Stereoselective synthesis of analogues of deoxyfebrifugine

Abstract

The preparation of six new optically active analogues of the natural product febrifugine (1) is reported. These analogues, lacking the hydroxy group from the natural product, were prepared from optically active N-protected S-pelletierine (7) and differ in terms of the specific quinazolinone portion included. The required S-7 (80% enantiomeric excess) was prepared from an asymmetric Mannich reaction between piperideine (8) and acetone in the presence of l-proline. The differently substituted quinazolinone used in this study (10a–10g) was either commercially available or was prepared from the corresponding substituted anthranilic acid and were installed via a bromination–alkylation sequence. N-Deprotection of the subsequent adducts (12a–12g) gave target compounds 13a–13f and completed the synthetic sequence.

Introduction

Febrifugine (1) is a naturally occurring substance found in Dichroafebrifuga (sometimes colloquially known as Chinese quinine) and a species from the Hydrangea family of plants.^1–5D. febrifuga has been used for centuries in traditional Chinese medicine for the treatment of a variety of ailments and since the first scientific papers concerning the plant-based active ingredient appeared (approximately 60years ago) febrifugine (1) and its analogues have received attention^6–8 from both a biological^9–22 and a chemical standpoint.^23–48 In relation to the latter, the precise structure of naturally occurring febrifugine (1) was only finally confirmed in 1999—partly due to complications in structure analysis as a result of its isomerisation into isofebrifugine (2) (Scheme 1).^24,49 Despite the potent antimalarial activity of febrifugine (1), it proved too toxic to develop as an antimalarial medicine and attempts to mitigate this toxicity led to halofuginone (3), an analogue in which the metabolically vulnerable aromatic protons are masked.^50–53 This compound has been used as a racemate in veterinary medicine [to combat protozoal infections in livestock (particularly cattle and poultry)], and during this period, the antifibrotic activity of 3 was serendipitously uncovered.^54–56

Scheme 1.

Structures of febrifugine (1), isofebrifugine (2) and their isomerisation and the structure of the dihalogenatedfebrifugine analogue halofuginone (3) and their 3-deoxy analogues 4 and 5.

Since this latter discovery, a great deal of effort has been dedicated to investigating and understanding halofuginone’s properties in animal models for numerous diseases.^57–61 These studies have ultimately supported the development of 3 as a human drug candidate, and racemic halofuginone has been the subject of clinical trials associated with its anticancer activity ⁶² and its ability to improve the clinical indications in muscular dystrophy patients. ⁶³ We have previously synthesised both (+)- and (−)-febrifugine (1) and halofuginone (3)^44,46 and linked to this were keen to investigate how the presence of the 3-hydroxy group effected biological activity. In part, this was because the epimerisation event, likely occurring via a retro-conjugate addition, is complicated by hemiketal formation to generate the hemiketal group in isofebrifugine (2).^6–8 In addition, recent work indicates that, at least in part, the activity of 3 is due to inhibition of human prolyl-tRNA synthetase.^12,13 In this context, X-ray crystallography has provided insight into the mode of inhibition and the piperidine portion of 3mimics the pyrrolidine ring in proline and binds to the site occupied by this amino acid.^15,16 We consequently felt that the presence of the hydroxy group in 3 might not be required for action and that a synthetically simpler 3-deoxy analogue of 1 and 3 (i.e. compounds 4 and 5 in Scheme 1) would be of great interest.

‘Deoxyfebrifugine’ (4) has previously been prepared in racemic form three times,^64–66 and in one report, ⁶⁵ its antimalarial activity was compared with febrifugine itself. Although this was less potent, it did show comparable activity to quinine. In 2015, we reported the first asymmetric synthesis of both enantiomers of deoxyfebrifugine (4) and deoxyhalofuginone (5). ⁶⁷ This synthesis initially relied on the preparation of Cbz-protected pelletierine 7, prepared from piperidine 6 using a proline-based asymmetric Mannich reaction sequence (Scheme 2). ⁶⁸

Scheme 2.

Enantioselective synthesis of both enantiomers of Cbz-protected pelletierine (7).

Subsequently, the required quinazolinone was introduced to both enantiomers of 7 in a bromination-nucleophilic displacement sequence, before finally the Cbz-group was removed to form 4 and 5 as their dihydrobromide salts (Scheme 3).

Scheme 3.

Asymmetric synthesis of deoxyfebrifugine (4) and deoxyhalofuginone (5).

Based on the late-stage introduction of the quinazolinone portion, we recognised that divergence at this stage in the reaction sequence could be used to efficiently construct a library of analogues. Ultimately, these would be of value to gauge the impact structural changes in this region have on biological activity. Linked to this objective, the synthesis of quinazolinones from the corresponding anthranilic acids (9) is well-established, and many substituted anthranilic acids are commercially available. ⁶⁹ In this work, we wish to report the asymmetric synthesis of a variety of deoxyfebrifugine analogues.

