# Structure and Synthesis of Vindolicine and Derivatives

Yousuf Sammer, Laure Vendier, Georges Massiot

► To cite this version:

Yousuf Sammer, Laure Vendier, Georges Massiot. Structure and Synthesis of Vindolicine and Derivatives. Chemistry and Biodiversity, 2024, 21 (4), pp.e202301928. 10.1002/cbdv.202301928. hal-04479357

HAL Id: hal-04479357

https://hal.univ-reims.fr/hal-04479357

Submitted on 27 Feb 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Structure and Synthesis of Vindolicine and Derivatives

Yousuf Sammer, Laure Vendier, Georges Massiot

► To cite this version:

Yousuf Sammer, Laure Vendier, Georges Massiot. Structure and Synthesis of Vindolicine and Derivatives. Chemistry and Biodiversity, 2024, 21 (4), pp.e202301928. 10.1002/cbdv.202301928. hal-04479357

HAL Id: hal-04479357

https://univ-reims.hal.science/hal-04479357v1

Submitted on 27 Feb 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

# Structure and Synthesis of Vindolicine and Derivatives

Asia,[a] Yousuf Sammer,[a] Laure Vendier,[b] and Georges Massiot*[c]

This article describes the reaction of vindoline with formaldehyde and trimethyl orthoformate to prepare vindoline, tris-vindolicinyl methane and higher molecular weight homologues. The synthesis of 10-formyl vindoline as an intermediate allowed further exploration of its chemistry, in particular the reaction with acetone which yielded a symmetrical dimer, which was further reacted with vindoline to give molecules containing three and four vindoline units. These

molecules were characterized by NMR and for some of them (vindolicine, 10-formyl vindoline, 10-(1'-(but-1'-en-3'-one))-vindoline) by X-ray crystallography. Depending on the substitution and on the absence of axes of symmetry, the NMR spectra displayed non-equivalent spin systems for the vindoline moieties. The dimer formed from the double condensation of 10-formyl vindoline with acetone showed cytotoxic activity in the micromolar range.

## Introduction

Vindolicine (1) is a very special compound among the many alkaloids from the periwinkle, *Catharanthus roseus*. It was first considered as a monomer, related to vindoline (2),[1] then a bisindole structure was proposed at a symposium in Kyoto,[2] but never published in a regular journal until 1976, where it appeared in an article dealing with ¹³C NMR of alkaloids.[3] In the meantime, the compound was isolated from *Catharanthus longifolius* and *ovalis*.[4] The ¹³C NMR spectrum of vindolicine was again published in 1983, with a double set of signals corresponding to each of the moieties.[5] As noticed in a patent, vindolicine has long been the indole alkaloid with the highest molecular weight.[6] It consists of two vindoline units linked by a methylene between the C-10s and thus possesses a symmetry axis. Consequently, the ¹H and ¹³C NMR spectra are simplified and show a single set of signals for the two vindoline parts and a singlet for the methylene. The "double set" assignment seems odd and may be due to spectrometer instability or to the sample not being vindolicine. Unlike other dimeric indole alkaloids from *C. roseus*, vindolicine is not cytotoxic but it was recently demonstrated to possess antidiabetic properties.[7]

Vindoline (2), the monomer constituent of vindolicine is, by far, the most abundant alkaloid of *C. roseus* and is isolated on

an industrial scale along with catharanthine, as part of the synthesis of the drug navelbine and of related compounds.[8] Since vindoline is in excess compared to the accompanying catharanthine, it is readily available for further experimentation. The most frequently used sequences of derivatization take advantage of nucleophilicity of vindoline with electrophiles.[9] To the best of our knowledge, the only alternative to nucleophilic addition is the addition of radicals to vindoline developed by Boger et al.[10] Among single electrophiles, we reported additions of vindoline to benzoquinone[8] and as a follow-up of this approach, we wish to describe here the reaction of vindoline with formaldehyde and trimethyl orthoformate and hence, the synthesis of vindolicine and of related compounds.

## Results and Discussion

### Synthesis of vindolicine

Even though the biosynthetic pathway for vindoline was long ago deciphered in great details,[11] nothing is known on the biosynthesis of vindolicine. The hypothesis of its formation during methylene chloride extraction is ruled out since the molecule is highly stable in this solvent.[12] From a biosynthetic standpoint, formaldehyde, produced through the serine pathway, is the best candidate for the supernumerary carbon atom introduction and it was found to be the reagent of choice to perform the one-carbon atom homologation of vindoline and "dimerization". Solid paraformaldehyde, beforehand depolymerized in acidic water, was preferred over formalin to avoid the presence of methanol. The reaction was conducted in acidic water, at room temperature, and led to the formation of (1). Long reaction times and higher temperatures led to the concomitant formation of high molecular weight oligomers of vindoline and formaldehyde, such as, for example, "trimer" (3) and "tetramer" (4) (Scheme 1). Vindolicine was purified by medium pressure liquid chromatography and obtained in a crystalline state.

[a] Asia, Y. Sammer
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Sindh, Pakistan

[b] L. Vendier
UPR CNRS 8241, Laboratoire de Chimie de Coordination, 205 Route de Narbonne, 31077 Toulouse Cedex 04, France

[c] G. Massiot
Institut de Chimie Moléculaire de Reims, UMR CNRS 7312, Université Reims-Champagne-Ardenne, UFR Sciences, BP 1039, CEDEX 2, 51687 Reims, France
E-mail: georges.massiot@univ-reims.fr

Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbdv.202301928

© 2024 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AG.
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

# Structure and Synthesis of Vindolicine and Derivatives

Asia,[a] Yousuf Sammer,[a] Laure Vendier,[b] and Georges Massiot*[c]

This article describes the reaction of vindoline with formaldehyde and trimethyl orthoformate to prepare vindoline, tris-vindolicinyl methane and higher molecular weight homologues. The synthesis of 10-formyl vindoline as an intermediate allowed further exploration of its chemistry, in particular the reaction with acetone which yielded a symmetrical dimer, which was further reacted with vindoline to give molecules containing three and four vindoline units. These

molecules were characterized by NMR and for some of them (vindolicine, 10-formyl vindoline, 10-(1'-(but-1'-en-3'-one))-vindoline) by X-ray crystallography. Depending on the substitution and on the absence of axes of symmetry, the NMR spectra displayed non-equivalent spin systems for the vindoline moieties. The dimer formed from the double condensation of 10-formyl vindoline with acetone showed cytotoxic activity in the micromolar range.

## Introduction

Vindolicine (1) is a very special compound among the many alkaloids from the periwinkle, *Catharanthus roseus*. It was first considered as a monomer, related to vindoline (2),[1] then a bisindole structure was proposed at a symposium in Kyoto,[2] but never published in a regular journal until 1976, where it appeared in an article dealing with ¹³C NMR of alkaloids.[3] In the meantime, the compound was isolated from *Catharanthus longifolius* and *ovalis*.[4] The ¹³C NMR spectrum of vindolicine was again published in 1983, with a double set of signals corresponding to each of the moieties.[5] As noticed in a patent, vindolicine has long been the indole alkaloid with the highest molecular weight.[6] It consists of two vindoline units linked by a methylene between the C-10s and thus possesses a symmetry axis. Consequently, the ¹H and ¹³C NMR spectra are simplified and show a single set of signals for the two vindoline parts and a singlet for the methylene. The "double set" assignment seems odd and may be due to spectrometer instability or to the sample not being vindolicine. Unlike other dimeric indole alkaloids from *C. roseus*, vindolicine is not cytotoxic but it was recently demonstrated to possess antidiabetic properties.[7]

Vindoline (2), the monomer constituent of vindolicine is, by far, the most abundant alkaloid of *C. roseus* and is isolated on

an industrial scale along with catharanthine, as part of the synthesis of the drug navelbine and of related compounds.[8] Since vindoline is in excess compared to the accompanying catharanthine, it is readily available for further experimentation. The most frequently used sequences of derivatization take advantage of nucleophilicity of vindoline with electrophiles.[9] To the best of our knowledge, the only alternative to nucleophilic addition is the addition of radicals to vindoline developed by Boger et al.[10] Among single electrophiles, we reported additions of vindoline to benzoquinone[8] and as a follow-up of this approach, we wish to describe here the reaction of vindoline with formaldehyde and trimethyl orthoformate and hence, the synthesis of vindolicine and of related compounds.

## Results and Discussion

### Synthesis of vindolicine

Even though the biosynthetic pathway for vindoline was long ago deciphered in great details,[11] nothing is known on the biosynthesis of vindolicine. The hypothesis of its formation during methylene chloride extraction is ruled out since the molecule is highly stable in this solvent.[12] From a biosynthetic standpoint, formaldehyde, produced through the serine pathway, is the best candidate for the supernumerary carbon atom introduction and it was found to be the reagent of choice to perform the one-carbon atom homologation of vindoline and "dimerization". Solid paraformaldehyde, beforehand depolymerized in acidic water, was preferred over formalin to avoid the presence of methanol. The reaction was conducted in acidic water, at room temperature, and led to the formation of (1). Long reaction times and higher temperatures led to the concomitant formation of high molecular weight oligomers of vindoline and formaldehyde, such as, for example, "trimer" (3) and "tetramer" (4) (Scheme 1). Vindolicine was purified by medium pressure liquid chromatography and obtained in a crystalline state.

[a] Asia, Y. Sammer
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Sindh, Pakistan

[b] L. Vendier
UPR CNRS 8241, Laboratoire de Chimie de Coordination, 205 Route de Narbonne, 31077 Toulouse Cedex 04, France

[c] G. Massiot
Institut de Chimie Moléculaire de Reims, UMR CNRS 7312, Université Reims-Champagne-Ardenne, UFR Sciences, BP 1039, CEDEX 2, 51687 Reims, France
E-mail: georges.massiot@univ-reims.fr

Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbdv.202301928

© 2024 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AG.
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
<figure><img src="image_4.png" /><figcaption>**Scheme 1.** Synthesis of vindolicine (1) and of oligomers.</figcaption></figure>

The NMR spectra of vindolicine showed a single set of signals for the vindolines and a singlet at δ 3.70 for the supplementary methylene (δ ¹³C 30.2). An X-ray crystal structure was obtained, and it showed the integrity of the two vindoline moieties (Figure 1). In the crystal, the aromatic rings are not coplanar, their dihedral angle is ca 115° and the distance between the two methyl groups of the ethyl side chains is 3 Å. This short distance explains the shielding of these protons in the NMR spectrum (δ 0.33 ppm). The most unexpected information obtained from the NMR experiments is the observation of an NOE between the aromatic H-9 and the methyl triplet of CH₃-18. The distance between these atoms is far too long for an intra-vindoline effect and this corresponds to an NOE between H-9 of one of the vindolines and CH₃-18 of the other.

Structure of oligomers 3 and 4

The second more polar compound from the condensation of formaldehyde with vindoline contained three vindoline units as shown by the ¹H-NMR spectrum, which displayed distinct sets of signals for each of the sub-units. The aromatic part of the spectrum consisted of five singlets, three H-9s (δ 6.72, 6.59, 6.48) and two H-12s (δ 6.07, 6.05), according to the HSQC, indicating that one of the vindolines in vindolicine was substituted by another 10-methylene-vindoline. The third substitution by a methylene-vindoline had thus occurred on one of the C-12, thus establishing the structure of (3) (Figure 2).

<figure><img src="image_5.png" /><figcaption>**Figure 1.** ORTEP representation of vindolicine.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Structure of trimer (3).</figcaption></figure>

For sake of simplicity, compound (3) may be designated as V₁₀CH₂₁₀·V₁₂ CH₂₁₀V with V standing for vindoline. Vindoline "trimers" are uncommon and the only examples present in the literature are the "natural" trimer described in ref [6] and of unsolved structure, and the trimers produced by Fe-III oxidation of vindoline.[13a] Although possible with ultra-high field spectrometers,[14] complete NMR assignment of (3), where each signal appears as a triad, is beyond the scope of this article and only tentative assignments are proposed. The starting point for the analysis is the assignment of the aromatic protons with the observation of a long-range coupling for the sole pair of singlets at δ 6.48 and 6.07, assigned to either H-9 and -12 or to H-9" and -12". The other two H-9s showed a ROE interaction compatible only with the H-9 and H-9' of structure (3) represented below. The COSY and NOESY information allowed to unequivocally assign the aromatic protons as: H-9 (6.72), H-9' (6.59), H-9" (6.48), H-12 (6.07) and H-12" (6.05). From there on, the H-12 allowed to recognize the N-CH₃ and therefrom the H-2s and the rest of the vindolines.

