 1. Structures of cis-khellactone with (a) 3'R,4'R or (b) 3'S,4'S configuration.

Khellactone coumarins with 3'S,4'S configuration exist mainly in the plants Peucedanum praeruptorum Dunn and Peucedanum japonicum [10]. Recently, more and more researchers have paisd close attention to the calcium antagonist activity and P-glycoprotein inhibitory ability of the (3'S,4'S)-cis-khellactone coumarins [11,12], whereas, other activities of (3'S,4'S)-cis-khellactone coumarins, for example, antitumor activity, have been rarely reported.

Figure 2. Structures of 4-methyl-(3'S,4'S)-cis-khellactone derivatives 3ao.

Figure 2. Structures of 4-methyl-(3'S,4'S)-cis-khellactone derivatives 3ao.

In the present study, a series of 4-methyl-(3'S,4'S)-cis-khellactone derivatives 3ao (Figure 2) were designed and asymmetrically synthesized. Furthermore, all the synthesized compounds were screened against three cultured human cancer cell lines (HEPG-2, SGC-7901, LS174T). The results showed that some compounds showed potent cytotoxicity and compound 3a in particular exhibited the most significant cytotoxicity against these cancer cell lines, especially against HEPG-2 cells.

2. Results and Discussion

2.1. Chemistry

Our synthetic strategy was first to obtain 4-methylseselin (1), and then to stereoselectively synthesize 4-methyl-(−)-cis-khellactone (2) and its derivatives 3ao. The corresponding synthetic routes are shown in Scheme 1.

Scheme 1. Synthesis of (3'S,4'S)-4-methyl-cis-khellactone derivatives 3ao.

Scheme 1. Synthesis of (3'S,4'S)-4-methyl-cis-khellactone derivatives 3ao.

Reagent and conditions: (a) 3-chloro-3-methyl-1-butyne, K2CO3, KI in DMF, 70–80 °C, 3–4 days; (b) N,N-diethylaniline, reflux, 15 h; (c) K3Fe(CN)6, K2CO3, (DHQD)2-PYR, K2OsO2(OH)4, methane-sulfonamide, in t-BuOH/H2O (v/v, 1:1) at 0 °C, 1 day; (d) Various organic acids, DCC, DMAP in anhydrous CH2Cl2, reflux.

7-Hydroxy-4-methylcoumarin, a commercially available compound, was reacted with 3-chloro-3-methyl-1-butyne in DMF in the presence of anhydrous potassium carbonate and potassium iodide and then thermal rearrangement occurred in boiling diethylaniline to form 4-methylseselin (1) by following the procedures published previously [13,14].

4-Methylseselin (1) was asymmetrically dihdroxylated using (DHQD)2-PYR (hydroquinidine 2,5-diphenyl-4,6-pyrimidinediyl diether) as a chiral catalyst to give 4-methyl-(−)-cis-khellactone (2) [15]. Without further purification, compound 2 was directly esterified in anhydrous CH2Cl2 with various organic acids in the presence of dimethylaminopyridine (DMAP) and N,N'-dicyclohexylcarbodiimide (DCC), respectively, to produce 4-methyl-cis-khellactone derivatives 3ao [16].

For the determination of enantiomeric excess, 4-methylseselin (1) was oxidized with OsO4 to give racemic 4-methyl-cis-khellactones 2' [17] (Scheme 2). The asymmetric dihydroxylation for 2 is highly stereoselective, with good enantiomeric excess (74% e.e.) by chiral HPLC analysis. (DHQD)2-PYR leads primarily to the cis-diol with S, S configuration [15].

Scheme 2. Syntheses of 4-methyl-(±)-cis-khellactone 2'.

Scheme 2. Syntheses of 4-methyl-(±)-cis-khellactone 2'.

2.2. In Vitro Biological Evaluation

All of the 4-methyl-(3'S,4'S)-cis-khellactone derivatives were evaluated for cytotoxic activity in vitro against three human cancer cell lines (HEPG-2, SGC-7901, LS174T) using the MTT assay. The antitumor activity was indicated in terms of IC50 (μM) values and the results are presented in Table 1.

Table 1. Cytotoxic activity of the synthesized compounds against three human cancer cell lines a.

