Cyclopropanation of Some Alkaloids

New derivatives of natural compounds, galanthamine using in treatment of Alzheimer’s disease and antitumor dimer alkaloids vinblastine and vincristine were synthesized. In the course of the reaction between galanthamine and diazomethane in the presence of a catalyst, such as palladium(II) acetate or copper(I) bromide, methylene insertion into the aromatic ring was observed instead of the expected cyclopropanation of the carbon-carbon double bond. New vindoline derivatives conjugated with amino acid esters were prepared. In SimmonsSmith reaction vinblastine and vincristine cyclopropanated in the carbon carbon double bond in position 14 and 15 of the vindoline monomer were obtained. New dimer alkaloids showed significant inhibiting effects in several tumor cell lines.


Introduction
Among the alkaloids and alkaloid-like compounds there are many biologically active derivatives with useful effects on some diseases.By structural modification of that compounds we can hopefully have even more useful derivatives.Several possibilities can be investigated.We tried the effect of introduction of cyclopropane ring into some alkaloids or alkaloidlike compounds.
Many reports have described different synthetic routes to prepare galanthamine [2,[7][8][9][10][11][12][13][14], and with systematic transformations of its cyclic structure a number of derivatives were synthesized; e.g. by changing the position of the nitrogen atom in the azepine d ring [15], building in a sulfone group in ring d in place of the nitrogen atom [16], and substituting the furan b ring with pyrrole [17].Derivatives without a furan b ring [18], compounds containing a nitrogen heteroatom in the aromatic ring a [19] and galanthamine with an opened azepine d ring were also investigated [20].The last result in the chemistry of galanthamine was the synthesis of homogalanthamine (2) with a larger azepine ring [21].
A study of the literature data presented above led us to the concept of attempting to change the c ring by considering the carbon-carbon double bond as a target.The saturated derivative of galanthamine is known as lycoramine [3] and has a weaker biological effect, but the cyclopropano derivative (3) is unknown in the literature.
The cyclopropane subunit has always been considered as an electronically unique [22,23] structural element not only in the chemistry of artificial molecular structures [24] but in natural compounds as well [25][26][27].Synthetic methods for the cyclopropanation of carbon-carbon double bond were reviewed recently [28], the Simmons-Smith reaction [29,30], reaction with dimethyloxosulfonium methylide [31], and cyclopropanation with diazomethane [32] in the presence of a catalyst are frequently used.
In our first approach to the cyclopropanation reaction of galanthamine (1) we used the Simmons-Smith method by allowing 1 to react with diiodomethane [29] in the presence of diethylzinc at 0 o C or using trifluoroacetic acid [30] besides the aforementioned reagents at -15 o C.However, from these reactions only decomposition products were isolated.Secondly, a reaction between 1 and diazomethane -generated from Nmethyl-N-nitrosourea in dichloromethane solution in the presence of palladium(II) acetate [32] at 0 o C -resulted in a new derivative of galanthamine (Scheme 1).Cyclopropanation has not taken place but from the reaction mixture we could isolate a cycloheptatriene derivative (4) in 8% yield.Besides 4 a sideproduct with an open d ring (5) could also be detected.
The formation of compound 4 occurs with insertion of a methylene group into the aromatic ring.The reaction is known in the literature and was mainly investigated in relatively simple aromatics to prepare tropylium salts with different substituents [33][34][35][36].(The formation of compound 5 can be explained with an oxidative ring opening of 1; N-formylation reaction with diazomethane is known in the literature [37].) According to the literature [33] the Rh/C catalyst gives the best yields in these types of reaction and in some cases copper(I) halogenides, copper(I) chloride [34] and copper(I) bromide [35,36] were also effective.Using Rh/C catalyst no reaction was observed between 1 and diazomethane.Performing the reaction in the presence of copper(I) bromide, however, the cycloheptatriene derivative 4 was isolated in 13% yield.Replacing diazomethane with trimethylsilyldiazomethane [38] proved to be unsuccessful.Based on literature results [39] a possible mechanism is presented in Scheme 2. Carbene formed from diazomethane attaches to the aromatic a ring (1a) producing the cycloheptatriene derivative (4) via a norcaradiene-type intermediate (1b).
Formation of an aromatic methyl derivative (1c) in this reaction is also possible, but no traces of this product were detected.
In summary, an attempted cyclopropanation of the carboncarbon double bond in the c ring of galanthamine (1) with diazomethane resulted in a new derivative (4) with a cycloheptatriene structure in place of the aromatic a ring.Such a reaction with methylene insertion into an aromatic ring has not yet been described in the field of natural compounds.