The most polar compound (4) was assigned a tetrameric structure based on its mass spectrum. It might be assumed that compound (4) corresponds to a double elongation of vindoline or to addition of formaldehyde and vindoline to compound (3). Two structural possibilities are left corresponding to additions on the remaining reactive centers C-12s. In the above notation, (4) is either: V-₁₀ CH₂₁₀·V₁₂ CH₂₁₀·V₁₂ CH₂₁₀ V or V₁₀ CH₂₁₂ V₁₀ CH₂₁₀·V₁₂ CH₂₁₀·V, the latter formula being characterized by a symmetry center. The ¹H-NMR spectrum of (4) showed signals for two sets of vindolines, corresponding to a symmetrical molecule with some of them, but not all, assignable to "inner" and "outer" vindolines. Two singlets were observed for the four H-9 and those belonging to the outer vindolines H-9s (δ 6.43) were distinguished by the coupling with H-12s (δ 6.06). The ROESY experiment allowed to assign the "outer" aromatic OMe to the most deshielded signals (δ 3.85) while the inner OMe appeared as shielded in ¹H-NMR (δ 3.44) and deshielded in ¹³C NMR (δ 61.2). The first effect is due to the magnetic anisotropy linked to the neighboring aromatic ring, while the second one is due to steric encumbrance. Compound (4) is thus (V₁₀ CH₂₁₂ V-₁₀)₂CH₂ (Figure 3). The formation of (3) and (4) demonstrates that although electrophilic addition to C-10 is preferred, addition to C-12 is possible when the C-10 position is occupied. We never observed additions to C-9, for stereo-electronic reasons and because of
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Scheme 1. Synthesis of vindolicine (1) and of oligomers.</figcaption></figure>

The NMR spectra of vindolicine showed a single set of signals for the vindolines and a singlet at δ 3.70 for the supplementary methylene (δ ¹³C 30.2). An X-ray crystal structure was obtained, and it showed the integrity of the two vindoline moieties (Figure 1). In the crystal, the aromatic rings are not coplanar, their dihedral angle is ca 115° and the distance between the two methyl groups of the ethyl side chains is 3 Å. This short distance explains the shielding of these protons in the NMR spectrum (δ 0.33 ppm). The most unexpected information obtained from the NMR experiments is the observation of an NOE between the aromatic H-9 and the methyl triplet of CH₃-18. The distance between these atoms is far too long for an intra-vindoline effect and this corresponds to an NOE between H-9 of one of the vindolines and CH₃-18 of the other.

Structure of oligomers 3 and 4

The second more polar compound from the condensation of formaldehyde with vindoline contained three vindoline units as shown by the ¹H-NMR spectrum, which displayed distinct sets of signals for each of the sub-units. The aromatic part of the spectrum consisted of five singlets, three H-9s (δ 6.72, 6.59, 6.48) and two H-12s (δ 6.07, 6.05), according to the HSQC, indicating that one of the vindolines in vindolicine was substituted by another 10-methylene-vindoline. The third substitution by a methylene-vindoline had thus occurred on one of the C-12, thus establishing the structure of (3) (Figure 2).

<figure><img src="image_6.png" /><figcaption>Figure 1. ORTEP representation of vindolicine.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Figure 2. Structure of trimer (3).</figcaption></figure>

For sake of simplicity, compound (3) may be designated as V₁₀CH₂₁₀·V₁₂ CH₂₁₀V with V standing for vindoline. Vindoline "trimers" are uncommon and the only examples present in the literature are the "natural" trimer described in ref [6] and of unsolved structure, and the trimers produced by Fe-III oxidation of vindoline. [13a] Although possible with ultra-high field spectrometers,[14] complete NMR assignment of (3), where each signal appears as a triad, is beyond the scope of this article and only tentative assignments are proposed. The starting point for the analysis is the assignment of the aromatic protons with the observation of a long-range coupling for the sole pair of singlets at δ 6.48 and 6.07, assigned to either H-9 and -12 or to H-9" and -12". The other two H-9s showed a ROE interaction compatible only with the H-9 and H-9' of structure (3) represented below. The COSY and NOESY information allowed to unequivocally assign the aromatic protons as: H-9 (6.72), H-9' (6.59), H-9" (6.48), H-12 (6.07) and H-12" (6.05). From there on, the H-12 allowed to recognize the N-CH₃ and therefrom the H-2s and the rest of the vindolines.

The most polar compound (4) was assigned a tetrameric structure based on its mass spectrum. It might be assumed that compound (4) corresponds to a double elongation of vindoline or to addition of formaldehyde and vindoline to compound (3). Two structural possibilities are left corresponding to additions on the remaining reactive centers C-12s. In the above notation, (4) is either: V-₁₀ CH₂₁₀·V₁₂ CH₂₁₀·V₁₂ CH₂₁₀ V or V₁₀ CH₂₁₂ V₁₀ CH₂₁₀·V₁₂ CH₂₁₀·V, the latter formula being characterized by a symmetry center. The ¹H-NMR spectrum of (4) showed signals for two sets of vindolines, corresponding to a symmetrical molecule with some of them, but not all, assignable to "inner" and "outer" vindolines. Two singlets were observed for the four H-9 and those belonging to the outer vindolines H-9s (δ 6.43) were distinguished by the coupling with H-12s (δ 6.06). The ROESY experiment allowed to assign the "outer" aromatic OMe to the most deshielded signals (δ 3.85) while the inner OMe appeared as shielded in ¹H-NMR (δ 3.44) and deshielded in ¹³C NMR (δ 61.2). The first effect is due to the magnetic anisotropy linked to the neighboring aromatic ring, while the second one is due to steric encumbrance. Compound (4) is thus (V₁₀ CH₂₁₂ V-₁₀)₂CH₂ (Figure 3). The formation of (3) and (4) demonstrates that although electrophilic addition to C-10 is preferred, addition to C-12 is possible when the C-10 position is occupied. We never observed additions to C-9, for stereo-electronic reasons and because of
<figure><img src="image_7.png" /><figcaption>Figure 3. Structure of tetramer (4).</figcaption></figure>

the presence of a near-by encumbered quaternary carbon atom.

Reaction mechanism

It is likely that 10-hydroxymethyl vindoline (5) is an intermedi-
ate in the sequence. Despite numerous attempts, it could not
be isolated from the direct reaction of vindoline with
formaldehyde and when an excess of this reagent was used, an
intractable mixture was obtained. The primary alcohol was
however prepared by an indirect route through reduction of
aldehyde (6). Compound (6), 10-formyl vindoline, has been
obtained on attempted dichlorocarbene addition on vindoline
and by Palladium catalyzed formylation of vindoline.[13] We
found it convenient to prepare it by formylation of vindoline
with a threefold excess of trimethyl orthoformate under acidic
conditions (Scheme 2). As expected, (6) shows an extended

<figure><img src="image_8.png" /><figcaption>Scheme 2. Synthesis of intermediate 10-hydroxymethyl-vindoline (5).</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Figure 4. X-ray crystal structures of (5) and (6).</figcaption></figure>

chromophore with λ<sub>max</sub> at 348 nm. Compounds (5) and (6) were
obtained in a crystalline state and their structures were
confirmed by X-ray crystallography (Figure 4).

The hypothesis of the intermediacy of 5 in the formation of vindolicine was tested by mixing equimolecular amounts of 5 and of vindoline in the presence of TFA. The ¹H-NMR spectrum of the crude mixture showed the exclusive formation of vindolicine with some remaining vindoline in an approximate 7 to 3 ratio. Pure vindolicine was isolated from this mixture in 50% yield. The non-quantitative yield of this reaction may be explained by a leak of formaldehyde due to the reversibility of the formylation step (Scheme 3). To support this hypothesis, 5 alone was treated with TFA and vindolicine and vindoline were formed.

Synthesis of tris-vindolinyl-methane (9)

The preparation of 10-formyl vindoline (6) led us to consider
trimethyl orthoformate as a template in the construction of tris-
vindolyl-methane. When used in excess over vindoline, this
reagent led to ketal (7), which yielded aldehyde (6) after
aqueous work-up. Under these conditions, no products of high
molecular weight were formed, but when a 3.5 to 1 ratio of
vindoline to trimethyl orthoformate was used, a bis adduct (8)
and a tri-adduct (9) were formed (Scheme 4). Their structures
were determined by NMR and mass spectrometry. Compound
(9) was the expected product of the triple substitution and it
showed a molecular ion at m/z 1379 in the positive ESI mode,
i.e. three vindolinyl units plus a methine. The molecule
possesses a three-fold axis of symmetry and therefore a single
set of signals is observed for the three vindolines. The triplet of
CH3-18 is the most shielded resonance at δ⊥0.25 and as in
vindolicine, it shows NOEs with the aromatic protons H-9 and
H-12 of adjacent sub-units. The methine appears as a sharp
singlet at δH 6.14, δC 37.3, compared to δH 3.71, δC 30.2 in

<figure><img src="image_10.png" /><figcaption>Scheme 3. Reversibility of the formation of (5) from (2).</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Scheme 4. Synthesis of tris-vindolinyl-methane (9).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 3. Structure of tetramer (4).</figcaption></figure>

the presence of a near-by encumbered quaternary carbon atom.

Reaction mechanism

It is likely that 10-hydroxymethyl vindoline (5) is an intermedi-
ate in the sequence. Despite numerous attempts, it could not
be isolated from the direct reaction of vindoline with
formaldehyde and when an excess of this reagent was used, an
intractable mixture was obtained. The primary alcohol was
however prepared by an indirect route through reduction of
aldehyde (6). Compound (6), 10-formyl vindoline, has been
obtained on attempted dichlorocarbene addition on vindoline
and by Palladium catalyzed formylation of vindoline.[13] We
found it convenient to prepare it by formylation of vindoline
with a threefold excess of trimethyl orthoformate under acidic
conditions (Scheme 2). As expected, (6) shows an extended

<figure><img src="image_9.png" /><figcaption>Scheme 2. Synthesis of intermediate 10-hydroxymethyl-vindoline (5).</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Figure 4. X-ray crystal structures of (5) and (6).</figcaption></figure>

chromophore with λ<sub>max</sub> at 348 nm. Compounds (5) and (6) were
obtained in a crystalline state and their structures were
confirmed by X-ray crystallography (Figure 4).

The hypothesis of the intermediacy of 5 in the formation of vindolicine was tested by mixing equimolecular amounts of 5 and of vindoline in the presence of TFA. The ¹H-NMR spectrum of the crude mixture showed the exclusive formation of vindolicine with some remaining vindoline in an approximate 7 to 3 ratio. Pure vindolicine was isolated from this mixture in 50% yield. The non-quantitative yield of this reaction may be explained by a leak of formaldehyde due to the reversibility of the formylation step (Scheme 3). To support this hypothesis, 5 alone was treated with TFA and vindolicine and vindoline were formed.

Synthesis of tris-vindolinyl-methane (9)

The preparation of 10-formyl vindoline (6) led us to consider
trimethyl orthoformate as a template in the construction of tris-
vindolyl-methane. When used in excess over vindoline, this
reagent led to ketal (7), which yielded aldehyde (6) after
aqueous work-up. Under these conditions, no products of high
molecular weight were formed, but when a 3.5 to 1 ratio of
vindoline to trimethyl orthoformate was used, a bis adduct (8)
and a tri-adduct (9) were formed (Scheme 4). Their structures
were determined by NMR and mass spectrometry. Compound
(9) was the expected product of the triple substitution and it
showed a molecular ion at m/z 1379 in the positive ESI mode,
i.e. three vindolinyl units plus a methine. The molecule
possesses a three-fold axis of symmetry and therefore a single
set of signals is observed for the three vindolines. The triplet of
CH3-18 is the most shielded resonance at δ⊥0.25 and as in
vindolicine, it shows NOEs with the aromatic protons H-9 and
H-12 of adjacent sub-units. The methine appears as a sharp
singlet at δH 6.14, δC 37.3, compared to δH 3.71, δC 30.2 in

<figure><img src="image_11.png" /><figcaption>Scheme 3. Reversibility of the formation of (5) from (2).</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>Scheme 4. Synthesis of tris-vindolinyl-methane (9).</figcaption></figure>
vindolicine. The second product (8) is the result of incomplete substitution and NMR and MS allow its identification as 10'-OMe vindolicine. The introduction of a methoxy group on the methylene bridge destroys the symmetry present in vindolicine, and the resonances of the two moieties become non-equivalent.

## Reactions of 10'-formyl vindoline with acetone

10'-Formyl vindoline (6) was reacted with acetone in the presence of a base to give butenone derivative (10) as the main product (Scheme 5). The new double bond was *trans* as shown by the large coupling constant ($J=16.2$ Hz) and its presence induced a downfield shift for H-9 (δ 7.47). No other damage occurred to the molecule, the structure of which was safely established by X-ray crystallography. Deprotonation of (10) with NaH and addition of a second molecule of 10'-formyl vindoline yielded the symmetrical bis adduct (11). Compounds (10) and (11) showed extended UV chromophores with $\lambda_{max}$ at 473 nm for (11) and 393 nm for (10). Owing to electron deficiency on the side chains, they were prone to further addition of vindoline and (10) gave a bis-vindolinyl adduct (12), and (11) gave a tris vindolinyl adduct (13) and tetra vindolinyl adduct (14). The formation of these adducts does not give rise to the creation of new stereogenic center and therefore these compounds are unique.

<figure><img src="image_12.png" /><figcaption>**Scheme 5.** Synthesis of acetone adduct (10–14) and ORTEP of (10).</figcaption></figure>

## Synthesis of vindolicinone

With vindolicine at hand, it was tempting to explore its chemistry and, in particular, the oxidation of the methylene bridge, which would open a route to olefins substituted by vindolines. The methylene bridge was easily oxidized by DDQ in the presence of water under Yonemitsu conditions (Scheme 6). Ketone 15, named here vindolicinone, was obtained as a pale-yellow solid in ca 60% yield. It showed an extended chromophore with $\lambda_{max}$ 370 nm.

