Concise total syntheses of lysergene, lysergine, isolysergine and festuclavine

A streamlined synthesis of the tetracyclic ergot alkaloid scaffold enabled the preparation of several clavine alkaloids, including lysergol, isolysergol, lysergine, isolysergine, lysergene, and festuclavine. The mesylation and subsquent elimination or reduction of lysergol and isolysergol building blocks complements and expands previous total syntheses of these natural products. In the course of the preparation of isolysergine, an interesting new cascade process was discovered that led to the formation of a cyclopropanated ergolin-2(3 H )- one as a spontaneous oxidation product of an intermediate 1,3-dihydrobenzo[ cd ]indole.


Introduction
The ergot alkaloid family continues to attract vivid interest from synthetic chemists for its structural diversity and complexity as well as for its broad range of physiological effects, often paired with exceptional potency at CNS receptors.Historically, ergot alkaloids have been associated with outbreaks of ergotism and mass hysteria in the middle ages (dancing plague, Salem witch trials) in the West, and, in the East, with lifestock poisonings (drunken horse disease), longevity, and spiritual transcendence. 1,2,3,4More recently, there is a surge of interest in the medical psychedelic properties of ergot alkaloids, in conjunction with their clinical success in the treatment of depression, anxiety, substance abuse, and obsessive-compulsive disorders. 5In October 2023, there were 71 active clinical trials with psilocybin, 25 with N,N-dimethyltryptamine (DMT) and 5-hydroxytryptophan (5-HTP), and 14 trials with lysergic acid diethylamide (LSD). 6he first total synthesis of lysergic acid and ergonovine by Woodward in 1954, 7 followed by numerous reports on the total syntheses of these and other ergot metabolites, 1,8,9 clearly established the feasibility of a nature-independent access to these alkaloids that could be used for analog preparation and biological testing.However, the medicinal chemistry field was slow to transfer these advances to medical breakthroughs in the development of psychedelic drugs with attractive pharmacokinetic (PK) and ADME/Tox profiles, undoubtedly in part due to major legal and societal hurdles after lysergic acid and its analogs were removed from lawful circulation in 1966.Furthermore, the accomplished total syntheses were frequently mainly of academic significance and lacked practical value due to their linear nature, number of steps, and limited scope and robustness.Synthetic advances in this field therefore remain highly significant.Clinical research and development of psychedelics is currently mainly limited to legacy compounds, i.e. nature-derived or structurally simple synthetic products from the public domain.In fact, the "paucity of structure-activity data for ergolines principally results from the synthetic difficulty attendant to chemical transformations of the ergolines". 10ased on a new approach to indole synthesis 11,12,13 and the application of novel strategic disconnections, 14,15 our group has developed a rapid access to ergot alkaloids, including the synthesis of unnatural stereoisomers, enabling iterative structure-activity relationship (SAR) investigations. 16Initially, both enantiomers of cycloclavine were prepared in eight steps with two sequential Diels-Alder reactions.Their CNS receptor profiles were compared to the three main agonist chemotypes at the serotonin (5-HT) receptor: tryptamines (and inverse tryptamines), ergolines related to LSD (essentially rigidified tryptamines), and phenethylamines (Figure 1), showcasing the strong stereoselectivity and a distinct structure-activity relationship of the cycloclavines. 16or example, this work demonstrated for the first time the increased selectivity for serotonin receptors (5-HTRs) of the clavine alkaloid subclass that lacks the carboxyl group that is typical for lysergic acid and LSD derivatives. 16 key aspect of our synthetic approach was the rapid access to the tetracyclic scaffolds of these natural products.For example, an unprecedented Rh-catalyzed asymmetric cyclopropanation of allene (1) with the pentafluoro diazoester 2 was used, followed by aminolysis with vinylogous amide 3 to generate the precursor 4 for a novel intramolecular Diels-Alder reaction of a methylene cyclopropane (IMDAMC) to yield tricyclic indolinone 5 (Scheme 1). 16  Overview of an 8-step asymmetric total synthesis of (+)-cycloclavine. 16r recent 5-step synthesis of lysergol and isolysergol enables the production of both natural products and synthetic analogs. 17This process takes advantage of a transition metal-free hydrogen autotransfer (HA) alkylation reaction with commercially available 10 and 4-bromoindole 9 to generate intermediate 11 (Scheme 2). 18Further optimization of this reaction led to a 79% yield on a 4-g reaction scale.After N-alkylation of 11 and reduction of the resulting pyridinium salt, 12 proved to be a valuable intermediate for scaffold variation. 17somerization of the alkene results in 13 that is subjected to an intramolecular Heck coupling to provide both methyl lysergate (14) and isolysergate (15) configurations at C (8). Reduction with LiAlH4 then generates lysergol 16 and isolysergol 17.The latter two compounds are valuable precursors for the synthesis of other ergot alkaloids 18 (vide infra).
A panel of 13 pertinent CNS GPCRs was profiled at 10 μM and 1 μM concentration for a comparison of the properties of cycloclavine (CCV), lysergol, and isolysergol enantiomeric pairs (Table 1). 16The cycloclavines were significantly more selective in this panel than D-LSD, the bioactive LSD stereoisomer, as well as psilocin, the active substance derived from the natural phosphate prodrug psilocybin that is currently being investigated clinically as a treatment for anxiety and depression in cancer care, as well as for enhancement of cognitive flexibility and creativity. 19(+)-Cycloclavine ((+)-CCV) proved considerably more potent at 5-HT1AR than (-)cycloclavine (EC50=0.14μM vs ca. 5 μM for (-)-cycloclavine ((-)-CCV); Table 1).Both CCV stereoisomers are poor activators at 5-HT2A, suggesting that hallucinogenic or strongly euphoric effects in humans might be significantly reduced in comparison to D-LSD.(+)-CCV also displays a potent activation (EC50=16 nM) at 5-HT2C, a receptor that is thought to contribute to the desirable mental effects of psychedelics.None of these agents activated 5-HT2B, a subtype that has been associated with cardiotoxicity.Overall, the 5-HTR profile of (+)-CCV mirrors that of psilocin with the exception of agonist effects at 5-HT2A and is substantially different from the strongly hallucinogenic D-LSD, which bodes well for future therapeutic investigations of the clavine compound class.Substitution at the indole ring, or stereochemical variations in the scaffold of the clavine alkaloid (+)-lysergol can further enhance the selectivity among 5-HT receptors compared to D-LSD.Strong stereochemical preferences are also apparent in the receptor profile of the (iso)lysergols (Table 1), and the data confirm that further structural modifications to decrease 5-HT2C binding and increase 5-HT1A affinity are feasible.These and other structure-activity relationships are currently being investigated, and the potential utility of these compounds in an exploratory disease model will be reported in due course.Scheme 2. Overview of a 5-step total synthesis of lysergols that serves as a segue to the preparation of other clavine alkaloids 18. 17