Results and discussion

The asymmetric Mannich reaction between an imine and an enolisable ketone, proceeding via enamine-based organocatalysis, ⁷⁰ has been reported.^71–74 The range of imines utilised in this type of process has been widened to incorporate Δ ¹ -piperideine (8), ⁷⁵ which Bella and co-workers have demonstrated reacts with several methyl ketones in the presence of several secondary amines possessing a Brønsted acidic group. ⁶⁸ Proline itself performs well in this chemistry and in this manner unnatural pelletierine was prepared in good yield and enantioselectivity. As Scheme 4 illustrates, at room temperature,8 undergoes a rapid Mannich reaction with l-proline and an excess of acetone in a dimethylsulfoxide (DMSO (–water mixture (8:1)). The crude material obtained after aqueous work-up was directly converted into the corresponding carbamate, and (−)-(S)-7 was isolated in reasonable to good yields with enantiomeric ratios 90:10 in a reproducible process performed on gramme-scale (performing the reaction at 5 °C, under otherwise identical conditions, led to the isolation of (−)-7 in 23% and 75% ee). In relation to this sequence, it should be noted that to minimise the loss of enantiomeric purity of the intermediate β-amino ketone, ⁷⁶ it was important not to prolong the period for the Mannich reaction and to directly convert the free-base pelletierine to its Cbz-protected form – which, it should be noted, proved to be stable stereochemically.

Scheme 4.

Synthesis of (−)-(S)-Cbz-protected pelletierine 7 (80% ee) via an asymmetric Mannich reaction.

With access to multi-gramme quantities of the enantioenriched 2-substituted piperidine (−)-(S)-7 (80% ee), its conversion into a range of deoxyfebrifugine analogues was considered. To achieve this, several quinazolinone derivatives, 10a–10g, were identified. Compounds 10a–10f were prepared from the corresponding, commercially available, 2-aminobenzoic (anthranilic) acids 9a–9f in a modified Niementowski reaction involving heating the anthranilic acids with formamide(Scheme 5). ⁷⁷ In this manner, halo-containing compounds 9a and 9b gave 10a and 10b in reasonable to good yield. 2-Amino-4,5-dimethoxybenzoic acid (9c)only gave 10c in only low yield (19%) which, despite several attempts, could not be improved. We speculate that this is due to the limited stability of 9c at the reaction temperatures.Naphtho-based anthranilic acid 9d smoothly gave benzo[g]quinazolin-4(3H)-one (10d), and 2-amino nicotinic acid (9e) was also successfully employed to accesspyrido[2,3-d]pyrimidin-4(3H)-one (10e), albeit in a low yielding process reflecting the reduced nucleophilicity of the amino group in this instance.

Scheme 5.

The Niementowski-type reaction for the synthesis of substituted quinazolin-4(3H)-ones 10a–10f (with isolated yields given and conditions employed in brackets).

An additional quinazolinone derivative of interest was 2-methylquinazolin-4(3H)-one (10f) (compound 10f has been isolated from a culture of the microorganism Bacillus cereus). ⁷⁸ Employing a method reported by Guiry ⁷⁹ and Okano, ⁸⁰ anthranilic acid 9f was first treated with acetic anhydride at 120 °C. Ammonia solution (28%) was then added and the mixture heated to reflux before standing at room temperature overnight. This gave 2-methyl substituted quinazolinone 10f in 38% yield. The final aromatic heterocycle identified was benzo[d][1,2,3]triazin-4(3H)-one (10g), whichis commercially available.

With the range of quinazolinones in hand (10a–10g) their conversion into deoxyfebrifugine analogues was next considered (Scheme 6). In each case, the methyl group in (−)-(S)-7 was brominated using a two-step, one-pot procedure involving the formation of an intermediate trimethylsilyl enol ether. The crude α-bromo ketone (11) obtained after work up was then separately treated with each of the derivatives 10a–10g. Following this operation, adducts 12a–12g were isolated and purified by flash column chromatography and moderate to reasonable yields were obtained for the three-step sequence. It should be noted that the efficiency of this sequence was improved by inclusion of activated 4 Å molecular sieves during the silyl enol ether formation. In our previous work, ⁶⁷ we demonstrated that the enantiomeric excess from Cbz-protected 7 was not altered in this sequence (i.e. these adducts 12a–12g are formed in 80% enantiomeric excess).

Scheme 6.

The bromination-quinazolinone substitution sequence for the synthesis of Cbz-protected deoxyfebrifugine analogues 12a–12g.

Finally, as shown in Scheme 7, removal of the Cbz-protecting group from compounds 12a–12g was considered. Using standard HBr-based deprotection conditions, ⁸¹ compounds 12a–12f all formed the desired compounds as their HBr salts. Mass recovery and previous microanalytical studies ⁶⁷ indicated that compounds 13a–13d and 13f were isolated as their dihydrobromide salts, whereas pyridino-compound 13e gave the trihydrobromide. Unfor-tunately, protected triazinone analogue 12g failed to generate the hoped-for compound 13g.

Scheme 7.

The Cbz-deprotection of deoxyfebrifugine analogues 12a–12g to generate deoxyfebrifugine analogues 13a–13f.

In summary, six optically active (2S)-deoxyfebrifugine analogues (13a–13g) were prepared using an enantioselective Mannich-based synthesis as a key step. These analogues differ in terms of their quinazolinone motif. In turn, the quinazolinones utilised were prepared following a classical Niementowski reaction. In the future, experiments concerning analysis of their ability to block protein synthesis in parasitic and mammalian cells will be conducted.