## Symmetry considerations

At the onset of the work on vindolicine, there have been discussions on the unicity or not of the NMR signals for the two vindoline moieties. These two "vindolines" are exchangeable through a rotation around an axis bisecting the central methylene and therefore show magnetic equivalence; the same considerations hold for vindolicinone, tris-vindolinyl-methane and bis-acetone adduct (11). In "trimer" (3), the three vindoline units are chemically different and give rise to three distinct but difficult-to-assign sets of signals. In "tetramer" (4), there is an axis of symmetry exchanging the inner vindolines on one hand and the outer ones on the other, producing two sets of signals. When the central methylene is substituted (viz compound (8)), the exchange after rotation is no longer possible and the signals of the two vindoline units become nonequivalent. This is the case of compounds (12) and (13) although in this later example the isolated vindoline shows a third set of signals. The tetrasubstituted acetone adduct (14) only shows two sets of signals because the VCHV radicals present on each side of the molecule are exchangeable through rotation around the carbonyl and therefore are equivalent. However, the two vindolines which substitute the same carbon atom are not exchangeable through rotation and display anisochronous signals.

## Biological properties of the new molecules

Although vindoline is not a cytotoxic molecule, we felt that the reactivity of 10-hydroxymethyl vindoline (5) would cause cell damages through reaction with electron rich biomolecules

<figure><img src="image_13.png" /><figcaption>**Scheme 6.** Synthesis of vindolicinone (15).</figcaption></figure>
vindolicine. The second product (8) is the result of incomplete substitution and NMR and MS allow its identification as 10'-OMe vindolicine. The introduction of a methoxy group on the methylene bridge destroys the symmetry present in vindolicine, and the resonances of the two moieties become non-equivalent.

## Reactions of 10'-formyl vindoline with acetone

10'-Formyl vindoline (6) was reacted with acetone in the presence of a base to give butenone derivative (10) as the main product (Scheme 5). The new double bond was *trans* as shown by the large coupling constant ($J=16.2$ Hz) and its presence induced a downfield shift for H-9 (δ 7.47). No other damage occurred to the molecule, the structure of which was safely established by X-ray crystallography. Deprotonation of (10) with NaH and addition of a second molecule of 10'-formyl vindoline yielded the symmetrical bis adduct (11). Compounds (10) and (11) showed extended UV chromophores with $\lambda_{max}$ at 473 nm for (11) and 393 nm for (10). Owing to electron deficiency on the side chains, they were prone to further addition of vindoline and (10) gave a bis-vindolinyl adduct (12), and (11) gave a tris vindolinyl adduct (13) and tetra vindolinyl adduct (14). The formation of these adducts does not give rise to the creation of new stereogenic center and therefore these compounds are unique.

<figure><img src="image_13.png" /><figcaption>Scheme 5. Synthesis of acetone adduct (10–14) and ORTEP of (10).</figcaption></figure>

## Synthesis of vindolicinone

With vindolicine at hand, it was tempting to explore its chemistry and, in particular, the oxidation of the methylene bridge, which would open a route to olefins substituted by vindolines. The methylene bridge was easily oxidized by DDQ in the presence of water under Yonemitsu conditions (Scheme 6). Ketone 15, named here vindolicinone, was obtained as a pale-yellow solid in ca 60% yield. It showed an extended chromophore with $\lambda_{max}$ 370 nm.

## Symmetry considerations

At the onset of the work on vindolicine, there have been discussions on the unicity or not of the NMR signals for the two vindoline moieties. These two "vindolines" are exchangeable through a rotation around an axis bisecting the central methylene and therefore show magnetic equivalence; the same considerations hold for vindolicinone, tris-vindolinyl-methane and bis-acetone adduct (11). In "trimer" (3), the three vindoline units are chemically different and give rise to three distinct but difficult-to-assign sets of signals. In "tetramer" (4), there is an axis of symmetry exchanging the inner vindolines on one hand and the outer ones on the other, producing two sets of signals. When the central methylene is substituted (viz compound (8)), the exchange after rotation is no longer possible and the signals of the two vindoline units become nonequivalent. This is the case of compounds (12) and (13) although in this later example the isolated vindoline shows a third set of signals. The tetrasubstituted acetone adduct (14) only shows two sets of signals because the VCHV radicals present on each side of the molecule are exchangeable through rotation around the carbonyl and therefore are equivalent. However, the two vindolines which substitute the same carbon atom are not exchangeable through rotation and display anisochronous signals.

## Biological properties of the new molecules

Although vindoline is not a cytotoxic molecule, we felt that the reactivity of 10-hydroxymethyl vindoline (5) would cause cell damages through reaction with electron rich biomolecules

<figure><img src="image_14.png" /><figcaption>Scheme 6. Synthesis of vindolicinone (15).</figcaption></figure>
(nucleic acids, peptides). This was not the case however and the molecule was found inactive against HeLa, PC3 and MB-23 cell lines at micromolar concentrations. Cytotoxicity at the micromolar level was found on the bis acetone adduct (11), but this maybe simply due to the Michael acceptor character of the molecule.[15] On the larvicidal assay, 10-hydroxymethyl vindoline (5) showed a 98% inhibition at 100 µM concentration. Compound (15) showed potent α-glucosidase inhibitory activity with 92% inhibition at 0.5 mM concentration and was inactive against 3T3 cell lines.

## Conclusions

The availability of vindoline makes it an attractive starting point for the construction of libraries of compounds for biological investigation. Its relatively high molecular weight allows the preparation of molecules in the 1000–1500 Dalton range, a domain which is rarely explored.[16] So far, the best and unique market for vindoline remains the preparation of semisynthetic navelbine but there remains a host of simple and elaborated electrophiles to investigate in order to give alternative value to this resource.

## Experimental Section

### General experimental procedures

Optical rotations were determined in MeOH with an Anton Paar MCP 5100 polarimeter at 25 °C and on JASCO P-2000 spectrometer in 1 mL cells. Fourier transform-infrared (FT-IR) measurements were carried out using a Perkin Elmer Spectrum Two spectrometer in the ATR mode and on Bruker Vector 22 FT-IR spectrometer (France) on KBr disk. HR-ESI-MS experiments were performed using a SYNAPT G2 si, Waters instrument (Manchester, UK). Thermo-scientific model-300 spectrometer was used to record the UV spectra. ¹H-(400, 500 or 600 MHz), and ¹³C-NMR (100, 125 or 150 MHz) spectra were recorded on Bruker Avance-or Neo spectrometers. Single-crystal X-ray diffraction data of compounds 5, 6 and 10 was collected on Bruker APEXII D8 Venture diffractometer, fitted with PHOTON 100 detector (CMOS technology), and fine-focus sealed tube having X-ray source [Cu Kα radiation λ = 1.54178 Å]. Reflection intensities were integrated using SAINT software. Absorption correction was done on M-multi-scan. Structures were solved on SHELXTL. X-ray data for compound 1 were collected at low temperature (180 K) on an Agilent Gemini diffractometer using a graphite-monochromated Cu–Kα Enhance radiation (λ = 1.54184 Å) and equipped with an Oxford Instrument Cooler Device. Deposition numbers 2310835 (for 1), 2310923 (for 5), 2310924 (for 6) and 2310925 (for 10) contain the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.

### Synthesis of vindolicine 1 and of oligomers 3 and 4

Vindoline (961 mg, 2.1 mmoles) is dissolved in 10 mL 2 N HCl and 0.4 mL of a solution prepared from 200 mg *p*-formaldehyde in 2 mL 2 N HCl is added (full dissolution requires sonication). The solution is stirred for 17 hours at room temperature and develops a blue

color. It is then neutralized with sodium bicarbonate in the presence of 50 mL DCM. The phases are separated, and the organic layer is dried over Na₂SO₄ and evaporated. One obtains 1.1 g of a blue solid which is purified by flash chromatography on 25 g of Sigel.

Vindolicine (1): off white powder; [α]₂D°-48° (c 0.014, CHCl₃); mp = 255–260 °C; UV (MeOH): λmax 219, 260 and 310 nm; IR (KBr): 3441, 2926, 2855, 1736, 1612, 1498, 1461, 1372, 1249, 1119, 1044, 813 cm⁻¹; ESI-MS (+ve) m/z: 925 (M+H)⁺; ¹H-NMR (500 MHz, CD₃OD): δ 6.86 (2H, s, H-9), 6.20 (2H, s-H-12), 5.83 (2H, ddd, *J* = 10.1, 4.5, 1.2 Hz, H-14), 5.35 (2H, s, H-17), 5.13 (2H, brd, *J* = 10.1 Hz, H-15), 3.79 (6H, s, C-11 OCH₃), 3.76 (6H, s, COOCH₃), 3.71 (2H, s, H-10'), 3.53 (2H, s, H-2), 3.47 (2H, dd, *J* = 16.5, 4.5 Hz, H-3β), 3.38 (2H, dt, *J* = 3.6, 18.2 Hz, H-5), 2.81 (2H, bd, *J* = 16.5 Hz, H-3α), 2.64 (6H, s, NCH₃), 2.59 (2H, s, H-21), 2.53 (2H, dd, *J* = 8.5 Hz, H-5), 2.27 (4H, m, H-6), 1.99 (6H, s, OCOCH₃), 1.50 (2H, dq, *J* = 14.5, 7.3 Hz, H-19), 1.01 (2H, dq, *J* = 14.5, 7.3 Hz, H-19), 0.33 (6H, t, *J* = 7.3 Hz, CH₃-18); ¹³C-NMR (125 MHz, CD₃OD): δ 173.6 (COOCH₃), 172.5 (OCOCH₃), 159.9 (C-11), 153.2 (C-13), 131.4 (C-15), 125.6 (C-14), 125.1 (C-9), 125.0 (C-8), 123.0 (C-10), 94.8 (C-12), 84.8 (C-2), 81.0 (C-16), 77.6 (C-17), 68.2 (C-21), 56.0 (C-11 OCH₃), 54.3 (C-7), 53.0 (C-5), 52.8 (COOCH₃), 52.1 (C-3), 44.8 (C-6), 44.4 (C-20), 39.7 (N-CH₃), 32.0 (C 19), 30.2 (C-10'), 20.8 (OCOCH₃), 8.4 (C-18).

X-ray analysis of vindolicine: data were collected at low temperature (180 K) on a Gemini Rigaku diffractometer using a graphite-monochromated Cu–Kα Enhance radiation (λ = 1.54184) and equipped with an Oxford Instrument Cooler Device. The final unit cell parameters have been obtained by means of a least square refinement. The structure has been solved by Direct methods using SIR92 and refined by means of least squares procedures on a F2 with the program SHELXL9 included in the software package WinGX version 1.63.[17–19] The Atomic Scattering Factors were taken from international tables for X-Ray Crystallography.[20] All hydrogen atoms were geometrically placed and refined by using a riding model, except for atom H3B, which has been localized by Fourier differences and isotropically refined. Although the molecule is an organic compound with light elements, it was possible to determine its absolute configuration under Cu–Kα radiation. All non-hydrogen atoms were anisotropically refined, and in the last cycles of refinement a weighting scheme was used, where weights are calculated from the following formula: w = 1/[σ²(Fo²) + (aP)² + bP] where P = (Fo² + 2Fc²)/3. Drawing was performed with the program ORTEP32 with 30% probability displacement ellipsoids for non-hydrogen atoms.

Compound 3: amorphous solid; [α]₂D°-108° (c 1.2, MeOH); UV (MeOH): λmax 212, 258 and 308 nm; IR (neat, ATR): 3617, 3455, 2949, 1738, 1615, 1371, 1222, 1039 cm⁻¹; ESI-MS (+ve) m/z: 1393.6848 (calc for C₇₇H₉₇N₆O₁₈: 1393.6859, M+H⁺), 697, 465; ¹H-NMR (500 MHz, CDCl₃): δ 9.75, 9.52 and 9.25 (3 OH), 6.72, 6.59 and 6.48 (3H, 3s, H-9), 6.07 and 6.05 (2H, 2s, H-12), 5.82 (3H, 3 brd, H-14), 5.44, 5.34 and 5.31 (3H, 3s, H-17), 5.24 (3H, 3brd, H-15), 3.85, 3.71 and 3.50 (9H, 3s, C-11 OCH₃), 3.85 and 3.74 (2H, AB system, *J* = 16 Hz, 2 H-10'), 3.77 and 3.69 (2H, AB system, 2 H-10''), 3.78, 3.75 and 3.70 (9H, 3s, COOCH₃), 3.66, 3.64 and 3.54 (3H, 3s, H-2), 2.68, 2.64 and 2.51 (9H, 3s, NCH₃), 2.64, 2.56 and 2.56 (3H, 3s, H-21), 2.04, 2.03 and 2.02 (9H, 3s, OCOCH₃), 0.42, 0.37 and 0.22 (9H, 3t, *J* = 7.3 Hz, CH₃-18); ¹³C-NMR (125 MHz, CDCl₃): δ 171.9, 171.7, 171.2, 170.8, 170.7, 170.5 (COOCH₃ and OCOCH₃), 158.8, 158.6, 157.9 (C-11), 152.0, 151.4, 151.3 (C-13), 130.7, 130.6, 130.4 (C-15), 128.9, 127.3 (C-8), 124.3, 124.1, 124.0 (C-14), 124.9, 122.3, 121.6 (C-9), 93.6, 92.9 (C-12), 84.4, 83.3, 83.1 (C-2), 80.1, 79.8, 79.5 (C-16), 76.5, 76.3, 76.0 (C-17), 66.7, 66.3, 65.1 (C-21), 61.0, 55.5, 55.4 (C-11 OCH₃), 52.2, 52.1, 52.0 (COOCH₃), 53.0 (C-7), 52.8 (C-5), 52.1 (C-3), 44.3 (C-6), 44.8 (C-20), 43.9, 39.0, 38.7 (N-CH₃), 30.7, 30.4, 30.1 (C 19), 29.8 (C-10'), 25.3 (C-10''), 21.0 (OCOCH₃), 8.1, 7.65, 7.3 (C-18).
(nucleic acids, peptides). This was not the case however and the molecule was found inactive against HeLa, PC3 and MB-23 cell lines at micromolar concentrations. Cytotoxicity at the micromolar level was found on the bis acetone adduct (11), but this maybe simply due to the Michael acceptor character of the molecule.[15] On the larvicidal assay, 10-hydroxymethyl vindoline (5) showed a 98% inhibition at 100 µM concentration. Compound (15) showed potent α-glucosidase inhibitory activity with 92% inhibition at 0.5 mM concentration and was inactive against 3T3 cell lines.