Results and Discussion
The efficient synthesis of ergolines 14-17 allowed for the possibility to extend the preparation of lysergols to other clavine alkaloids with the general structure 18.Specifically, mesylation of lysergol 16 provided 19 in quantitative yield, and subsequent elimination with KO-t-Bu led to lysergene (20) in 61% yield (Scheme 3).The mesylate 19 could also be reduced with LiAlH4 in THF at reflux to furnish lysergine (21) in 56% yield.This alkaloid was converted to festuclavine (22) by treatment with Pd/C and H2 gas, selectively installing the favored transring junction in 93% yield.
Previous preparations of lysergene, 20 lysergine 21 and festuclavine 22 used different precursors and reaction protocols, and access to these ergot alkaloids was mainly accomplished by means of formal syntheses from more readily available, isolated natural products.Lysergene, f. ex., has been prepared by a dehydration of the tertiary alcohol in setoclavine 23 and by a Wittig methylenation of a lysergic acid derivative. 24The reduction of agroclavine with sodium metal and n-butanol yielded pyroclavine, costaclavine, festuclavine, and lysergine. 25,26estuclavine was also obtained from chanoclavine-I aldehyde, 27 and through total syntheses using nitroaldol, 28,29 Larock indole, 30 and Giese coupling 31 approaches as key steps.Finally, Vollhardt et al. 32 and Ninomiya et al. used, respectively, a CpCo(CO)2 catalyzed cocyclization of two alkynes and a nitrile, and reductive enamide photocyclization to prepare racemic lysergene.
Analogous to the mesylation of 16, mesylation of isolysergol (17) 17 proceeded smoothly before workup and concentration of the reaction mixture; however, the resulting mesylate 23 was surprisingly unstable compared to the epimeric 19, complicating its purification and characterization (Scheme 4).Without purification, the mesylate was therefore subjected to nucleophilic displacement with LiAlH4 in THF at reflux, and the desired clavine alkaloid isolysergine (24) was isolated in 32% overall yield from 17. Interestingly, the reaction mixture also contained the novel benzo[cd]indol-2(1H)-one 25, which was isolated in 12% yield based on 17.A 1 H, 1 H-NOESY experiment was critical to support the structural assignment of 25.Namely, there were through-space correlations between the characteristic aromatic C(4) singlet and the N-Me group as well as between the C(12) proton and the cyclopropane methine hydrogens at C (9).The cyclopropane hydrogen were slightly shifted Page 7 of 13 © AUTHOR(S) downfield in the 1 H NMR, likely due to the conjugation to the benzo[cd]indol-2(1H)-one.The oxindole carbonyl carbon typically appears at ca. 171±2 ppm in the 13 C NMR, and product 25 also showed this characteristic carbon resonance at 170.2 ppm.
Mechanistically, the formation of ergolin-2(3H)-one 25 could be rationalized by a deprotonation of indole 23 with LiAlH4, followed by a vinylogous intramolecular displacement of the pendant pseudo-axial mesylate in 26 to provide 28 (Scheme 4). 14,33Upon workup and exposure to air, 34 the 1,3-dihydrobenzo[cd]indole is expected to be oxidized to the oxindole 25.Structurally related oxindole products have previously been observed as spontaneous air oxidation products and are also obtained from LSD and other lysergates through metabolic or chemical oxidation. 32,35In addition to the interesting structural similarity of the cyclopropanecontaining scaffold of 25 to cycloclavine and the general relevance of the oxidative conversion of the putative intermediate 28 to 25 for the chemistry of ergot alkaloids, substituted benzo[cd]indol-2(1H)-ones have also emerged as potent pharmacological agents, f. ex. in the discovery of BET bromodomain inhibitors, and could therefore be of broader medical significance. 36Furthermore, the indolo[4,3-fg]quinolin-5(4H)-one scaffold of 25 has been found in isolates from the medicinal fungus Xylaria nigripes (KL.)SACC. 37imilarly to the prior synthetic work toward festuclavine and lysergine, isolysergine 21 has previously been prepared from agroclavine by treatment with Na in n-BuOH, which also yielded pyroclavine and costaclavine. 26he latter work also explored the use of methyl lysergate as a source of lysergene, lysergine and isolysergine by LiAlH4 reduction, treatment with sodium butoxide, and catalytic hydrogenation.Finally, Ninomiya et al. used a reductive enamide photocyclization to prepare racemic lysergene and isolysergine, as well as agroclavine and fumigaclavine B through multi-step procedures. 38In comparison, our routes to lysergine, isolysergine, lysergene, and festuclavine from lysergol and isolysergol are considerably more concise due to the rapid preparation of the precursor alkaloids from readily available starting materials by the hydrogen autotransfer alkylation of 4bromoindole.