Experimental details

General directions: Starting materials were supplied from commercial sources and used without further purification. All commercially available solvents were used as supplied unless otherwise stated. All ‘dry’ solvents were dried and distilled by standard procedures or were processed through a Grubbs type still. Oxygen-free nitrogen was obtained from BOCgases and was used without further drying. Infrared spectroscopy was performed on a Fourier-transform infrared(FTIR) spectrometer. Routine electrospray mass spectra and high-resolution mass spectra (HRMS) were run on electrospray ionisation (ESI). Chiral HPLC was performed on chiralpakIB, IC or AS-H columns as stated. The nuclear magnetic resonance (NMR) spectra were recorded at 25 °C on 300 or 400MHz spectrometers, as indicated. All peak assignments are confirmed by 2D experiments ( ¹ H- ¹ HgCOSY, ¹H- ¹³ CHSQC). TLC (thin layer chromatography) was performed on 60F₂₅₄ aluminium-backed plates with realisation by ultraviolet (UV) irradiation and/or chemical staining. Flash column chromatography was performed with silica, particle size 0.040–0.063 mm.

Δ ¹ -Piperideine 8: ⁶⁸ Piperidine 6 (5.17 g, 60.72 mmol) was added to a solution of N-chlorosuccinimide (15.00 g, 112.33 mmol) in diethyl ether (400 mL). The solution was stirred at rt for 1h. After filtration and rinsing of the precipitate with diethyl ether (40 mL), the diethyl ether solution was washed with water (2 × 400 mL) and brine (200 mL) and then dried with MgSO₄. The N-chloropiperidine solution was then filtered and concentrated under reduced pressure to roughly 80mL volume. The resultant solution was added dropwise to a solution of KOH (4.00 g, 71.20 mmol) in absolute ethanol (64 mL). During this process, the temperature was maintained between 5 °C and 10 °C. After addition, the mixture was stirred at room temperature for 24h and filtered. The precipitate was rinsed with absolute ethanol (60 mL), and the combined solution was concentrated to about 25mL. Diethyl ether (200 mL) and water (20 mL) were added and the mixture was extracted. The phases were separated and the aqueous phase was then further extracted with diethyl ether (2 × 100 mL). The combined organic phases were washed with brine and dried with anhydrous MgSO₄, filtered, and concentrated in vacuo to afford 8 (2.5 g, 60%) as yellowish oil, which gradually solidified. This material was subsequently used without further purification and can be stored indefinitely at low temperature.

(−)-(2S)-(2-Oxopropyl) piperidine-1-carbamic acid benzyl ester (Cbz-pelletierine) 7: ⁶⁸ Δ ¹ -Piperideine 8 (Typical experiments used the solid 1 | -piperideine trimeric form of 8. However, the initially isolated oil form of 8 can also be used with similar results)^67,82 (670 mg, 8.06 mmol, 1 eq.), acetone (27 mL, 367.59 mmol, 46 eq.), DMSO (27 mL), water (3.4 mL), and l-proline (185 mg, 1.61 mmol, 0.2 eq.) were mixed and stirred for 1 h at room temperature. An aqueous saturated solution of sodium bicarbonate (50 mL) was added to the mixture, which was extracted with CH₂Cl₂ (2 × 50 mL). The organic layer was washed with brine (50 mL), filtered and dried over anhydrous MgSO₄. After filtration, the solvent was removed under reduced pressure, and the crude pelletierine was used immediately without purification [HRMS (ES) calcd for [(C₈H₁₅NO + H)]⁺ 142.1232, found 142.1232 (0.1 ppm)]. The residue obtained above was diluted in CH₂Cl₂ (25 mL) and a 0.4 M solution of aqueous sodium carbonate (18 mL, 7.20 mmol, 0.9 eq.) was added. The mixture was cooled to 0 °C and benzyl chloroformate (0.9 mL, 6.30 mmol, 0.8 eq.) was added dropwise. The ice bath was removed and the solution was stirred at rt overnight. The solution was diluted with CH₂Cl₂ (25 mL) and extracted. The resultant organic layer was washed with water (50 mL), and then with brine (50 mL), dried over anhydrous MgSO₄, filtered and concentrated in vacuo. This gave the crude product which was purified by flash column chromatography (CH₂Cl₂/EtOAc; 6:1) to afford the title compound 7 (1.25 g, 72 % yield over two steps) as light-yellow oil. R_f = 0.55 (EtOAc/CH₂Cl₂; 1:6); [α]_D ²⁰  = –10.5 (c = 1.00, CHCl₃) {lit., ⁸³ [α]_D ²⁸  = –10.0 (c = 0.50, CHCl₃)}; HRMS (ES+) calcd. for [(C₁₆H₂₁NO₃+Na)]⁺ 298.1491, found 298.1430 (+3.8 ppm); IR: 3032, 2938, 2862, 1688, 1497, 1353, 1258, 1165, 1049, 735, 697 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.42–7.32 (m, 5H), 5.11 (d, J = 12.5 Hz, 1H), 5.08 (d, J = 12.5 Hz, 1H), 4.84 (br. s, 1H), 4.07 (br. s, 1H), 2.87 (app. t, J = 12.5 Hz, 1H), 2.73–2.61 (m, 2H), 2.15 (s, 3H), 1.78–1.34 (m, 6H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 206.9 (CO), 155.3 (CO), 136.7 (C), 128.4 (CH), 127.9 (CH), 127.8 (CH), 67.1 (CH₂), 47.5 (CH), 44.3 (CH₂), 39.8 (CH₂), 30.0 (CH₃), 28.3 (CH₂), 25.2 (CH₂), 18.8 (CH₂) ppm; HPLC analysis (Chiralpak IB), heptane/EtOH; 98:2 (1mL/min.): (−)-7 tr = 12.1 min., (+)-7 tr = 13.6; 90:10 e.r.