Conclusions

The availability of vindoline makes it an attractive starting point for the construction of libraries of compounds for biological investigation. Its relatively high molecular weight allows the preparation of molecules in the 1000–1500 Dalton range, a domain which is rarely explored.[16] So far, the best and unique market for vindoline remains the preparation of semisynthetic navelbine but there remains a host of simple and elaborated electrophiles to investigate in order to give alternative value to this resource.

Experimental Section

General experimental procedures

Optical rotations were determined in MeOH with an Anton Paar
MCP 5100 polarimeter at 25 °C and on JASCO P-2000 spectrometer
in 1 mL cells. Fourier transform-infrared (FT-IR) measurements were
carried out using a Perkin Elmer Spectrum Two spectrometer in the
ATR mode and on Bruker Vector 22 FT-IR spectrometer (France) on
KBr disk. HR-ESI-MS experiments were performed using a SYNAPT
G2 si, Waters instrument (Manchester, UK). Thermo-scientific
model-300 spectrometer was used to record the UV spectra. ¹H-
(400, 500 or 600 MHz), and ¹³C-NMR (100, 125 or 150 MHz) spectra
were recorded on Bruker Avance-or Neo spectrometers. Single-
crystal X-ray diffraction data of compounds 5, 6 and 10 was
collected on Bruker APEXII D8 Venture diffractometer, fitted with
PHOTON 100 detector (CMOS technology), and fine-focus sealed
tube having X-ray source [Cu Kα radiation λ = 1.54178 Å]. Reflection
intensities were integrated using SAINT software. Absorption
correction was done on M-multi-scan. Structures were solved on
SHELXTL. X-ray data for compound 1 were collected at low
temperature (180 K) on an Agilent Gemini diffractometer using a
graphite-monochromated Cu–Kα Enhance radiation (λ = 1.54184 Å)
and equipped with an Oxford Instrument Cooler Device. Deposition
numbers 2310835 (for 1), 2310923 (for 5), 2310924 (for 6) and
2310925 (for 10) contain the supplementary crystallographic data
for this paper. These data are provided free of charge by the joint
Cambridge Crystallographic Data Centre and Fachinformationszen-
trum Karlsruhe Access Structures service.

**Synthesis of vindolicine 1 and of oligomers 3 and 4**

Vindoline (961 mg, 2.1 mmoles) is dissolved in 10 mL 2 N HCl and 0.4 mL of a solution prepared from 200 mg *p*-formaldehyde in 2 mL 2 N HCl is added (full dissolution requires sonication). The solution is stirred for 17 hours at room temperature and develops a blue

color. It is then neutralized with sodium bicarbonate in the
presence of 50 mL DCM. The phases are separated, and the organic
layer is dried over Na₂SO₄ and evaporated. One obtains 1.1 g of a
blue solid which is purified by flash chromatography on 25 g of
Sigel.

Vindolicine (1): off white powder; [α]<sup>20°</sup><sub>D</sub>-48° (c 0.014, CHCl<sub>3</sub>); mp = 255–260 °C; UV (MeOH): λ<sub>max</sub> 219, 260 and 310 nm; IR (KBr): 3441, 2926, 2855, 1736, 1612, 1498, 1461, 1372, 1249, 1119, 1044, 813 cm<sup>-1</sup>; ESI-MS (+ve) m/z: 925 (M+H)<sup>+</sup>; ¹H-NMR (500 MHz, CD<sub>3</sub>OD): δ 6.86 (2H, s, H-9), 6.20 (2H, s-H-12), 5.83 (2H, ddd, *J* = 10.1, 4.5, 1.2 Hz, H-14), 5.35 (2H, s, H-17), 5.13 (2H, brd, *J* = 10.1 Hz, H-15), 3.79 (6H, s, C-11 OCH<sub>3</sub>), 3.76 (6H, s, COOCH<sub>3</sub>), 3.71 (2H, s, H-10'), 3.53 (2H, s, H-2), 3.47 (2H, dd, *J* = 16.5, 4.5 Hz, H-3β), 3.38 (2H, dt, *J* = 3.6, 18.2 Hz, H-5), 2.81 (2H, bd, *J* = 16.5 Hz, H-3α), 2.64 (6H, s, NCH<sub>3</sub>), 2.59 (2H, s, H-21), 2.53 (2H, dd, *J* = 8.5 Hz, H-5), 2.27 (4H, m, H-6), 1.99 (6H, s, OCOCH<sub>3</sub>), 1.50 (2H, dq, *J* = 14.5, 7.3 Hz, H-19), 1.01 (2H, dq, *J* = 14.5, 7.3 Hz, H-19), 0.33 (6H, t, *J* = 7.3 Hz, CH<sub>3</sub>-18); <sup>13</sup>C-NMR (125 MHz, CD<sub>3</sub>OD): δ 173.6 (COOCH<sub>3</sub>), 172.5 (OCOCH<sub>3</sub>), 159.9 (C-11), 153.2 (C-13), 131.4 (C-15), 125.6 (C-14), 125.1 (C-9), 125.0 (C-8), 123.0 (C-10), 94.8 (C-12), 84.8 (C-2), 81.0 (C-16), 77.6 (C-17), 68.2 (C-21), 56.0 (C-11 OCH<sub>3</sub>), 54.3 (C-7), 53.0 (C-5), 52.8 (COOCH<sub>3</sub>), 52.1 (C-3), 44.8 (C-6), 44.4 (C-20), 39.7 (N-CH<sub>3</sub>), 32.0 (C 19), 30.2 (C-10'), 20.8 (OCOCH<sub>3</sub>), 8.4 (C-18).

X-ray analysis of vindolicine: data were collected at low temper-
ature (180 K) on a Gemini Rigaku diffractometer using a graphite-
monochromated Cu–Kα Enhance radiation (λ=1.54184) and
equipped with an Oxford Instrument Cooler Device. The final unit
cell parameters have been obtained by means of a least square
refinement. The structure has been solved by Direct methods using
SIR92 and refined by means of least squares procedures on a F2
with the program SHELXL9 included in the software package
WinGX version 1.63.[17–19] The Atomic Scattering Factors were taken
from international tables for X-Ray Crystallography.[20] All hydrogen
atoms were geometrically placed and refined by using a riding
model, except for atom H3B, which has been localized by Fourier
differences and isotropically refined. Although the molecule is an
organic compound with light elements, it was possible to
determine its absolute configuration under Cu–Kα radiation. All
non-hydrogen atoms were anisotropically refined, and in the last
cycles of refinement a weighting scheme was used, where weights
are calculated from the following formula: w = 1/[σ²(Fo)² + (aP)² +
bP] where P = (Fo² + 2Fc²)/3. Drawing was performed with the
program ORTEP32 with 30% probability displacement ellipsoids for
non-hydrogen atoms.

Compound 3: amorphous solid; [α]<sup>20°</sup><sub>D</sub>-108° (c 1.2, MeOH); UV (MeOH): λ<sub>max</sub> 212, 258 and 308 nm; IR (neat, ATR): 3617, 3455, 2949, 1738, 1615, 1371, 1222, 1039 cm<sup>-1</sup>; ESI-MS (+ve) m/z: 1393.6848 (calc for C<sub>77</sub>H<sub>97</sub>N<sub>6</sub>O<sub>18</sub>: 1393.6859, M+H<sup>+</sup>), 697, 465; ¹H-NMR (500 MHz, CDCl<sub>3</sub>): δ 9.75, 9.52 and 9.25 (3 OH), 6.72, 6.59 and 6.48 (3H, 3s, H-9), 6.07 and 6.05 (2H, 2s, H-12), 5.82 (3H, 3 brd, H-14), 5.44, 5.34 and 5.31 (3H, 3s, H-17), 5.24 (3H, 3brd, H-15), 3.85, 3.71 and 3.50 (9H, 3s, C-11 OCH<sub>3</sub>), 3.85 and 3.74 (2H, AB system, *J* = 16 Hz, 2 H-10'), 3.77 and 3.69 (2H, AB system, 2 H-10''), 3.78, 3.75 and 3.70 (9H, 3s, COOCH<sub>3</sub>), 3.66, 3.64 and 3.54 (3H, 3s, H-2), 2.68, 2.64 and 2.51 (9H,3s, NCH<sub>3</sub>), 2.64, 2.56 and 2.56 (3H, 3s, H-21), 2.04, 2.03 and 2.02 (9H, 3s, OCOCH<sub>3</sub>), 0.42, 0.37 and 0.22 (9H, 3t, *J* = 7.3 Hz, CH<sub>3</sub>-18); <sup>13</sup>C-NMR (125 MHz, CDCl<sub>3</sub>): δ 171.9, 171.7, 171.2, 170.8, 170.7, 170.5 (COOCH<sub>3</sub> and OCOCH<sub>3</sub>), 158.8, 158.6, 157.9 (C-11), 152.0, 151.4, 151.3 (C-13), 130.7, 130.6, 130.4 (C-15), 128.9, 127.3 (C-8), 124.3, 124.1, 124.0 (C-14), 124.9, 122.3, 121.6 (C-9), 93.6, 92.9 (C-12), 84.4, 83.3, 83.1 (C-2), 80.1, 79.8, 79.5 (C-16), 76.5, 76.3, 76.0 (C-17), 66.7, 66.3, 65.1 (C-21), 61.0, 55.5, 55.4 (C-11 OCH<sub>3</sub>), 52.2, 52.1, 52.0 (COOCH<sub>3</sub>), 53.0 (C-7), 52.8 (C-5), 52.1 (C-3), 44.3 (C-6), 44.8 (C-20), 43.9, 39.0, 38.7 (N-CH<sub>3</sub>), 30.7, 30.4, 30.1 (C 19), 29.8 (C-10'), 25.3 (C-10''), 21.0 (OCOCH<sub>3</sub>), 8.1, 7.65, 7.3 (C-18).
**Compound 4:** amorphous solid; $[\alpha]_D^{20}-470°$ (c 0.61, MeOH); UV (MeOH): $\lambda_{max}$ 210, 258 and 306 nm; IR (neat, ATR): 3463, 2949, 1739, 1619, 1222, 1040 cm$^{-1}$; ESI-MS (+ve) m/z 1861.9148 (calc for $C_{103}H_{128}N_8O_{24}$: 1861.9197, M + H$^+$, 931, 621, 466; $^1$H-NMR (500 MHz, CDCl$_3$): $\delta$ 9.63 and 9.23 (4H, 2brs, OH), 6.73 (2H, s, H-9'), 6.43 (2H, s, H-9), 6.06 (2H, s, H-12), 5.86, 5.80 (4H, dd, H-14), 5.33, 5.31 (4H, 2s, H-17), 5.29, 5.23 (4H, brd, H-15), 3.85 (6H, s, Ar-OCH$_3$), 3.73, 3.77 (12 H, s, COOCH$_3$), 3.44 (6H, s, Ar-OCH$_3$), 3.84 and 3.71 (2H, AB system, J=16 Hz, H-10'), 3.77 and 3.67 (2H, AB system, H-10''), 3.64, 3.57 (4H, s, H-2), 2.67, 2.48 (12H, s, NCH$_3$), 2.68, 2.56 (4H, s, H-21), 2.06, 2.05 (12H, s, OCOCH$_3$), 0.51, 0.23 (12H, t, J=7.3 Hz, H$_3$-18); $^{13}$C-NMR (125 MHz, CDCl$_3$): $\delta$ 171.7, (COOCH$_3$), 170.7 (OCOCH$_3$), 159.3 (inner C-11), 157.8 (outer C-11), 152.1 (C-13), 151.7 (C-13), 130.6 (C-15), 125.6 (C-8), 124.3 (C-14), 122.4 (inner C-9), 122.2 (outer C-9), 122.9 (C-10), 92.9 (C-12), 84.8, 83.2 (2 C-2) 80.1 (C-16), 76.1 (C-17), 66.6, 65.3 (2 C-21), 61.2, 55.5 (2 C-11 OCH$_3$), 52.3 (COOCH$_3$), 53.0 (C-7), 50.9 (C-5), 50.6 (C-3), 44.3 (C-6), 42.8 (C-20), 43.6, 39.0 (2 N-CH$_3$), 31.5, 25.7 (2 C-10 CH$_2$), 30.6, 30.3 (2 C 19), 21.2 (OCOCH$_3$), 8.2, 7.7 (2 C-18).