Conclusions
Ergot alkaloids are of high interest for their historical significance, structural complexity and inspiration for organic synthesis strategic and tactical development studies.Their therapeutic potential for the treatment of mental health challenges as well as cancer co-morbidities is under active re-investigation in many biotech companies.We were able to leverage our recent streamlined synthesis of all four stereoisomers of lysergol and isolysergol to establish new approaches to the ergot alkaloids lysergene, lysergine, isolysergine and festuclavine.This pathway complements and expands previous total syntheses of these natural products.In the course of the preparation of isolysergine, we also discovered an interesting new cascade process leading to formation of a cyclopropanated ergolin-2(3H)-one as a spontaneous oxidation product of an intermediate 1,3dihydrobenzo[cd]indole that was formed in an intramolecular SN2-reaction.After resolution of these compounds, we plan to characterize the pharmacological properties of the resulting ten products in comparison to the cycloclavine and lysergol stereoisomers, and further develop a structure-activity relationship that might support the development of non-hallucinogenic ergot alkaloid analogs as alternatives to psilocin and LSD.

Experimental Section
General.Unless stated otherwise, all reactions were performed under an atmosphere of N2 that was passed through a column (10 × 2 cm) of Drierite®.Prior to use, THF was freshly distilled over sodium/benzophenone, and CH2Cl2 was freshly distilled over CaH2.Et3N and i-PrNEt2 were distilled over CaH2 and stored over KOH.All glassware and stir bars were dried in an oven for 3 h prior to use.When necessary, degassed solvents were prepared by sparging with N2 for 1 h.Reactions were monitored by TLC analysis (pre-coated silica gel 60 F254) and spots were visualized (UV lamp 254 nm and 395 nm).C NMR) used as the internal standard.Chemical shifts were tabulated as follows: chemical shift, multiplicity (s singlet, d doublet, t triplet, q quartet, dd doublet of doublet, dt doublet of triplet, td triplet of doublets, dq doublet of quartets, ddd doublet of doublet of doublet, m multiplet, br s broad singlet), coupling constant(s), and integration.IR spectra were obtained using neat samples on a PerkinElmer 100 IR-ATR spectrometer.Melting points were obtained using a Mel-Temp instrument and are uncorrected.The spectral information of all natural products was checked against the literature references in the isolation papers, and was consistent with this information.