Quinazolinone synthesis: General Procedure: A mixture of the anthranilic acid (8.0 g, 0.06 mol) and formamide (10.8 g, 0.24 mol, 4 eq.) was stirred at 165 °C (oil bath temperature) for 4h. The mixture was cooled before water (15 mL) was added. At this stage copious solid material appeared. The mixture was then warmed to 60 °C and water (30 mL) was added. The mixture was stirred for another 30 min at 60 °C. The resulting precipitate was filtered and then recrystallised with ethanol to give the quinazolinone compounds 10a–10f.

6-Chloroquinazolin-4(3H)-one10a: 5-Chloroanthranilic acid 9a gave 10a as a white solid. Yield: 61%. M.p. 274 °C–275 °C (EtOH) {lit., ⁷⁷ 259 °C–261 °C}. ¹H NMR (300 MHz, (CD₃)₂SO): δ 12.45 (br. s, 1H), 8.13 (s, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.85 (dd, J = 8.5, 2.5 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H).

6-Bromoquinazolin-4(3H)-one10b: 5-Bromoanthranilic acid 9b gave 10b as a white solid. Yield: 70%. M.p.267 °C–269 °C (EtOH) {lit., ⁷⁷ 261 °C–267 °C}. ¹H NMR (300 MHz, (CD₃)₂SO): δ 8.17 (d, J = 2.5 Hz, 1H), 8.12 (s, 1H), 7.94 (dd, J = 8.5, 2.5 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H).

6,7-Dimethoxquinazolin-4(3H)-one10c: 4,5-Dimethoxy-anthranilic acid 9c gave 10c as brownish solid. Yield: 19%. M.p.290 °C–291 °C (EtOH) {lit., ⁸⁴ >260 °C}. ¹H NMR (300 MHz, (CD₃)₂SO): δ 7.96 (s, 1H), 7.42 (s, 1H), 7.11 (s, 1H), 3.88 (s, 3H), 3.85 (s, 3H).

Benzo[g]quinazolin-4(3H)-one10d: 3-Amino-2-naphthoic acid 9d gave 10d as a yellow solid. Yield: 61%. M.p. > 270 °C (EtOH) {lit., ⁸⁴ >260 °C}. ¹H NMR (300 MHz, (CD₃)₂SO): δ 12.05 (br. s, 1H), 8.82 (s, 1H), 8.45–8.16 (m, 2H), 8.12–8.02 (m, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H).

Pyrido[2,3-d]pyrimidin-4(3H)-one10e: 2-Amino nicotinic acid 9e gave 10e as a yellowish solid. Yield: 18%. M.p.256 °C–258 °C (EtOH) {lit., ⁸⁴ 258 °C}. ¹H NMR (300 MHz, (CD₃)₂SO): δ 12.50 (br. s, 1H), 8.92 (dd, J = 4.5, 2.0 Hz, 1H), 8.47 (dd, J = 8.0, 2.0 Hz, 1H), 8.28 (s, 1H), 7.52 (dd, J = 8.0, 4.5 Hz, 1H).

2-Methylquinazolin-4(3H)-one10f: Using a modified literature procedure,^79,80 a solution of anthranilic acid 9f (454 mg, 3.3 mmol) in acetic anhydride (2.0 mL) was heated at 120 °C for 3 h and the reaction mixture was evaporated to dryness. Ammonia solution (28% w/v; 4.0 mL) was added and the mixture was heated at reflux for 4 h and then left overnight at rt. The precipitate was collected by filtration, washed successively with water, then methanol and dried under reduced pressure, to give the quinazolinone 10f (200 mg, 38%) as white powder. M.p. 228 °C–230 °C {lit., ⁸⁰ 230 °C–232 °C}. ¹H NMR (300 MHz, (CD₃)₂SO): δ 12.14 (br. s, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.75 (t, J = 7.5 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 2.20 (s, 3H).