## Synthesis of compound 6

To a stirred suspension of vindoline (100 mg, 0.21 mmol) in acetonitrile (0.3 mL), trimethyl orthoformate (0.1 mL, 0.60 mmol) was added. The solution was stirred at room temperature for 5 minutes and 0.2 mL TFA was rapidly added. The deep blue solution was stirred for 30 minutes at room temperature and then neutralized with 0.1 N aqueous sodium carbonate, which was extracted with DCM, dried over sodium sulfate and evaporated. A gummy material was obtained, which was dissolved in minimum amount of DCM followed by dropwise addition of hexane. The precipitate was filtered washed with hexane and dried at room temperature to obtain a white powder (95 mg, 95%).

**Compound 6:** $[\alpha]_D^{20}-83°$ (c 10, CHCl$_3$); mp = 250-253 °C, $R_f$ = 0.4 (silica gel, MeOH/DCM 99/1); UV (MeOH); $\lambda_{max}$ (log ε) 220, 261, 303 and 348 (4.79) nm; IR (KBr): 3431, 3027, 2968, 2933, 2877, 2851, 1736, 1658, 1607, 1474, 1433, 1375, 1242, 1149, 1118, 1089, 1041, 953, 845, 815, 744 cm$^{-1}$; ESI-MS (+ve) m/z 485 (M+H)$^+$; $^1$H-NMR (600 MHz, CD$_3$OD): $\delta$ 10.0 (1H, s, H-10'), 7.54 (1H, s, H-9), 6.20 (1H, s,H-12), 5.89 (1H, dd, J=10.0, 4.3 Hz, H-14), 5.22 (1H, m, H-15), 5.22 (1H, s, H-17), 3.95 (3H, s, C-11 OCH$_3$), 3.90 (1H, s, H-2), 3.78 (3H, s, COOCH$_3$), 3.47 (1H, dd, J=16.0, 4.9 Hz, H-3β), 3.41 (1H, dt, J=4.5, 9.0 Hz, H-5), 2.94 (1H, bd, J=16.0 Hz, H-3α), 2.92 (1H, s, H-21), 2.84 (3H, s, N-CH$_3$), 2.72 (1H, dt, J=6.4, 10.2 Hz, H-5), 2.28 (2H, m, H-6), 2.01 (3H, s, OCOCH$_3$), 1.57 (1H, dq, J=14.7, 7.2 Hz, H-19), 1.10 (1H, dq, J=14.7, 7.2 Hz, H-19), 0.57 (3H, t, J=7.2 Hz, H-18); $^{13}$C-NMR (150 MHz, CD$_3$OD): $\delta$ 188.8 (C-10'), 173.2 (COOCH$_3$), 172.3 (OCOCH$_3$), 167.6 (C-11), 160.7 (C-13), 131.0 (C-15), 127.0 (C-8), 126.0 (C-14), 123.6 (C-9), 117.5 (C-10), 91.6 (C-12), 83.8 (C-2), 80.6 (C-16), 77.3 (C-17), 67.4 (C-21), 56.5 (C-11 OCH$_3$), 53.4 (C-7), 53.0 (COOCH$_3$), 51.9 (C-5), 51.6 (C-3), 44.4 (C-6), 44.2 (C-20), 36.6 (N-CH$_3$), 32.1 (C 19), 29.7), 20.7 (OCOCH$_3$), 7.8 (C-18).

A solution of 6 (100 mg, 0.20 mmol) in acetonitrile (0.5 mL) was stirred at room temperature for 5 minutes and then NaBH$_4$ (14.9 mg, 0.40 mmol) was added. After stirring at room temperature for 3 hours, the reaction mixture was quenched with water. The precipitate was filtered on paper and the aqueous layer was extracted with CH$_2$Cl$_2$. The extract was dried over sodium sulfate. filtered, concentrated under reduced pressure and purified by column chromatography on silica gel (ethyl acetate: hexane, 7:3) to give compound 5 (80 mg, 80%).

**Compound 5:** white powder $[\alpha]_D^{20}-51°$ (c 10, MeOH); mp = 222-225 °C, $R_f$ = 0.3 (silica gel, MeOH/DCM 98/2); UV (MeOH); $\lambda_{max}$: 221, 260, 307 nm; IR (KBr): 3436, 2941, 2872, 1738, 1611, 1495, 1462, 1219,

1191, 1362, 1143, 1130, 1106, 1044, 1025, 930, 900, 872, 790, 741, 705 cm$^{-1}$; ESI-MS (+ve) m/z 487.4 (M+H)$^+$; $^1$H-NMR (500 MHz, CD$_3$OD): $\delta$ 7.08 (1H, s, H-9), 6.25 (1H, s, H-12), 5.86 (1H, ddd, J=10.1, 4.8, 1.5 Hz, H-14), 5.37 (1H, s, H-17), 5.18 (1H, brd, J=10.1 Hz, H-15), 4.53 (d, J=12.5 Hz, H-10'), 4.48 (d, J=12.5 Hz, H-10'), 3.83 (3H, s, C-11 OCH$_3$), 3.77 (3H, s, COOCH$_3$), 3.59 (1H, s, H-2), 3.48 (1H, ddd, J=16.2, 4.8, 1 Hz, H-3β), 3.40 (1H, dt, J=4.8, 9.3 Hz, H-5), 2.89 (1H, bd, J=16.2 Hz, H-3α), 2.75 (1H, s, H-21), 2.67 (3H, s, N-CH$_3$), 2.62 (1H, m, H-5), 2.37, (1H, m, H-6), 2.26 (1H, m, H-6), 2.00 (3H, s, OCOCH$_3$), 1.57 (1H, dq, J=14.4, 7.4 Hz, H-19), 1.08 (1H, dq, J=14.4, 7.4 Hz, H-19), 0.49 (3H, t, J=7.4 Hz, H-18); $^{13}$C-NMR (125 MHz, CD$_3$OD): $\delta$ 173.6 (COOCH$_3$), 172.5 (OCOCH$_3$), 160.2 (C-11), 154.6 (C-13), 131.3 (C-15), 125.7 (C-14), 125.0 (C-8), 124.2 (C-9), 122.0 (C-10), 94.5 (C-12), 84.8 (C-2), 81.0 (C-16), 77.7 (C-17), 68.0 (C-21), 60.6 (C-10'), 56.0 (C-11 OCH$_3$), 54.3 (C-7), 52.82 (COOCH$_3$), 52.80 (C-5), 52.0 (C-3), 44.9 (C-6), 44.3 (C-20), 39.3 (NCH$_3$), 32.0 (C-19), 20.8 (OCOCH$_3$), 8.1 (C-18).

## Synthesis of vindolicine from vindoline and 10-hydroxymethyl vindoline

A mixture of 5 (100 mg, 0.20 mmol) and 2 (100 mg, 0.21 mmol) was dissolved in 1 mL of acetonitrile at room temperature. After 5 minutes stirring, TFA (0.5 mL) was added. The mixture was stirred at room temperature for 30 minutes, then neutralized with 0.1 N sodium carbonate, extracted with CH$_2$Cl$_2$ (3×20 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude reaction mixture was purified on alumina (MeOH/DCM 99.8/0.2) to give vindolicine in 85% yield.

## Synthesis of trisvindolicine 9

Trimethyl orthoformate (0.01 mL, 0.06 mmol) was added to a stirred solution of vindoline (100 mg, 0.219 mmol) in 0.1 mL acetonitrile, followed by 0.01 mL of TFA. The reaction mixture turned deep blue. Stirring was pursued for 24 h at room temperature. The reaction mixture was neutralized with 0.1 N sodium carbonate and extracted with DCM (3×20 mL), dried over sodium sulfate, and concentrated under reduced pressure. Purification of the crude reaction mixture on alumina gave compounds 8 (20 mg, 20%, eluted with MeOH/DCM 99.8/0.2) and 9 (50 mg, 50%, eluted with MeOH/DCM 99/1). Compound 8: white powder $[\alpha]_D^{20}-70°$ (c 10, MeOH); mp 185-190 °C; $R_f$ = 0.5 (silica gel, MeOH/DCM 97/3); UV (MeOH); $\lambda_{max}$: 229, 261, 310 nm; IR (KBr): 3308, 2930, 2865, 2358, 2344, 2183, 2155, 2100, 2028, 20114, 1998, 17734, 1615, 1463, 1373, 1142, 955, 907, 889, 799, 741 cm$^{-1}$; ESI-MS (+ve) m/z 955 (M+H)$^+$; $^1$H-NMR (600 MHz, CD$_3$OD): $\delta$ 7.09, 6.80 (2H, 2s, H-9), 6.23, 6.21 (2H, 2s, H-12), 5.87, 5.84 (2H, m, H-14), 5.87 (1H, s, C-10'), 5.36, 5.26 (2H, 2s, H-17), 5.20, 5.14 (2H, brd, J=10.1 Hz, H-15), 3.81, 3.72 (6H, s, C-11 OCH$_3$), 3.765, 3.757 (6H, 2s, COOCH$_3$), 3.58, 3.56 (2H, 2s, H-2), 2.78, 2.51 (2H, 2s, H-21), 2.69, 2.65, (6H, 2s, N-CH$_3$), 2.00, 1.98 (6H, 2s, OCOCH$_3$), 0.54, 0.20 (3H, 2t, J=7.2 Hz, H-18); $^{13}$C-NMR (150 MHz, CD$_3$OD): $\delta$ 173.5, 173.4 (COOCH$_3$), 172.5, 172.4 (OCOCH$_3$), 160.4, 160.2 (C-11), 154.2, 154.1 (C-13), 131.4. 131.3 (C-15), 125.8, 125.7 (C-14), 124.9, 124.7 (C-8), 123.3, 122.1 (C-9), 122.9 (C-10'), 95.0, 94.6 (C-12), 84.6, 84.4 (C-2) 81.1, 81.0 (C-16), 77.6, 77.5 (C-17), 74.6 (C-10'), 67.7, 67.6 (C-21), 57.0(C-10' OCH$_3$),56.4, 56.2 (C-11 OCH$_3$), 54.4, 54.2 (C-7), 52.8 (COOCH$_3$), 52.6, 52.3 (C-5), 52.0, 51.9 (C-3), 45.3, 44.8 (C-6), 44.3, 44.2 (C-20), 39.4, 39.0 (N-CH$_3$), 31.9, 31.7 (C 19), 20.8, (OCOCH$_3$), 8.5, 8.3 (C-18). Compound 9: white powder. $[\alpha]_D^{20}-194°$ (c 10, MeOH); mp = 200-205 °C; $R_f$ = 0.2 (silica gel, MeOH/DCM 96/3); UV (MeOH): $\lambda_{max}$: 221, 261 and, 311 nm; IR (KBr): 3308, 2930, 2865, 2358, 2344, 2183, 2155, 2100, 2028, 20114, 1998, 17734, 1615, 1463, 1373, 1142, 955, 907, 889, 799, 741 cm$^{-1}$; ESI-MS (+ve) m/z 1379 (M+H)$^+$; $^1$H-NMR (400 MHz, CD$_3$OD): $\delta$ 6.50 (3H, s, H-9), 6.21 (3H, s, H-12), 6.14 (1H, s, H-10'), 5.82 (3H, br dd, J=10.2, 4.1 Hz, H-14), 5.30
**Compound 4:** amorphous solid; [$\alpha$]<sup>20</sup><sub>D</sub>-470° (c 0.61, MeOH); UV (MeOH): $\lambda_{max}$ 210, 258 and 306 nm; IR (neat, ATR): 3463, 2949, 1739, 1619, 1222, 1040 cm<sup>-1</sup>; ESI-MS (+ve) m/z 1861.9148 (calc for C<sub>103</sub>H<sub>128</sub>N<sub>8</sub>O<sub>24</sub>: 1861.9197, M + H<sup>+</sup>, 931, 621, 466; ¹H-NMR (500 MHz, CDCl<sub>3</sub>): $\delta$ 9.63 and 9.23 (4H, 2brs, OH), 6.73 (2H, s, H-9'), 6.43 (2H, s, H-9), 6.06 (2H, s, H-12), 5.86, 5.80 (4H, dd, H-14), 5.33, 5.31 (4H, 2s, H-17), 5.29, 5.23 (4H, brd, H-15), 3.85 (6H, s, Ar-OCH<sub>3</sub>), 3.73, 3.77 (12 H, s, COOCH<sub>3</sub>), 3.44 (6H, s, Ar-OCH<sub>3</sub>), 3.84 and 3.71 (2H, AB system, J=16 Hz, H-10'), 3.77 and 3.67 (2H, AB system, H-10''), 3.64, 3.57 (4H, s, H-2), 2.67, 2.48 (12H, s, NCH<sub>3</sub>), 2.68, 2.56 (4H, s, H-21), 2.06, 2.05 (12H, s, OCOCH<sub>3</sub>), 0.51, 0.23 (12H, t, J=7.3 Hz, H<sub>3</sub>-18); <sup>13</sup>C-NMR (125 MHz, CDCl<sub>3</sub>): $\delta$ 171.7, (COOCH<sub>3</sub>), 170.7 (OCOCH<sub>3</sub>), 159.3 (inner C-11), 157.8 (outer C-11), 152.1 (C-13), 151.7 (C-13), 130.6 (C-15), 125.6 (C-8), 124.3 (C-14), 122.4 (inner C-9), 122.2 (outer C-9), 122.9 (C-10), 92.9 (C-12), 84.8, 83.2 (2 C-2) 80.1 (C-16), 76.1 (C-17), 66.6, 65.3 (2 C-21), 61.2, 55.5 (2 C-11 OCH<sub>3</sub>), 52.3 (COOCH<sub>3</sub>), 53.0 (C-7), 50.9 (C-5), 50.6 (C-3), 44.3 (C-6), 42.8 (C-20), 43.6, 39.0 (2 N-CH<sub>3</sub>), 31.5, 25.7 (2 C-10 CH<sub>2</sub>), 30.6, 30.3 (2 C 19), 21.2 (OCOCH<sub>3</sub>), 8.2, 7.7 (2 C-18).

