A new subgroup of the Amaryllidaceae alkaloids having a 10b,4a-ethanoiminodibenzo[b,d]pyrane (2-benzopyrano[3,4-b]indole) nucleus is isolated from two different Galanthus species as three novel monomeric alkaloids, (+)-graciline (1), (+)-11-acetoxygraciline (2) and (+)-3,4-dihydro-3-hydroxygraciline (3). The fourth new compound, (-)-digracine (4) is a dimer of two such moieties. The Amaryllidaceae alkaloids constitute an exclusive group of basic compounds elaborated almost solely by species of the specified plant family. These alkaloids, some of which display remarkable physiological and pharmacological activities, have so far been classified on structural considerations into eight subgroups. A thorough literature survey has revealed that the members of the genus Galanthus have so far been reported to contain alkaloids from almost all of these subgroups only with the exception of simple phenanthridines. During our ongoing phytochemical studies on Galanthus species of Turkey, we have isolated from Galanthus gracilis a strongly dextrorotatory basic compound, (+)-graciline (1). The ¹H NMR spectrum of 1 taken in CDCl₃ (300 K), accounting for seventeen hydrogens, displayed relevant signals for a basic N-methyl group, a methylenedioxy (OCH₂O) substituent, two aromatic protons appearing as singlets, an isolated deshielded methylene as two well-defined doublets along with four aliphatic protons in the δ 2.2–3.0 region. A striking finding, however, was the presence of four olefinic protons. The ¹³C NMR and DEPT spectra revealed the presence of seventeen carbons: one methyl, four methylene, six methine, and six nonprotonated carbons. Among these, the noteworthy signals were of the quaternary carbons resonating at δ 47.2 and δ 92.6. Since these spectral findings conformed to none of the previously described subgroups and strongly suggested that we were dealing with an unknown structure, extensive 2D NMR experiments were performed. Using information from ¹H, ¹H DQF COSY, HSQC, and HMBC experiments, the skeleton construction was initiated from ring A, which incorporated the methylenedioxy substitution. In the HMBC spectrum, H-7 (δ 6.45) showed a prominent three-bond correlation with the δ 64.2 carbon carrying the isolated geminal hydrogens (H-6), which in turn were connected to the quaternary carbon at δ 92.6 through a ³JCH coupling. The informative three-bond coupling of H-10 (δ 6.73) beyond ring A was with the quaternary carbon at δ 47.2. In the ¹H NMR spectrum, signals for two of the olefinic protons were almost superimposed, while another was buried under the methylenedioxy signals, making it impossible to calculate individual coupling constants. A ¹H NMR spectrum in benzene-d₆ yielded more complex results with three olefinic protons having nearly identical chemical shifts. However, ¹H, ¹H COSY and TOCSY data (CDCl₃) confirmed that these four olefinic protons belonged to a conjugated system. The relative positions of these hydrogens on ring C were assigned via HMBC, particularly three-bond connectivities from δ 5.90 and 5.78 protons to δ 47.2 carbon, and from δ 6.10 and 6.09 signals to δ 92.6 quaternary carbon, allowing construction of a tentative dibenzo[b,d]pyrane nucleus partially saturated in ring C. ¹H,¹H DQF COSY and TOCSY spectra indicated the remaining four aliphatic protons formed a four-spin system. In HMBC, the more deshielded carbon (δ 49.4) of this ethylene chain showed a three-bond correlation with N-methyl protons, which also connected to the central δ 92.6 carbon via ³JCH, establishing one end of the ethanoimino bridge. Shielded methylene protons (δ 2.34 and 2.30) of this bridge displayed expected three-bond correlations with C-1 (δ 136.9), C-10a (δ 133.5), and C-4a (δ 92.6), confirming the ethanoimino bridge linkage between C-10b and C-4a of the dibenzo[b,d]pyrane skeleton. These results add a new tetracyclic ring system (2-benzopyrano[3,4-b]indole) to Amaryllidaceae alkaloids. EI MS of 1 showed a low-abundance molecular ion at m/z 283 (4%), verifying the molecular formula C₁₇H₁₇NO₃. Facile ethanoimino bridge rupture (expelling CH₃CH₂NCH₃) yielded a stable aromatic cation base peak at m/z 225. NOESY (1.2 s mixing time) showed strong spatial relationships between H-1 (δ 6.09), H-10 (δ 6.73), and H-11β (δ 2.30). N-methyl protons displayed cross signals with C-12 methylene hydrogens and H-4 (δ 5.90). A notable interaction was H-4 with the β-oriented hydrogen of the C-6 geminal pair (δ 4.67). Dreiding models favored ring C projecting in front of the A/B ring mean