Synthesis and in vitro antitumor effect of new vindoline derivatives coupled with amino acid esters [40]
The dimeric alkaloids vinblastine (6) and vincristine (7) (Scheme 3) were isolated from the Madagascar periwinkle Catharanthus roseus.Both materials have been used in anticancer chemotherapy for more than forty years [41,42].Vinblastine ( 6) is an antimicrotubule drug used to treat certain kinds of cancer, including Hodgkin's lymphoma, non-small cell lung cancer, breast cancer, head and neck cancer, and testicular cancer.Vincristine (7), the formyl derivative of vinblastine, can be used in various types of chemotherapy regimens.Its main uses are in non-Hodgkin's and Hodgkin's lymphoma, acute lymphoblastic leukemia, and in the treatment of nephroblastoma (Wilms tumor, a kidney tumor most common in young children).These dimeric alkaloids have two monomer alkaloid parts: vindoline (8) and catharanthine (9) (Scheme 3).Although the chemistry and pharmacology of vinblastine and vincristine are well known, and a number of their derivatives were synthesized to improve their therapeutic properties [43], however, vindoline and its derivatives were found to be biologically insignificant, Scheme 2 therefore there is much fewer data on their chemistry and biological effect in the literature [44].
Our research project was conceived on the basis of two interesting experiences in connection with dimeric alkaloids.
Firstly, some amino acid derivatives of vinblastine and vincristine were synthesized by coupling amino acid esters with the vindoline part in position 16 [45].The obtained conjugates exhibited significant antitumor effect against P388 and L1210 leukemia in mice.At the same time (D)-and (L)-tryptophan derivatives at the 16-position of desacetylvinblastine were conjugated through the carboxyl group with oligoarginine octapeptide as a carrier peptide at the N-terminus by Bánóczi et al. [46] One of the obtained stereoisomers showed a selective cytotoxic effect against the HL-60 human leukemia cells of higher proliferation rate.
Secondly, for dihydrovinblastine (i.e.vinblastine saturated in the 14,15-position of the vindoline part) a decreased antitumor activity and toxicity was observed, probably due to a different mechanism of action from vinblastine [47].These observations suggest that amino acid conjugation and reduction of the 14,15-position both alter the mechanism of action.Based on these observations our goal was to couple vindoline (8), 14,15-dihydrovindoline, and vindoline condensed with a cyclopropane ring in position 14,15, with (L)-and (D)-tryptophan methyl ester at position 16 with a view to screening their biological effect.The azide coupling method known from peptide chemistry was used as the key step in the coupling of vindoline with the amino group of the amino acid ester.
The first task was to protect position 10 of vindoline, because in the presence of sodium nitrite used in the preparation of the corresponding azide, a nitrosation reaction took place in position 10, resulting in 10-nitrosovindoline [44].In this procedure a bromo substituent was used to protect position 10 (Scheme 4).
Coupling the vindoline derivatives with tryptophan methyl esters was achieved by preparation of the azides (Scheme 6).The reaction of hydrazide 14a with sodium nitrite in methanol in the presence of hydrochloric acid resulted in azide 15a, which was then allowed to react without isolation with (L)tryptophan methyl ester to yield the vindoline conjugate 16a.
The influence of the different modifications of the ring system and/or the conjugation with tryptophan on the in vitro cytostasis of HL-60 human leukemia cells was analysed by using MTT assay [49] (Table 1).