## Synthesis of compound 6

To a stirred suspension of vindoline (100 mg, 0.21 mmol) in acetonitrile (0.3 mL), trimethyl orthoformate (0.1 mL, 0.60 mmol) was added. The solution was stirred at room temperature for 5 minutes and 0.2 mL TFA was rapidly added. The deep blue solution was stirred for 30 minutes at room temperature and then neutralized with 0.1 N aqueous sodium carbonate, which was extracted with DCM, dried over sodium sulfate and evaporated. A gummy material was obtained, which was dissolved in minimum amount of DCM followed by dropwise addition of hexane. The precipitate was filtered washed with hexane and dried at room temperature to obtain a white powder (95 mg, 95%).

**Compound 6:** [$\alpha$]<sup>20</sup><sub>D</sub>-83° (c 10, CHCl<sub>3</sub>); mp = 250-253 °C, Rf = 0.4 (silica gel, MeOH/DCM 99/1); UV (MeOH); $\lambda_{max}$ (log ε)220, 261, 303 and 348 (4.79) nm; IR (KBr): 3431, 3027, 2968, 2933, 2877, 2851, 1736, 1658, 1607, 1474, 1433, 1375, 1242, 1149, 1118, 1089, 1041, 953, 845, 815, 744 cm<sup>-1</sup>; ESI-MS (+ve) m/z 485 (M+H)<sup>+</sup>; ¹H-NMR (600 MHz, CD<sub>3</sub>OD): $\delta$ 10.0 (1H, s, H-10'), 7.54 (1H, s, H-9), 6.20 (1H, s,H-12), 5.89 (1H, dd, J=10.0, 4.3 Hz, H-14), 5.22 (1H, m, H-15), 5.22 (1H, s, H-17), 3.95 (3H, s, C-11 OCH<sub>3</sub>), 3.90 (1H, s, H-2), 3.78 (3H, s, COOCH<sub>3</sub>), 3.47 (1H, dd, J=16.0, 4.9 Hz, H-3β), 3.41 (1H, dt, J=4.5, 9.0 Hz, H-5), 2.94 (1H, bd, J=16.0 Hz, H-3α), 2.92 (1H, s, H-21), 2.84 (3H, s, N-CH<sub>3</sub>), 2.72 (1H, dt, J=6.4, 10.2 Hz, H-5), 2.28 (2H, m, H-6), 2.01 (3H, s, OCOCH<sub>3</sub>), ) 1.57 (1H, dq, J=14.7, 7.2 Hz, H-19), 1.10 (1H, dq, J=14.7, 7.2 Hz, H-19), 0.57 (3H, t, J=7.2 Hz, H-18); <sup>13</sup>C-NMR (150 MHz, CD<sub>3</sub>OD): $\delta$ 188.8 (C-10'), 173.2 (COOCH<sub>3</sub>), 172.3 (OCOCH<sub>3</sub>), 167.6 (C-11), 160.7 (C-13), 131.0 (C-15), 127.0 (C-8), 126.0 (C-14), 123.6 (C-9), 117.5 (C-10), 91.6 (C-12), 83.8 (C-2), 80.6 (C-16), 77.3 (C-17), 67.4 (C-21), 56.5 (C-11 OCH<sub>3</sub>), 53.4 (C-7), 53.0 (COOCH<sub>3</sub>), 51.9 (C-5), 51.6 (C-3), 44.4 (C-6), 44.2 (C-20), 36.6 (N-CH<sub>3</sub>), 32.1 (C 19), 29.7), 20.7 (OCOCH<sub>3</sub>), 7.8 (C-18).

A solution of **6** (100 mg, 0.20 mmol) in acetonitrile (0.5 mL) was stirred at room temperature for 5 minutes and then NaBH<sub>4</sub> (14.9 mg, 0.40 mmol) was added. After stirring at room temperature for 3 hours, the reaction mixture was quenched with water. The precipitate was filtered on paper and the aqueous layer was extracted with CH<sub>2</sub>Cl<sub>2</sub>. The extract was dried over sodium sulfate. filtered, concentrated under reduced pressure and purified by column chromatography on silica gel (ethyl acetate: hexane, 7:3) to give compound **5** (80 mg, 80%).

**Compound 5:** white powder [$\alpha$]<sup>20</sup><sub>D</sub>-51° (c 10, MeOH); mp = 222-225 °C, Rf = 0.3 (silica gel, MeOH/DCM 98/2); UV (MeOH); $\lambda_{max}$: 221, 260, 307 nm; IR (KBr): 3436, 2941, 2872, 1738, 1611, 1495, 1462, 1219,

1191, 1362, 1143, 1130, 1106, 1044, 1025, 930, 900, 872, 790, 741, 705 cm<sup>-1</sup>; ESI-MS (+ve) m/z 487.4 (M+H)<sup>+</sup>; ¹H-NMR (500 MHz, CD<sub>3</sub>OD): $\delta$ 7.08 (1H, s, H-9), 6.25 (1H, s, H-12), 5.86 (1H, ddd, J=10.1, 4.8, 1.5 Hz, H-14), 5.37 (1H, s, H-17), 5.18 (1H, brd, J=10.1 Hz, H-15), 4.53 (d, J=12.5 Hz, H-10'), 4.48 (d, J=12.5 Hz, H-10'), 3.83 (3H, s, C-11 OCH<sub>3</sub>), 3.77 (3H, s, COOCH<sub>3</sub>), 3.59 (1H, s, H-2), 3.48 (1H, ddd, J=16.2, 4.8, 1 Hz, H-3β), 3.40 (1H, dt, J=4.8, 9.3 Hz, H-5), 2.89 (1H, bd, J=16.2 Hz, H-3α), 2.75 (1H, s, H-21), 2.67 (3H, s, N-CH<sub>3</sub>), 2.62 (1H, m, H-5), 2.37, (1H, m, H-6), 2.26 (1H, m, H-6), 2.00 (3H, s, OCOCH<sub>3</sub>), 1.57 (1H, dq, J=14.4, 7.4 Hz, H-19), 1.08 (1H, dq, J=14.4, 7.4 Hz, H-19), 0.49 (3H, t, J=7.4 Hz, H-18); <sup>13</sup>C-NMR (125 MHz, CD<sub>3</sub>OD): $\delta$ 173.6 (COOCH<sub>3</sub>), 172.5 (OCOCH<sub>3</sub>), 160.2 (C-11), 154.6 (C-13), 131.3 (C-15), 125.7 (C-14), 125.0 (C-8), 124.2 (C-9), 122.0 (C-10), 94.5 (C-12), 84.8 (C-2), 81.0 (C-16), 77.7 (C-17), 68.0 (C-21), 60.6 (C-10'), 56.0 (C-11 OCH<sub>3</sub>), 54.3 (C-7), 52.82 (COOCH<sub>3</sub>), 52.80 (C-5), 52.0 (C-3), 44.9 (C-6), 44.3 (C-20), 39.3 (NCH<sub>3</sub>), 32.0 (C-19), 20.8 (OCOCH<sub>3</sub>), 8.1 (C-18).

## Synthesis of vindolicine from vindoline and 10-hydroxymethyl vindoline

A mixture of **5** (100 mg, 0.20 mmol) and **2** (100 mg, 0.21 mmol) was dissolved in 1 mL of acetonitrile at room temperature. After 5 minutes stirring, TFA (0.5 mL) was added. The mixture was stirred at room temperature for 30 minutes, then neutralized with 0.1 N sodium carbonate, extracted with CH<sub>2</sub>Cl<sub>2</sub> (3×20 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude reaction mixture was purified on alumina (MeOH/DCM 99.8/0.2) to give vindolicine in 85% yield.

## Synthesis of trisvindolicine 9

Trimethyl orthoformate (0.01 mL, 0.06 mmol) was added to a stirred solution of vindoline (100 mg, 0.219 mmol) in 0.1 mL acetonitrile, followed by 0.01 mL of TFA. The reaction mixture turned deep blue. Stirring was pursued for 24 h at room temperature. The reaction mixture was neutralized with 0.1 N sodium carbonate and extracted with DCM (3×20 mL), dried over sodium sulfate, and concentrated under reduced pressure. Purification of the crude reaction mixture on alumina gave compounds **8** (20 mg, 20%, eluted with MeOH/DCM 99.8/0.2) and **9** (50 mg, 50%, eluted with MeOH/DCM 99/1). Compound **8**: white powder [$\alpha$]<sup>20</sup><sub>D</sub>-70° (c 10, MeOH); mp 185-190 °C; Rf = 0.5 (silica gel, MeOH/DCM 97/3); UV (MeOH); $\lambda_{max}$: 229, 261, 310 nm; IR (KBr): 3308, 2930, 2865, 2358, 2344, 2183, 2155, 2100, 2028, 20114, 1998, 17734, 1615, 1463, 1373, 1142, 955, 907, 889, 799, 741 cm<sup>-1</sup>; ESI-MS (+ve) m/z 955 (M+H)<sup>+</sup>; ¹H-NMR (600 MHz, CD<sub>3</sub>OD): $\delta$ 7.09, 6.80 (2H, 2s, H-9), 6.23, 6.21 (2H, 2s, H-12), 5.87, 5.84 (2H, m, H-14), 5.87 (1H, s, C-10'), 5.36, 5.26 (2H, 2s, H-17), 5.20, 5.14 (2H, brd, J=10.1 Hz, H-15), 3.81, 3.72 (6H, s, C-11 OCH<sub>3</sub>), 3.765, 3.757 (6H, 2s, COOCH<sub>3</sub>), 3.58, 3.56 (2H, 2s, H-2), 2.78, 2.51 (2H, 2s, H-21), 2.69, 2.65, (6H, 2s, N-CH<sub>3</sub>), 2.00, 1.98 (6H, 2s, OCOCH<sub>3</sub>), 0.54, 0.20 (3H, 2t, J=7.2 Hz, H-18); <sup>13</sup>C-NMR (150 MHz, CD<sub>3</sub>OD): $\delta$ 173.5, 173.4 (COOCH<sub>3</sub>), 172.5, 172.4 (OCOCH<sub>3</sub>), 160.4, 160.2 (C-11), 154.2, 154.1 (C-13), 131.4. 131.3 (C-15), 125.8, 125.7 (C-14), 124.9, 124.7 (C-8), 123.3, 122.1 (C-9), 122.9 (C-10'), 95.0, 94.6 (C-12), 84.6, 84.4 (C-2) 81.1, 81.0 (C-16), 77.6, 77.5 (C-17), 74.6 (C-10'), 67.7, 67.6 (C-21), 57.0(C-10' OCH<sub>3</sub>),56.4, 56.2 (C-11 OCH<sub>3</sub>), 54.4, 54.2 (C-7), 52.8 (COOCH<sub>3</sub>), 52.6, 52.3 (C-5), 52.0, 51.9 (C-3), 45.3, 44.8 (C-6), 44.3, 44.2 (C-20), 39.4, 39.0 (N-CH<sub>3</sub>), 31.9, 31.7 (C 19), 20.8, (OCOCH<sub>3</sub>), 8.5, 8.3 (C-18). Compound **9**: white powder. [$\alpha$]<sup>20</sup><sub>D</sub>-194° (c 10, MeOH); mp = 200-205 °C; Rf = 0.2 (silica gel, MeOH/DCM 96/3); UV (MeOH): $\lambda_{max}$: 221, 261 and, 311 nm; IR (KBr): 3308, 2930, 2865, 2358, 2344, 2183, 2155, 2100, 2028, 20114, 1998, 17734, 1615, 1463, 1373, 1142, 955, 907, 889, 799, 741 cm<sup>-1</sup>; ESI-MS (+ve) m/z 1379 (M+H)<sup>+</sup>; ¹H-NMR (400 MHz, CD<sub>3</sub>OD): $\delta$ 6.50 (3H, s, H-9), 6.21 (3H, s, H-12), 6.14 (1H, s, H-10'), 5.82 (3H, br dd, J=10.2, 4.1 Hz, H-14), 5.30
7.2 Hz, H-18); ¹³C-NMR (125 MHz CD₃OD): δ 202.8 (CHCHCO), 173.6, 173.4, 173.3 (COOCH₃), 172.5, 172.4, 172.3 (OCOCH₃), 162.8, 159.8, 159.7 (C-11), 157.7, 153.2, 153.1 (C-13), 140.8 (CH=CHCO), 131.3, 131.3, 131.2 (C-15), 122.0, 123.7, 123.6 (C-9), 125.8, 125.1, 126.7 (C-8), 125.7, 125.7, 125.9 (C-14), 124.4, 124.9, 115.0 (C-10), 123.7 (CH=CHCO), 95.5, 95.1, 93.0 (C-12), 84.8, 84.4, 84.1 (C-2), 81.2, 80.9, 80.8 (C-16), 77.6, 77.5, 77.4 (C-17), 68.2, 67.7, 67.2 (C-21), 56.4, 56.3, 56.3 (C-11 OCH₃), 54.3, 53.9 (C-7), 53.0, 52.9, 52.8, (COOCH₃), 53.0, 52.8, 52.6 (C-5), 52.1, 51.9, 51.7 (C-3), 45.1 (CHCH₂CO), 44.8, 44.6 (C-6), 44.2, 44.2, 44.4 (C-20), 35.8 (C-10) CHCH₂CO, 37.5, 39.3, 39.5 (C-1), 32.2, 32.1, 31.8 (C-19), 20.8 (OCOCH₃), 8.75, 8.73, 8.03 (C-18).