plane, leading to an α-oriented ethanoimino bridge. The CD curve of 1 closely resembled (+)-crinine bases with α-oriented ethanoimino bridges, suggesting 4aR and 10bR absolute configuration for (+)-graciline (1). The CDCl₃ ¹H NMR spectrum of (2) (isolated from G. plicatus subsp. byzantinus) showed similarities to 1, including two aromatic p-protons, methylenedioxy, deshielded isolated methylene, and N-methyl signals. Four olefinic protons indicated structural analogy to 1. However, a three-spin system (deshielded methine at δ 5.42) and a three-proton singlet (δ 2.13) replaced 1’s four-spin system, suggesting an acetoxy substituent on the ethanoimino bridge. Ester carbonyl was confirmed by IR (1730 cm⁻¹) and ¹³C NMR (δ 171.0). ¹³C NMR (19 carbons, DEPT) showed δ 21.2 (acetoxy methyl) and δ 82.0 (acetoxy-bearing methine). ¹H,¹H DQF COSY, HSQC, and HMBC allowed most assignments, except olefinic protons (two obscured by methylenedioxy). Repeating NMR in benzene-d₆ improved olefinic signal resolution. Coupling constants (e.g., J₂,₃ = 5.3 Hz, J₃,₄ = 9.4 Hz) confirmed olefinic proton positions on ring C, assigning 11-acetoxygraciline to (2). EI MS of 2 had a base peak at m/z 43 (acetyl cation) and m/z 225 (53%, [M - CH₃N(CH₃)OCOCH₃]⁺). Molecular ion (m/z 341, low abundance) was confirmed by CI MS (C₁₉H₁₉NO₅). Peaks at m/z 298 ([M - 43]⁺) and 281 ([M - 60]⁺) were also observed. CD similarity to 1 suggested 2 had 4aR and 10bR stereochemistry. NOESY (CDCl₃) indicated α-acetoxy orientation at C-11 (cross signals from H-10 (δ 7.04) to H-1 (δ 6.06) and H-11α (δ 5.42)) and spatial proximity of H-4 to N-methyl and H-6β (δ 4.61). The ¹H and ¹³C NMR spectra of (3) (isolated from G. gracilis, MeOH-d₄ due to polarity) showed 17 carbons with characteristic quaternary shifts (δ 48.3 and 97.7), confirming the 10b,4a-ethanoiminodibenzo[b,d]pyrane nucleus. Similarities to 1/2 included two aromatic protons, methylenedioxy, deshielded methylene, and N-methyl, but the aliphatic region (nine protons) was distinct, with unresolved signals suggesting conformational flexibility. Only two broadened olefinic doublets were present. 2D NMR (280 K) showed two geminal proton pairs (δ 3.83/3.40 and 2.35/2.44) forming a four-spin system. HMBC confirmed the ethanoimino bridge (C-4a/C-10b). NOESY (400 ms) showed H-10 (δ 6.96), H-11β (δ 2.44), and δ 6.35 (H-1) interactions, establishing conformation. ¹H,¹H DQF COSY assigned H-1 (δ 6.35) → H-2 (δ 6.02) → H-3 (δ 4.40) → H-4 (δ 2.30/2.24), with H-3’s deshielding suggesting a C-3 hydroxyl (IR 3370 cm⁻¹). NOESY indicated β-hydroxyl orientation at C-3 (cross signals H-4β (δ 2.30) to H-6β (δ 4.93), H-4α (δ 2.24) to N-methyl (δ 2.93)). Ring C adopted a quasi-chair conformation. EI MS showed m/z 301 (M⁺, 54%, C₁₇H₁₉NO₄) with fragments m/z 283 ([M - H₂O]⁺), 243 ([225 + H₂O]⁺), and 225, confirming the hydroxyl group was not on the nitrogen bridge. Compound (4) (-)-digracine (isolated from G. gracilis) had ¹H/¹³C NMR indicating two 10b,4a-ethanoiminodibenzo[b,d]pyrane moieties (duplicated key resonances: δ 44.2/46.8 for C-10b, 91.1/99.3 for C-4a; four olefinic carbons). ¹H NMR showed four aromatic protons, two methylenedioxy, two N-methyls, and two isolated high-frequency methylenes. ¹H,¹H DQF COSY and TOCSY sorted proton shifts for both moieties, confirming ethanoimino bridges (C-10b/C-4a) and ring C with one double bond and two low-frequency methines. HSQC assigned protonated carbons. HMBC was key, showing unambiguous ²JCH/³JCH correlations between the two moieties, confirming a dimer (not epimers). Diagnostic correlations included H-1 (δ 2.82) to C-1' (δ 44.2), C-2' (δ 134.2), C-10'b (δ 52.3); H-2 (δ 2.61) to C-4' (δ 45.0); H-3 (δ 5.69) to C-4' (δ 45.0); H-1' (δ 3.25) to C-1 (δ 41.2), C-10b (δ 46.8); H-4' (δ 2.80) to C-1 (δ 41.2). These confirmed dimerization via C-1:C-1' and C-2:C-4' bonds. NOESY (850 ms) showed inter-moiety spatial proximities (e.g., H-1' (δ 3.25) to H-2' (δ 5.89), H-10' (δ 6.89), H-12b (δ 2.67), H-1 (δ 2.82), H-10 (δ 7.01); H-1 (δ 2.82) to H-2 (δ 2.61), H-10 (δ 7.01), H-11'a (δ 2.12), H-12'a (δ 2.95), H-1' (δ 3.25)). Dreiding models supported the proposed structure (4). ESI MS showed m/z 567 (M+H⁺, C₃₄H₃₄N₂O₆) and m/z 284 (monomeric fragment, C₁₇H₁₇NO₃⁺). Compound (4) is levorotatory, with a CD curve having reversed maxima compared to 1/2. Named gracilines after (+)-graciline (1), this novel subgroup is found in two Galanthus species, suggesting commonality in Amaryllidaceae. A possible biogenetic pathway from (+)-6-hydroxycrinine is proposed (Scheme I).