General procedure for synthesis of (–)-(2S)-2-[2-Oxo-3-(6-chloro-4-oxoquinazolin-4(3H)-yl)propyl]piperidine-1-carbamic acid benzyl ester 12a: A mixture of commercially activated 4Å molecular sieves (0.5 g) and (−)-(S)-7 (300 mg, 1.09 mmol, 1.0 eq.) in dry CH₂Cl₂ (8 mL) was cooled to 0 °C, and left for 30 min. DIPEA (0.28mL, 1.61 mmol, 1.5 eq.) and TMSOTf (0.27 mL, 1.49 mmol, 1.4 eq.) were added, and the mixture was stirred at room temperature for 1 h. NBS (248 mg, 1.39 mmol, 1.3 eq.) was then added in one portion. The mixture was stirred at room temperature for 2h then poured into water (20 mL) and extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with saturated aqueous NaHCO₃ (30 mL) and brine (30 mL), dried over anhydrous MgSO₄, filtered and concentrated. A solution of the residue in dimethylformamide (DMF) (10 mL) was treated with 6-chloroquinazolin-4(3H)-one10a (273 mg, 1.51 mmol, 1.4 eq.) and K₂CO₃ (220 mg, 1.59 mmol, 1.5 eq.) and was stirred overnight at rt. The mixture was poured into H₂O (60 mL) and extracted with EtOAc (2 × 60 mL). The combined organic layers were washed with H₂O (60 mL) and brine (60 mL), dried over anhydrous MgSO₄, filtered, and concentrated in vacuo to afford the crude product. This was purified by column chromatography (EtOAc), which afforded 12a as a white crystalline solid (58% over three steps). M.p.130 °C–133 °C; R_f = 0.45 (EtOAc); [α]_D ²⁰  = −12.0 (c = 1.00, acetone); HRMS (ES) calcd for [(C₂₄H₂₄N₃O₄ ³⁵ Cl + Na)]⁺476.1353, found 476.1335 (−3.8 ppm); IR: 3072, 2947, 1729, 1673, 1614, 1470, 1353, 1269, 1070, 850, 769, 733cm⁻¹; ¹H NMR (400 MHz, (CD₃)₂SO): δ 8.17 (s, 1H), 8.04 (d, J = 2.5 Hz, 1H), 7.86 (dd, J = 8.5, 2.5 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.32–7.24 (m, 5H), 5.06 (d, J = 12.5 Hz, 1H), 5.01 (d, J = 12.5 Hz, 1H), 4.96 (s, 2H), 4.72–4.64 (m, 1H), 3.87 (d, J = 14.0 Hz, 1H), 3.11 (dd, J = 16.0 Hz, 8.5 Hz, 1H), 2.88 (t, J = 12.5 Hz, 1H), 2.75 (dd, J = 16.0, 6.0 Hz, 1H), 1.63–1.47 (m, 5H), 1.34–1.21 (m, 1H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO) δ 202.2, 159.4, 154.9, 148.9, 147.1, 137.4, 135.1, 131.9, 130.0, 128.8, 128.2, 127.8, 125.5, 123.0, 66.6, 54.9, 47.2, 41.0, 40.0, 28.2, 25.4, 18.7 ppm.

(–)-(2S)-2-[2-Oxo-3-(6-Bromo-4-oxoquinazolin-4(3H)-yl)propyl]piperidine-1-carbamic acid benzyl ester 12b: Following the general procedure outlined above 6-bromoquinazolin-4(3H)-one10b gave 12b as a white crystalline solid (53% overthree steps). M.p.128 °C–130 °C; R_f = 0.40 (EtOAc); [α]_D ²⁰ = –13.6 (c = 1.00, acetone); HRMS (ES) calcd for [(C₂₄H₂₄N₃O₄ ⁷⁹ Br+Na)]⁺520.0848, found 520.0856 (1.6 ppm); IR: 3069, 2944, 1729, 1674, 1614, 1468, 1353, 1269, 1073, 850, 769, 734cm^-1; ¹H NMR (400 MHz, (CD₃)₂SO): δ 8.18 (s, 1H), 8.17 (s, 1H), 7.97 (dd, J = 8.5, 2.5, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.33–7.22 (m, 5H), 5.06 (d, J = 12.5 Hz, 1H), 5.01 (d, J = 12.5 Hz, 1H), 4.96 (s, 2H), 4.72–4.65 (m, 1H), 3.87 (d, J = 13.0 Hz, 1H), 3.10 (dd, J = 16.0, 8.5 Hz, 1H), 2.87 (t, J = 12.0 Hz, 1H), 2.75 (dd, J = 16.0 Hz, 6.0 Hz, 1H), 1.63–1.46 (m, 5H), 1.33–1.18 (m, 1H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO)) δ 202.2, 159.3, 155.1, 149.0, 147.4, 137.9, 137.4, 130.1, 128.8, 128.6, 128.2, 127.8, 123.4, 120.1, 66.6, 54.9, 47.2, 41.0, 40.0, 28.2, 25.4, 18.7 ppm.

(–)-(2S)-2-[2-Oxo-3-(6,7-dimethoxy-4-oxoquinazolin-4(3H)-yl)propyl]piperidine-1-carbamic acid benzyl ester 12c: Following the general procedure, 6,7-dimethoxyquinazolin-4(3H)-one10c gave 12c as a yellow solid (31% overthree steps). M.p. 96 °C–100 °C; R_f = 0.25 (EtOAc); [α]_D ²⁰  = –3.2 (c = 1.00, acetone); HRMS (ES) calcd for [(C₂₆H₂₉N₃O₆ + Na)]⁺502.1954, found 502.1973 (3.8 ppm); IR: 3059, 2921, 1667, 1607, 1498, 1370, 1271, 1018, 857, 782, 697cm^-1; ¹H NMR (400 MHz, (CD₃)₂SO): δ 8.03 (s, 1H), 7.4 (s, 1H), 7.35–7.24 (m, 5H), 7.13 (s, 1H), 5.06 (d, J = 12.5 Hz, 1H), 5.02 (d, J = 12.5 Hz, 1H), 4.91 (s, 2H), 4.72–4.65 (m, 1H), 3.88 (s, H, 3H), 3.87–3.84 (m, 1H), 3.83 (s, 3H), 3.11 (dd, J = 16.0, 8.5 Hz, 1H), 2.87 (t, J = 11.0 Hz, 1H), 2.72 (dd, J = 16.0 Hz, 5.8 Hz, 1H), 1.64–1.46 (m, 5H), 1.34–1.22 (m, 1H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 202.6, 159.7, 155.0, 149.2, 146.9, 144.6, 138.8, 137.4, 128.8, 128.2, 127.8, 114.9, 108.4, 105.5, 66.6, 56.4, 56.0, 54.8, 47.2, 41.0, 40.0, 28.2, 25.4, 18.7 ppm.