## Synthesis of compound 14

Trifluoroacetic acid (4 mL) was added to a stirred solution of 13 (300 mg, 0.30 mmol) and 2 (600 mg, 1.31 mmol) in DCM (12 mL) and H₂O (6 mL). The green mixture was stirred for 48 hours at room temperature. The reaction mixture was neutralized with sodium carbonate and extracted with DCM. The extract dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified on alumina to give compounds 14 (150 mg, 37%): white powder; [α]₂D = -120 (c 0.3, MeOH); mp = 290-300 °C; Rf = 0.1 (silica gel, MeOH/DCM 96/4); UV (λmax MeOH): 219, 261, 311; IR (KBr): 3305, 2935, 2871, 2360, 2341, 2073, 2079, 2002, 1735, 1615, 1498, 1455, 1432, 1371, 1223, 929, 872, 817, 742, 699, 683, 669, cm⁻¹; ESI-MS (+ ve) m/z 1905.9 (M + H); ¹H-NMR (600 MHz, CD₃OD): δ 6.77, 6.75 (4H, 2s, H-9), 6.22, 6.20 (4H, s, H-12), 5.85 (4H, m, H-14), 5.34, 5.29 (4H, 2s, H-17), 5.16 (6 H, m, CHCH₂CO H-15), 3.77, 3.73 (12H, 2s, C-11 OCH₃), 3.76 (12H, s, COOCH₃), 3.54, 3.51 (4H, 2s, H-2), 3.47 (4H, m, H-3b), 3.45 (4H, m, H-5), 2.88 (4H, m, CHCH₂CO), 2.79 (4H, m, H-3a), 2.67, 2.62 (12H, 2s, N-CH₃), 2.62, 2.56 (4H, 2s, H-21), 2.53 (4H, m, H-5), 2.22 (8H, m, H-6), 1.99, 1.98 (12H, 2s, OCOCH₃), 1.52 (4H, m, H-19), 1.04, (4H, m, H-19), 0.41, 0.30 (12H, 2t, J₁₈,₁₉ = 7.2 Hz, H-18); ¹³C-NMR (150 MHz, CD₃OD): δ 213 (CHCH₂CO), 173.5, 173.4 (COOCH₃), 172.4, 172.4 (OCOCH₃), 159.8, 159.6 (C-11), 153.3, 153.2 (C-13), 131.3, 131.2 (C-15), 125.6, 124.6 (C-8), 125.7, 125.6 (C-14), 123.4, 122.7 (C-9), 124.7, 124.6 (C-10), 95.6, 95.0 (C-12), 84.7, 84.4 (C-2) 80.9 (C-16), 77.6, 77.4 (C-17), 67.8, 67.7 (C-21), 56.2 (C-11 OCH₃), 52.8 (COOCH₃), 54.4, 54.3 (C-7), 52.0, 51.9 (C-5), 52.6 (C-3), 48.8 (CHCH₂CO), 44.9, 44.8 (C-6), 44.9, 44.2 (C-20), 35.2 (CHCH₂CO), 39.6, 39.2 (N-CH₃), 32.0, 31.8 (C 19), 20.7 (OCOCH₃), 8.81, 8.68 (C-18).

## Synthesis of vindolicinone 15

Vindolicine 1 (100 mg, 0.10 mmol) and 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) (181 mg, 0.79 mmol) were dissolved in 0.8 mL diethyl ether at 0 °C under N₂. Water (100 µL) and acetic acid (100 µL) were added, and the solution was stirred for 6 hours at 0 °C, and 24 h at room temperature. The reaction mixture was neutralized with aqueous sodium carbonate and extracted with DCM Purification of the crude reaction mixture on alumina gave compound 15 (60 mg, 60%, eluted with hexane/acetone 3:7) as a lemon yellow powder; [α]₂D = -135° (c 0.007, MeOH); mp = 232-235 °C, UV (MeOH): λmax(log ε) 229, 261 and 370 (3.99) nm; IR (KBr): 3440, 2965, 2855, 1735, 1610, 1498, 1461, 1372, 1249, 1119, 1172, 1044, 740 cm⁻¹; ESI-MS (+ ve) m/z: 939 (M + H)⁺; ¹H-NMR (600 MHz, CD₃OD): δ 7.24 (2H, s, H-9), 6.18 (2H, s,H-12), 5.87 (2H, brdd, J = 9.5, 4.5 Hz, H-14), 5.33 (2H, s, H-17), 5.20 (2H, brd, J = 9.5 Hz, H-15), 3.79 (8H, s, COOCH₃, H-2), 3.62 (6H, s, C-11 OCH₃), 3.46 (2H, dd, J = 16.2, 4.5 Hz, H-3), 3.42 (2H, m, H-5), 2.90 (2H, bd, J = 16.2 Hz, H-3), 2.83 (2H, s, H-21), 2.78 (6H, s, N-CH₂), 2.66 (2H, m, H-5), 2.33, (4H, m, 2 H-6), 2.02 (6H, s, OCOCH₃), 1.62 (2H, dq, J = 14.7, 7.2 Hz, H-19), 1.21 (2H, dq, J = 14.7, J = 7.2 Hz, H-19), 0.55 (6H, t, J = 7.2 Hz, H-18); ¹³C-NMR (150 MHz, CD₃OD): δ 195.6 (C-10), 173.4 (COOCH₃), 172.5 (OCOCH₃), 163.1 (C-11), 157.9 (C-13), 131.2 (C-15), 126.2 (C-9), 125.9

(C-14), 125.4 (C-8), 123.3 (C-10), 93.0 (C-12), 84.2 (C-2), 80.9 (C-16), 77.5 (C-17), 67.8 (C-21), 56.2, (C-11 OCH₃), 53.8 (C-7), 53.0 (COOCH₃), 52.3 (C-5), 51.8 (C-3), 44.4 (C-6), 44.3 (C-20), 37.7 (N-CH₃), 32.0 (C 19), 20.8 (OCOCH₃), 8.1 (C-18).

## Author Contributions

This project was devised and conducted by G.M. who also wrote most of the article. Experiments were done by Asia who also wrote a preliminary draft of the article; The X-ray structure elucidations were performed by Y. S. and L.V.

## Acknowledgements

Supervision and guidance by Prof. Dr. M. Iqbal Choudhary are gratefully acknowledged. Interactions with Prof. Dr. Atta-ur-Rahman were greatly appreciated. Invaluable help from Prof. Dr. Hina Siddiqui during the last stages of the redaction permitted to achieve the project. We wish to thank the technical staff of ICBSS for all the analytical and biochemical measurements. Prof. J. D. Connolly, the University of Glasgow is thanked for bringing a final touch to the article.

## Conflict of Interests

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

**Keywords:** vindoline · vindolicine · cytotoxicity · symmetry

[1] G. H. Svoboda, M. Gorman, N. Neuss, A. J. Barnes Jr., *J. Pharm. Sci.* **1961**, *50*, 409–413.

[2] M. Gorman, J. Sweeny, **1964**, C-6-3 Vinca Alkaloids XXIII Vindolicine (Alkaloids and alkaloid biosynthesis). In International Symposium on the Chemistry of Natural Products (pp. 99–100). the Science Council of Japan under the Auspices of the International Union of Pure and Applied Chemistry.

[3] D. E. Dorman, J. W. Paschal, *Org. Magn. Reson.* **1976**, *8*, 413–418.

[4] a) P. Rasoanaivo, N. Langlois, P. Potier, *Phytochemistry* **1972**, *11*, 2616–2617; b) N. Langlois, P. Potier, *Phytochemistry* **1972**, *11*, 2617–2618; c) A. Rabaron, M. Plat, P. Potier, *Plant. Med. Phytother.* **1973**, *7*, 53–58.

[5] A. El-Sayed, G. A. Handy, G. A. Cordell, *J. Nat. Prod.* **1983**, *46*, 517–527.

[6] R. N. Bowman, R. J. Jondle, **1999**. Trimeric and polymeric alkaloids, PCT/US99/17177.

[7] S. H. Tiong, C. Y. Looi, H. Hazni, A. Arya, M. Paydar, W. F. Wong, S.-C. Cheah, M. R. Mustafa, K. Awang, *Molecules* **2013**, *18*(8), 9770–9784.

[8] R. Z. Andriamialisoa, N. Langlois, Y. Langlois, P. Potier, *Tetrahedron* **1980**, *36*, 3053–3060.

[9] a) S. Ali, E. Hénon, R. Leroy, G. Massiot, *Molecules* **2021**, *26*, 6395; b) P. Keglevich, L. Hazaj, Á. Gorka-Kereskényi, L. Péter, J. Gyenese, Zs. Lengyel, Gy. Kalaus, Zs. Dubrovay, M. Dékány, E. Orbán, Z. Bánóczi, Cs. Szántay Jr., Cs. Szántay, *Heterocycles* **2013**, *87*, 2299–2317; c) A. Keglevich, Á. Szigetvári, M. Dékány, Cs. Szántay Jr., P. Keglevich, L. Hazaj, *Curr. Org. Chem.* **2019**, *23*, 852–858; d) A. Keglevich, V. Zsiros, P. Keglevich, Á.
7.2 Hz, H-18); ¹³C-NMR (125 MHz CD₃OD): δ 202.8 (CHCHCO), 173.6, 173.4, 173.3 (COOCH₃), 172.5, 172.4, 172.3 (OCOCH₃), 162.8, 159.8, 159.7 (C-11), 157.7, 153.2, 153.1 (C-13), 140.8 (CH=CHCO), 131.3, 131.3, 131.2 (C-15), 122.0, 123.7, 123.6 (C-9), 125.8, 125.1, 126.7 (C-8), 125.7, 125.7, 125.9 (C-14), 124.4, 124.9, 115.0 (C-10), 123.7 (CH=CHCO), 95.5, 95.1, 93.0 (C-12), 84.8, 84.4, 84.1 (C-2), 81.2, 80.9, 80.8 (C-16), 77.6, 77.5, 77.4 (C-17), 68.2, 67.7, 67.2 (C-21), 56.4, 56.3, 56.3 (C-11 OCH₃), 54.3, 53.9 (C-7), 53.0, 52.9, 52.8, (COOCH₃), 53.0, 52.8, 52.6 (C-5), 52.1, 51.9, 51.7 (C-3), 45.1 (CHCH₂CO), 44.8, 44.6 (C-6), 44.2, 44.2, 44.4 (C-20), 35.8 (C-10) CHCH₂CO, 37.5, 39.3, 39.5 (C-1), 32.2, 32.1, 31.8 (C-19), 20.8 (OCOCH₃), 8.75, 8.73, 8.03 (C-18).