CompoundIC50 ± SD (μM)
HEPG-2 SGC-7901LS174T
3a8.51 ± 3.0329.65 ± 6.1219.14 ± 3.68
3b23.64 ± 5.2959.81 ± 9.8547.17 ± 6.17
3c15.62 ± 4.1529.36 ± 6.5520.16 ± 4.79
3d78.23 ± 11.07>100>100
3e50.92 ± 8.0178.93 ± 16.2754.79 ± 9.45
3f67.54 ± 8.36>10055.20 ± 12.10
3g89.29 ± 17.8171.30 ± 12.76>100
3h22.69 ± 5.9031.14 ± 4.3219.60 ± 4.32
3i>10075.23 ± 8.09>100
3j>10089.57 ± 11.9291.13 ± 13.05
3k>100>10084.03 ± 9.98
3l60.92 ± 8.7322.64 ± 5.4438.51 ± 4.63
3m77.55 ± 10.6082.09 ± 13.76>100
3n86.97 ± 11.09>100>100
3o>10090.36 ± 15.27>100

As shown in Table 1, some compounds showed promising anticancer activity for certain cancer cell lines in vitro and the changes of 3' and 4' substituents on the pyran ring had a significant influence on the cytotoxicity. For example, both compounds 3a and 3c exhibited potent inhibitory effects on the indicated cell lines, meanwhile, 3a with tigloyl group at 3' and 4' position showed the most significant cytotoxicity against the HEPG-2 cell lines, with an IC50 value of 8.51 μM. Compound 3b, compared to 3a, displayed low cytotoxicity against these cell lines, especially against the SGC-7901 and LS174T cell lines. The results revealed that the Z isomer of the 2-methyl-2-butenoyl group is superior to the E isomer for the antitumor activity of these two compounds. Additionally, compound 3h with an o–methylbenzoyl group showed noteworthy inhibitory activity against all three human cancer cell lines among all the synthesized compounds with aromatic groups at the 3' and 4' positions. For SGC-7901 cells, the most highly active compound was 3l with a p-bromobenzoyl group, which had an IC50 value of 22.64 μM. The preliminary results revealed that the tigloyl group at the 3' and 4' position on the pyran ring was favorable structural moiety to retain the anticancer activity. Further investigation to search for more potent groups is under way.

3. Experimental

3.1. General

All chemicals were purchased from Aladdin Chemicals Co. (Shanghai, China) and Energy Chemicals Co. (Shanghai, China), whereas chiral catalyst (DHQD)2-PYR was obtained from Sigma-Aldrich (St. Louis, MO, USA). The reactions were monitored by thin-layer chromatography (TLC) with silica gel GF254 plates (Qingdao Jiyida Silica Reagent Manufacture, Qingdao, China), which were visualized by UV light. Melting points were measured on a XT-4 melting point apparatus (Shanghai Precision Scientific Instrument Co. Ltd., Shanghai, China) without correction. The 1H-NMR spectra were acquired on Bruker Avance 600 MHz spectrometer (Bruker Corporation, Karlsruhe, Germany) from solutions in either deuterated chloroform or deuterated dimethylsulfoxide (DMSO-d6) containing tetramethylsilane as internal reference, while the 13C-NMR spectra were recorded at 150 MHz. ESI mass spectra were obtained on an API QTRAP 3200 LC-MS spectrometer (AB SCIEX Corporation, Boston, MA, USA). Optical rotations were measured on a Perkin–Elmer 241 polarimeter (PE Corporation, Nolwalk, CT, USA) at room temperature. The enatiomeric excesses (ee values) of the compounds were determined by Chiralpak AS-H chiral HPLC analysis by using a Shimadzu LC-10A instrument (Shimadzu, Suzhou, China), using n-hexane/2-propanol as eluent.

3.2. Procedure for the Synthesis of 4-Methylseselin (1)

To a solution of 4-methyl-7-hydroxycoumarin (1.76 g, 10 mmol), K2CO3 (3.45 g, 25 mmol), KI (1.66 g, 10 mmol) in DMF (20 mL) was added excess 3-chloro-3-methyl-1-butyne (6 mL), then the mixture was heated to 70–80 °C for 3–4 days. After the solid K2CO3 was filtered, the brown filtrate was poured into EtOAc and washed with water three times and dried over anhydrous Na2SO4. The solvent was removed in vacuo. The residue, without purification, was directly heated to reflux in 20 mL of N,N-diethylaniline for 15 h. The reaction mixture was cooled to room temperature, poured into EtOAc, and washed with 10% aqueous HCl, water and brine. The organic layer was separated, and solvent was removed in vacuo. The residue was purified by column chromatography with an eluant of petroleum ether-EtOAc = 10:1 to afford compound 1. Molecular formula (MW): C15H14O3 (242.27 g/mol); white solid; 23% Yield; mp: 139–141 °C (lit. [7]. 141–143 °C); 1H-NMR (DMSO-d6) δ 1.43 (6H, s, 2 × C-2'-CH3), 2.38 (3H, s, C-4-CH3), 5.93 (1H, d, J = 10.04 Hz, H-3'), 6.23 (1H, s, H-3), 6.74 (1H, d, J = 10.04 Hz, H-4'), 6.82 (1H, d, J = 8 .70 Hz, H-6), 7.54 (1H, d, J = 8.70 Hz, H-5); MS (ESI) m/z 264.9 ([M+Na]+).

3.3. Procedure for the Synthesis of 4-Methyl-(-)-cis-khellactone (2)

K3Fe(CN)6 (246 mg, 0.75 mmol) and K2CO3 (105 mg, 0.75 mmol) were dissolved in t-BuOH/H2

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