(–)-(2S)-2-[2-Oxo-3-(Benzo[g]-4-oxoquinazolin-4(3H)-yl)propyl]piperidine-1-carbamic acid benzyl ester 12d: Following the general procedure, benzo[g]quinazolin-4(3H)-one10d gave 12d as a yellow solid (32% overthree steps). M.p.132 °C–134 °C; R_f = 0.40 (EtOAc); [α]_D ²⁰  = -2.2 (c = 1.00, acetone); HRMS (ES) calcd for [(C₂₈H₂₇N₃O₄+H)]⁺470.2080, found 470.2068 (–2.5 ppm); IR: 3060, 2929, 1721, 1667, 1618, 1428, 1355, 1268, 1070, 883, 742, 696cm^-1; ¹H NMR (400 MHz, (CD₃)₂SO): δ 8.83 (s, 1H), 8.26 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.11–8.08 (m, 2H), 7.67 (t, J = 8.0 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.34–7.23 (m, 5H), 5.07 (d, J = 12.5 Hz, 1H), 5.02 (d, J = 12.5 Hz, 1H), 4.97 (s, 2H), 4.75–4.67 (m, 1H), 3.88 (d, J = 16.5 Hz, 1H), 3.13 (dd, J = 16.0 Hz, 8.5 Hz, 1H), 2.89 (t, J = 13.5 Hz, 1H), 2.76 (dd, J = 16.0 Hz, 6.0 Hz, 1H), 1.64–1.47 (m, 5H), 1.35–1.19 (m, 1H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 202.7, 160.9, 156.0, 147.6, 143.8, 137.4, 136.5, 131.5, 129.7, 129.1, 128.8, 128.3, 128.2, 128.0, 127.8, 127.0, 125.4, 120.7, 66.6, 54.7, 47.3, 41.0, 40.0, 28.2, 25.4, 18.7 ppm.

(–)-(2S)-2-[2-Oxo-3-(Pyrido[2,3-d]pyrimidin-4(3H)-yl)propyl]piperidine-1-carbamic acid benzyl ester 12e: Following the general procedure, pyrido[2,3-d]pyrimidin-4(3H)-one10e gave 12e as a brown solid (30% overthree steps); R_f = 0.30 (EtOAc); [α]_D ²⁰ = –3.9 (c = 1.00, acetone); HRMS (ES) calcd for [(C₂₃H₂₄N₄O₄+H)] ⁺421.1876, found 421.1892 (3.8 ppm); ¹H NMR (400 MHz, (CD₃)₂SO): δ 8.97 (dd, J = 4.5, 2.0Hz, 1H), 8.50 (dd, J = 8.0, 2.0Hz, 1H), 8.36 (s, 1H), 7.57 (dd, J = 8.0, 4.5Hz, 1H), 7.35–7.24 (m, 5H), 5.07 (d, J = 12.5Hz, 1H), 5.02 (d, J = 12.5Hz, 1H), 4.98 (s, 2H), 4.70–4.64 (m, 1H), 3.87 (d, J = 14.5Hz, 1H), 3.12 (dd, J = 16.0Hz, 8.5Hz, 1H), 2.86 (t, J = 12.0Hz, 1H), 2.76 (dd, J = 16.0Hz, 5.5Hz, 1H), 1.63–1.45 (m, 5H), 1.33–1.18 (m, 1H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 202.2, 168.0, 160.0, 156.5, 151.5, 137.6, 137.4, 128.8, 128.2, 127.9, 123.3, 117.0, 66.6, 47.2, 41.0, 40.0, 31.1, 28.2, 25.4, 18.7 ppm.

(–)-(2S)-2-[2-Oxo-3-(2-methyl-4-oxoquinazolin-4(3H)-yl)propyl]piperidine-1-carbamic acid benzyl ester 12f: Following the general procedure, 2-methylquinazolin-4(3H)-one10f gave 12f as a white crystalline solid (50% overthree steps). M.p.128 °C–130 °C; R_f = 0.30 (EtOAc); [α]_D ²⁰ = –6.9 (c = 1.00, acetone); HRMS (ES) calcd for [(C₂₅H₂₈N₃O₄+H)]⁺434.2080, found 434.2097 (4.0 ppm); ¹H NMR (400MHz, (CD₃)₂SO): δ 8.05 (d, J = 8.0Hz, 1H), 7.80 (t, J = 8.0Hz, 1H), 7.59 (d, J = 8.0Hz, 1H), 7.48 (t, J = 8.0Hz, 1H), 7.39–7.24 (m, 5H), 5.08 (s, 2H), 5.05 (s, 2H), 4.71–4.68 (m, 1H), 3.90 (d, J = 12.5Hz, 1H), 3.12 (dd, J = 16.0Hz, 8.0Hz, 1H), 2.95–2.80 (m, 2H), 2.50 (s, 3H), 1.67–1.49 (m, 5H), 1.35–1.23 (m, 1H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 203.0, 161.3, 155.3, 154.8, 147.5, 137.4, 135.0, 128.8, 128.2, 127.8, 127.0, 126.9, 126.6, 119.9, 66.7, 53.5, 47.4, 41.0, 40.0, 28.4, 25.4, 23.1, 18.7 ppm.