Vinblastine and vincristine condensed with a cyclopropane ring [50]
In the course of the synthetic investigations of dimeric Vinca alkaloids various methods have been elaborated to synthesize new derivatives of vinblastine (6) and vincristine (7) with improved therapeutic effect exhibiting higher selectivity and lower toxicity [43,51].
One of the structural modifications was very simple and only a little part of the large molecule was changed; 14,15-dihydrovinblastine (17) having a saturated vindoline ring was simply prepared by catalytic hydrogenation of vinblastine (6) (Scheme 7), and the antitumor activity almost ceased [47].
The introduction of a cyclopropane ring in place of the C( 14)=C(15) carbon carbon double bond of the vindoline ring (18) can be regarded as a minor change within the overall molecular framework of vinblastine; however, the electronic nature of this small ring has entirely special properties.According to NMR and X-ray-crystallographyc studies, the electronic structure of the cyclopropane ring cannot be described in terms of classical methods [22,23].
Our enquiry was aimed at finding out how the introduction of this unique structural system into vinblastine and vincristine will influence their biological effects.To that end we Scheme 6 Table 1 Cytostatic activity of vindoline derivatives on HL-60 cells.set out to synthesize 14,15-cyclopropanovinblastine (18) and some derivatives thoseof.

Compound
A number of cyclopropanation reactions are known in the literature [28] such as the Simmon-Smith reaction [29,30] and its variants, e.g. that involving triisobutylaluminium [52] or diazomethane in the presence of a catalyst [32].
Therefore the coupling reaction traditionally used to obtain dimeric alkaloids was selected to synthesize the expected cyclopropanated derivatives.
Compound 27 proved to be suitable for the coupling reaction with catharanthine to give cyclopropano-vinblastine and cyclopropano-vincristine.
The next step in our synthetic work was to prepare the cyclopropanated anhydrovinblastine (28), which can be an important intermediate in the synthesis of further derivatives (Scheme 10).14,15-Cyclopropanovindoline was coupled with catharanthine (9) by the method of biomimetic iron(III)-promoted coupling reaction [59,60].The procedure used by us is the traditional coupling reaction for the preparation of dimeric alkaloids.
Catharanthine (9) and 14,15-cyclopropanovindoline (27) were dissolved in diluted aqueous hydrochloric acid solution together with the non-nucleophilic cosolvent 2,2,2-trifluoroethanol (Scheme 10).The reaction was carried out at room temperature.Sodium borohydride was used as the reducing agent in water at 0 o C. 14,15-Cyclopropanoanhydrovinblastine (28) was obtained in 51% yield and was isolated by preparative thin-layer chromatography.From 28 base the sulfate salt was prepared immediately.
The new cyclopropano derivative of vinblastine (29) was oxidized [61] to 14,15-cyclopropanovincristine (30) by chromium(VI)oxide in acetone-acetic acid solution in the presence of acetic anhydride at -60 o C. The crude product which contained some N-demethylvincristine was reformylated with formic acid/acetic anhydride.After isolation the product by preparative thin-layer chromatography, sulfate salt was prepared and the expected product (30) was obtained in 52% yield.
The new compounds synthesized by us were sent to the US National Institute of Health (NIH) where they were subjected to pharmacological investigations.These experiments on 56 different tumor cell lines embracing 9 frequently occurring tumor types demonstrated the therapeutic activity of these substances in comparison with other known effective catharanthus alkaloids.The NCI screening procedures were described [62] as were the origins and processing of the cell lines [62][63][64].In Table 2 percentages of growths are listed for the reference alkaloids and the appropriate cyclopropano derivatives at the concentration of 10 -5 M. The bigger negative numbers show more significant decrease of cell number or stronger inhibition effects.
According to Table 2 cyclopropano-vinblastine (29) has significant tumor cell inhibiting effect in leukemia, non-small-cell lung cancer, colon cancer, melanoma and breast cancer, cyclopropano-vincristine (30) has significant tumor cell inhibiting effect in colon cancer, melanoma, ovarian cancer and prostate cancer.