(–)-(2S)-2-[2-Oxo-3-(1,2,3-benzotriazin-4-oxo-4(3H)-yl)propyl]piperidine-1-carbamic acid benzyl ester 12g: Following the general procedure, 1,2,3-benzotriazin-4(3H)-one10g gave 12g as a white crystalline solid (26% over three steps). M.p.136 °C–138 °C; R_f = 0.75 (EtOAc); [α]_D ²⁰ = –3.0 (c = 0.5, acetone); HRMS (ES) calcd for [(C₂₃H₂₄N₄O₄+Na)]⁺443.1695, found 443.1680 (-3.4 ppm); IR: 3071, 2983, 1724, 1687, 1452, 1334, 1296, 1074, 889, 770, 748cm^-1; ¹H NMR (400 MHz, (CD₃)₂SO): δ 8.24 (d, J = 8.0 Hz, 2H), 8.11 (t, J = 8.0 Hz, 1H), 7.95 (t, J = 8.0 Hz, 1H), 7.38–7.23 (m, 5H), 5.39 (s, 2H), 5.12–5.01 (m, 2H), 4.77–4.68 (m, 1H), 3.89 (d, J = 16.5 Hz, 1H), 3.18 (dd, J = 16.5 Hz, 8.5 Hz, 1H), 2.78–2.95 (m, 2H), 1.44–1.68 (m, 5H), 1.37–1.20 (m, 1H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 202.8, 155.1, 154.8, 144.2, 137.4, 136.2, 133.7, 128.8, 128.6, 128.2, 127.9, 125.0, 119.6, 66.7, 58.5, 47.2, 41.0, 40.0, 28.3, 25.4, 18.7 ppm.

Deoxyfebrifugine analogues. General procedure: The Cbz-protected deoxyfebrifugine analogues (0.20 mmol) were dissolved in CH₂Cl₂ (1 mL) and cooled to 0 °C before 33% w/v HBr in acetic acid solution (1 mL) was added. The mixture was stirred at 0 °C for 15min before the cooling bath was removed and stirring was continued for 1h. Over this period rt was gradually reached. The mixture was diluted in methanol (5 mL), and the product was precipitated by addition of ether (30 mL). The supernatant was removed and this process was repeated three times. The product was dried under high vacuum to afford the desired salt.

(+)-Deoxychlorofuginonedihydrobromide13a: Following the procedure outlined above, 12a (50mg, 0.11 mmol) gave 13a·2HBr (45 mg, 85%) as a white solid powder. M.p.220 °C–222 °C (with dec.); HRMS (ESI) calcd for [(C₁₆H₂₀ ³⁵ ClN₃O₂–H)]⁺320.1166, found 320.1181 (4.7 ppm); IR: 3392, 2959, 2808, 2732, 1724, 1670, 1468, 1383, 1299, 1021, 839, 778, 639 cm^-1; ¹H NMR (400 MHz; (CD₃)₂SO): δ 8.56–8.40 (m, 2H), 8.30 (s, 1H), 8.05 (d, J = 2.5 Hz, 1H), 7.89 (dd, J = 8.5, 2.5 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 5.07 (d, J = 18.0 Hz, 1H), 5.00 (d, J = 18.0 Hz, 1H), 3.53–3.43 (m, 1H), 3.24 (d, J = 12.5 Hz, 1H), 3.02–2.87 (m, 3H), 1.80 (d, J = 10.0 Hz, 1H), 1.71 (app. d, J = 10.5 Hz, 2H), 1.64–1.38 (m, 3H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 201.2, 159.5, 148.9, 147.1, 135.3, 132.1, 130.1, 125.4, 123.0, 55.1, 51.9, 44.4, 42.9, 28.4, 22.2, 21.8 ppm.

(+)-Deoxybromofuginonedihydrobromide13b: Following the procedure outlined above, 12b (30 mg, 0.06 mmol) gave 13b·2HBr (25 mg, 80%) as a white solid powder. M.p.176 °C–178 °C (with dec.); HRMS (ES) calcd for [(C₁₆H₂₀ ⁷⁹ BrN₃O₂–H)]⁺364.0661, found 364.0678 (4.8 ppm); IR: 2925, 2720, 1711, 1658, 1492, 1386, 1294, 1073, 819, 695cm^-1; ¹H NMR (400 MHz; (CD₃)₂SO): δ 8.42 (br. s, 2H), 8.28 (s, 1H), 8.20 (d, J = 2.5 Hz, 1H), 8.01 (dd, J = 8.5, 2.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 5.06 (d, J = 18.0 Hz, 1H), 4.99 (d, J = 18.0 Hz, 1H), 3.53–3.43 (m, 1H), 3.24 (d, J = 11.5 Hz, 1H), 3.00–2.87 (m, 3H), 1.81 (d, J = 11.5 Hz, 1H), 1.71 (app. d, J = 11.5 Hz, 2H), 1.58–1.23 (m, 3H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 201.3, 159.4, 149.0, 147.4, 137.8, 130.2, 128.5, 123.3, 120.3, 55.1, 51.8, 44.4, 42.9, 28.5, 22.2, 21.7 ppm.

(+)-Deoxydimethoxyfebrifuginedihydrobromide13c: Following the procedure outlined above 12c (33 mg, 0.07 mmol) gave 13c·2HBr (22 mg, 62%) as a brownish solid powder. M.p.178 °C–180 °C (with dec.); HRMS (ES) calcd for [(C₁₈H₂₄N₃O₄)]⁺346.1767, found 346.1765 (-0.5 ppm); IR: 3358, 2942, 2799, 1698, 1609, 1452, 1385, 1287, 1193, 1002, 867, 767cm^-1; ¹H NMR (400 MHz; (CD₃)₂SO): 8.46 (br. s, 2H), 8.18 (s, 1H), 8.18 (s, 1H), 7.41 (s, 1H), 7.16 (s, 1H), 5.01 (d, J = 18.0 Hz, 1H), 4.95 (d, J = 18.0 Hz, 1H), 3.90 (s, 3H), 3.85 (s, 3H), 3.53–3.43 (m, 1H), 3.24 (d, J = 12.5 Hz, 1H), 3.02–2.87 (m, 3H), 1.81 (d, J = 15.5 Hz, 1H), 1.71 (app. d, J = 11.5 Hz, 2H), 1.62–1.36 (m, 3H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 201.6, 159.7, 155.2, 149.4, 147.0, 144.3, 114.8, 108.3, 105.4, 56.5, 56.3, 55.0, 51.9, 44.4, 42.9, 28.4, 22.2, 21.7 ppm.

(+)-Deoxynaphthafebrifuginedihydrobromide13d: Following the procedure outlined above 12d (70mg, 0.15 mmol) gave 13d·2HBr (62 mg, 83%) as a pale pink powder. M.p.229 °C–230 °C (with dec.); HRMS (ES) calcd for [(C₂₀H₂₃N₃O₂–H)]⁺336.1712, found 336.1697 (–4.5 ppm); ¹H NMR (400MHz; (CD₃)₂SO): δ 8.84 (s, 1H), 8.43 (br. s, 2H), 8.28 (s, 1H), 8.22 (d, J = 5.5Hz, 2H), 8.12 (d, J = 8.5Hz, 1H), 7.68 (t, J = 7.5Hz, 1H), 7.59 (t, J = 7.5Hz, 1H), 5.06 (d, J = 18.0Hz, 1H), 4.98 (d, J = 18.0Hz, 1H), 3.54–3.44 (m, 1H), 3.23 (d, J = 12.5Hz, 1H), 3.03–2.88 (m, 3H), 1.82 (d, J = 12.0Hz, 1H), 1.71 (app. d, J = 10.0Hz, 2H), 1.60–1.36 (m, 3H) ppm; ¹³C NMR (100MHz, (CD₃)₂SO) δ 201.7, 161.0, 147.6, 143.6, 136.5, 131.6, 129.7, 128.3, 128.0, 127.12,125.354.9, 51.9, 44.4, 42.9, 28.5, 22.2, 21.8 ppm.

(+)-Deoxypyridinofebrifuginetrihydrobromide13e: Following the procedure outlined above 12e (100 mg, 0.24 mmol) gave 13e·3HBr (90 mg, 71%) as a yellowish solid powder. M.p. 264 °C–266 °C (with dec.); HRMS (ES) calcd for [(C₁₅H₁₈N₄O₂+H)]⁺287.1508 found 287.1512 (1.4 ppm); IR: 3026, 2931, 2779, 1723, 1693, 1614, 1421, 1374, 1336, 1217, 1161, 960, 881, 784, 724, 613cm^-1; ¹H NMR (400 MHz; (CD₃)₂SO): δ 9.01 (dd, J = 4.5, 2.0 Hz, 1H), 8.56 (dd, J = 8.0, 2.0 Hz, 1H), 8.50 (s, 1H), 8.46 (br. s, 2H), 7.62 (dd, J = 8.0, 4.5 Hz, 1H), 5.10 (d, J = 18.0 Hz, 1H), 5.03 (d, J = 18.0 Hz, 1H), 3.55–3.43 (m, 1H), 3.24 (d, J = 12.5 Hz, 1H), 3.01 (app. d, J = 6.5 Hz, 2H), 2.93 (dd, J = 10.0 Hz, 1H), 1.81 (d, J = 10.0 Hz, 1H), 1.71 (app. d, J = 10.0 Hz, 2H), 1.63–1.38 (m, 3H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 201.1, 160.8, 157.9, 156.2, 151.8, 136.9, 123.6, 117.2, 55.1, 51.9, 44.4, 42.9, 28.4, 22.2, 21.8 ppm.

(+)-Deoxymethylfebrifuginedihydrobromide13f: Following the procedure outlined above, 12f (80 mg, 0.18 mmol) gave 13f·2HBr (70 mg, 84%) as a whiteish solid powder. M.p.236 °C–238 °C (with dec.); HRMS (ES) calcd for [(C₁₇H₂₂N₃O₂+H)]⁺300.1707 found 300.1710 (1.09 ppm); IR: 3376, 2946, 2784, 1703, 1649, 1449, 1382, 1297, 988, 762, 695cm^-1; ¹H NMR (400 MHz, (CD₃)₂SO): 8.48 (br. s, 2H), 8.07 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 5.18 (d, J = 18.5 Hz, 1H), 5.12 (d, J = 18.5 Hz, 1H), 3.54–3.44 (m, 1H), 3.24 (d, J = 13.0 Hz, 1H), 3.07 (app. dd, J = 6.0, 4.0 Hz, 2H), 2.99–2.87 (m, 1H), 2.45 (s, 3H), 1.80 (d, J = 9.0 Hz, 1H), 1.71 (app. d, J = 10.0 Hz, 2H), 1.61–1.40 (m, 3H) ppm; ¹³C NMR (100 MHz, (CD₃)₂SO): δ 201.7, 161.0, 156.3, 146.0, 135.5, 127.4, 126.8, 126.0, 119.6, 53.6, 51.8, 44.4, 43.1, 28.5, 23.0, 22.2, 21.8 ppm.