## Synthesis of compound 14

Trifluoroacetic acid (4 mL) was added to a stirred solution of 13 (300 mg, 0.30 mmol) and 2 (600 mg, 1.31 mmol) in DCM (12 mL) and H₂O (6 mL). The green mixture was stirred for 48 hours at room temperature. The reaction mixture was neutralized with sodium carbonate and extracted with DCM. The extract dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified on alumina to give compounds 14 (150 mg, 37%): white powder; [α]₂D = -120 (c 0.3, MeOH); mp = 290-300 °C; Rf = 0.1 (silica gel, MeOH/DCM 96/4); UV (λmax MeOH): 219, 261, 311; IR (KBr): 3305, 2935, 2871, 2360, 2341, 2073, 2079, 2002, 1735, 1615, 1498, 1455, 1432, 1371, 1223, 929, 872, 817, 742, 699, 683, 669, cm⁻¹; ESI-MS (+ ve) m/z 1905.9 (M + H); ¹H-NMR (600 MHz, CD₃OD): δ 6.77, 6.75 (4H, 2s, H-9), 6.22, 6.20 (4H, s, H-12), 5.85 (4H, m, H-14), 5.34, 5.29 (4H, 2s, H-17), 5.16 (6 H, m, CHCH₂CO H-15), 3.77, 3.73 (12H, 2s, C-11 OCH₃), 3.76 (12H, s, COOCH₃), 3.54, 3.51 (4H, 2s, H-2), 3.47 (4H, m, H-3b), 3.45 (4H, m, H-5), 2.88 (4H, m, CHCH₂CO), 2.79 (4H, m, H-3a), 2.67, 2.62 (12H, 2s, N-CH₃), 2.62, 2.56 (4H, 2s, H-21), 2.53 (4H, m, H-5), 2.22 (8H, m, H-6), 1.99, 1.98 (12H, 2s, OCOCH₃), 1.52 (4H, m, H-19), 1.04, (4H, m, H-19), 0.41, 0.30 (12H, 2t, J₁₈,₁₉ = 7.2 Hz, H-18); ¹³C-NMR (150 MHz, CD₃OD): δ 213 (CHCH₂CO), 173.5, 173.4 (COOCH₃), 172.4, 172.4 (OCOCH₃), 159.8, 159.6 (C-11), 153.3, 153.2 (C-13), 131.3, 131.2 (C-15), 125.6, 124.6 (C-8), 125.7, 125.6 (C-14), 123.4, 122.7 (C-9), 124.7, 124.6 (C-10), 95.6, 95.0 (C-12), 84.7, 84.4 (C-2) 80.9 (C-16), 77.6, 77.4 (C-17), 67.8, 67.7 (C-21), 56.2 (C-11 OCH₃), 52.8 (COOCH₃), 54.4, 54.3 (C-7), 52.0, 51.9 (C-5), 52.6 (C-3), 48.8 (CHCH₂CO), 44.9, 44.8 (C-6), 44.9, 44.2 (C-20), 35.2 (CHCH₂CO), 39.6, 39.2 (N-CH₃), 32.0, 31.8 (C 19), 20.7 (OCOCH₃), 8.81, 8.68 (C-18).

## Synthesis of vindolicinone 15

Vindolicine 1 (100 mg, 0.10 mmol) and 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) (181 mg, 0.79 mmol) were dissolved in 0.8 mL diethyl ether at 0 °C under N₂. Water (100 µL) and acetic acid (100 µL) were added, and the solution was stirred for 6 hours at 0 °C, and 24 h at room temperature. The reaction mixture was neutralized with aqueous sodium carbonate and extracted with DCM Purification of the crude reaction mixture on alumina gave compound 15 (60 mg, 60%, eluted with hexane/acetone 3:7) as a lemon yellow powder; [α]₂D = -135° (c 0.007, MeOH); mp = 232-235 °C, UV (MeOH): λmax(log ε) 229, 261 and 370 (3.99) nm; IR (KBr): 3440, 2965, 2855, 1735, 1610, 1498, 1461, 1372, 1249, 1119, 1172, 1044, 740 cm⁻¹; ESI-MS (+ ve) m/z: 939 (M + H)⁺; ¹H-NMR (600 MHz, CD₃OD): δ 7.24 (2H, s, H-9), 6.18 (2H, s,H-12), 5.87 (2H, brdd, J = 9.5, 4.5 Hz, H-14), 5.33 (2H, s, H-17), 5.20 (2H, brd, J = 9.5 Hz, H-15), 3.79 (8H, s, COOCH₃, H-2), 3.62 (6H, s, C-11 OCH₃), 3.46 (2H, dd, J = 16.2, 4.5 Hz, H-3), 3.42 (2H, m, H-5), 2.90 (2H, bd, J = 16.2 Hz, H-3), 2.83 (2H, s, H-21), 2.78 (6H, s, N-CH₂), 2.66 (2H, m, H-5), 2.33, (4H, m, 2 H-6), 2.02 (6H, s, OCOCH₃), 1.62 (2H, dq, J = 14.7, 7.2 Hz, H-19), 1.21 (2H, dq, J = 14.7, J = 7.2 Hz, H-19), 0.55 (6H, t, J = 7.2 Hz, H-18); ¹³C-NMR (150 MHz, CD₃OD): δ 195.6 (C-10), 173.4 (COOCH₃), 172.5 (OCOCH₃), 163.1 (C-11), 157.9 (C-13), 131.2 (C-15), 126.2 (C-9), 125.9

(C-14), 125.4 (C-8), 123.3 (C-10), 93.0 (C-12), 84.2 (C-2), 80.9 (C-16), 77.5 (C-17), 67.8 (C-21), 56.2, (C-11 OCH₃), 53.8 (C-7), 53.0 (COOCH₃), 52.3 (C-5), 51.8 (C-3), 44.4 (C-6), 44.3 (C-20), 37.7 (N-CH₃), 32.0 (C 19), 20.8 (OCOCH₃), 8.1 (C-18).

## Author Contributions

This project was devised and conducted by G.M. who also wrote most of the article. Experiments were done by Asia who also wrote a preliminary draft of the article; The X-ray structure elucidations were performed by Y. S. and L.V.

## Acknowledgements

Supervision and guidance by Prof. Dr. M. Iqbal Choudhary are gratefully acknowledged. Interactions with Prof. Dr. Atta-ur-Rahman were greatly appreciated. Invaluable help from Prof. Dr. Hina Siddiqui during the last stages of the redaction permitted to achieve the project. We wish to thank the technical staff of ICBSS for all the analytical and biochemical measurements. Prof. J. D. Connolly, the University of Glasgow is thanked for bringing a final touch to the article.

## Conflict of Interests

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

**Keywords:** vindoline • vindolicine • cytotoxicity • symmetry

[1] G. H. Svoboda, M. Gorman, N. Neuss, A. J. Barnes Jr., *J. Pharm. Sci.* **1961**, *50*, 409–413.

[2] M. Gorman, J. Sweeny, **1964**, C-6-3 Vinca Alkaloids XXIII Vindolicine (Alkaloids and alkaloid biosynthesis). In International Symposium on the Chemistry of Natural Products (pp. 99–100). the Science Council of Japan under the Auspices of the International Union of Pure and Applied Chemistry.

[3] D. E. Dorman, J. W. Paschal, *Org. Magn. Reson.* **1976**, *8*, 413–418.

[4] a) P. Rasoanaivo, N. Langlois, P. Potier, *Phytochemistry* **1972**, *11*, 2616–2617; b) N. Langlois, P. Potier, *Phytochemistry* **1972**, *11*, 2617–2618; c) A. Rabaron, M. Plat, P. Potier, *Plant. Med. Phytother.* **1973**, *7*, 53–58.

[5] A. El-Sayed, G. A. Handy, G. A. Cordell, *J. Nat. Prod.* **1983**, *46*, 517–527.

[6] R. N. Bowman, R. J. Jondle, **1999**. Trimeric and polymeric alkaloids, PCT/US99/17177.

[7] S. H. Tiong, C. Y. Looi, H. Hazni, A. Arya, M. Paydar, W. F. Wong, S.-C. Cheah, M. R. Mustafa, K. Awang, *Molecules* **2013**, *18*(8), 9770–9784.

[8] R. Z. Andriamialisoa, N. Langlois, Y. Langlois, P. Potier, *Tetrahedron* **1980**, *36*, 3053–3060.

[9] a) S. Ali, E. Hénon, R. Leroy, G. Massiot, *Molecules* **2021**, *26*, 6395; b) P. Keglevich, L. Hazaj, Á. Gorka-Kereskényi, L. Péter, J. Gyenese, Zs. Lengyel, Gy. Kalaus, Zs. Dubrovay, M. Dékány, E. Orbán, Z. Bánóczi, Cs. Szántay Jr., Cs. Szántay, *Heterocycles* **2013**, *87*, 2299–2317; c) A. Keglevich, Á. Szigetvári, M. Dékány, Cs. Szántay Jr., P. Keglevich, L. Hazaj, *Curr. Org. Chem.* **2019**, *23*, 852–858; d) A. Keglevich, V. Zsiros, P. Keglevich, Á.
Szigetvári, M. Dékány, Cs. Szántay Jr., E. Mernyák, J. Wölfling, L. Hazai, *Curr. Org. Chem.* **2019**, *23*, 959–967; e) A. Keglevich, L. Dányi, A. Rieder, D. Horváth, Á. Szigetvári, M. Dékány, Cs. Szántay Jr., A. D. Latif, A. Hunyadi, I. Zupkó, P. Keglevich, L. Hazai, *Molecules* **2020**, *25*, 1010; f) D. Passarella, A. Giardini, B. Peretto, G. Fontana, A. Sacchetti, A. Silvani, C. Ronchi, G. Cappelletti, D. Cartelli, J. Borlak, B. Danielli, *Bioorg. Med. Chem.* **2008**, *16*, 6269–6285.

[10] a) T. C. Turner, K. Shibayama, D. L. Boger, *Org. Lett.* **2013**, *15*, 1100–1103;
b) J. Zhang, S. R. Paladugu, R. M. Gillard, A. Sarkar, D. L. Boger, *J. Am. Chem. Soc.* **2022**, *144*, 495–502.

[11] G. Guirimand, A. Guihur, P. Poutrain, F. Héricourt, S. Mahroug, B. St-Pierre, V. Burlat, V. Courdavault, *J. Plant Physiol.* **2011**, *168*, 549–557.

[12] R. Besselièvre, N. Langlois, P. Potier, *Bull. Soc. Chim. Fr.* **1972**, 1477–1478.

[13] a) A. Keglevich, Sz. Mayer, R. Pápai, Á. Szigetvári, Zs. Sánta, M. Dékány, Cs. Szántay Jr., P. Keglevich, L. Hazai, *Molecules* **2018**, *23*, 2574; b) G. Sun, X. Lv, Y. Zhang, M. Lei, L. Hu, *Org. Lett.* **2017**, *19*, 4235–4238.

[14] Á. Szigetvári, A. Keglevich, P. Keglevich, M. Dékány, L. Hazai, Cs. Szántay Jr., *Struct. Chem.* **2019**, *30*, 795–804.

[15] J. B. Baell, *J. Nat. Prod.* **2016**, *79*, 616–628.

[16] a) Y. Chen, M. G. de Lomana, N.-O. Friedrich, J. Kirchmair, *J. Chem. Inf. Model.* **2018**, *58*, 1518–1532; b) S. Wetzel, A. Schuffenhauer, S. Roggo, P. Ertl, H. Waldmann, *Chimia* **2007**, *61*, 355–355.

[17] A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, *J. Appl. Crystallogr.* **1993**, *26*, 343–350.

[18] SHELX97 [Includes SHELXS97, SHELXL97, CIFTAB] - Programs for Crystal Structure Analysis (Release 97-2). G. M. Sheldrick, Institut für Anorganische Chemie der Universität, Tammanstrasse 4, D-3400 Göttingen, Germany, 1998.

[19] L. J. Farrugia, *J. Appl. Crystallogr.* **1999**, *32*, 837–838.

[20] INTERNATIONAL tables for X-Ray crystallography, 1974, Vol IV, Kynoch press, Birmingham, England.

<footnote>Manuscript received: December 6, 2023</footnote>

<footnote>Version of record online: ■■, ■■</footnote>
Szigetvári, M. Dékány, Cs. Szántay Jr., E. Mernyák, J. Wölfling, L. Hazai, *Curr. Org. Chem.* **2019**, *23*, 959–967; e) A. Keglevich, L. Dányi, A. Rieder, D. Horváth, Á. Szigetvári, M. Dékány, Cs. Szántay Jr., A. D. Latif, A. Hunyadi, I. Zupkó, P. Keglevich, L. Hazai, *Molecules* **2020**, *25*, 1010; f) D. Passarella, A. Giardini, B. Peretto, G. Fontana, A. Sacchetti, A. Silvani, C. Ronchi, G. Cappelletti, D. Cartelli, J. Borlak, B. Danielli, *Bioorg. Med. Chem.* **2008**, *16*, 6269–6285.

[10] a) T. C. Turner, K. Shibayama, D. L. Boger, *Org. Lett.* **2013**, *15*, 1100–1103;
b) J. Zhang, S. R. Paladugu, R. M. Gillard, A. Sarkar, D. L. Boger, *J. Am. Chem. Soc.* **2022**, *144*, 495–502.

[11] G. Guirimand, A. Guihur, P. Poutrain, F. Héricourt, S. Mahroug, B. St-Pierre, V. Burlat, V. Courdavault, *J. Plant Physiol.* **2011**, *168*, 549–557.

[12] R. Besselièvre, N. Langlois, P. Potier, *Bull. Soc. Chim. Fr.* **1972**, 1477–1478.

[13] a) A. Keglevich, Sz. Mayer, R. Pápai, Á. Szigetvári, Zs. Sánta, M. Dékány, Cs. Szántay Jr., P. Keglevich, L. Hazai, *Molecules* **2018**, *23*, 2574; b) G. Sun, X. Lv, Y. Zhang, M. Lei, L. Hu, *Org. Lett.* **2017**, *19*, 4235–4238.

[14] Á. Szigetvári, A. Keglevich, P. Keglevich, M. Dékány, L. Hazai, Cs. Szántay Jr., *Struct. Chem.* **2019**, *30*, 795–804.

[15] J. B. Baell, *J. Nat. Prod.* **2016**, *79*, 616–628.

[16] a) Y. Chen, M. G. de Lomana, N.-O. Friedrich, J. Kirchmair, *J. Chem. Inf. Model.* **2018**, *58*, 1518–1532; b) S. Wetzel, A. Schuffenhauer, S. Roggo, P. Ertl, H. Waldmann, *Chimia* **2007**, *61*, 355–355.

[17] A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, *J. Appl. Crystallogr.* **1993**, *26*, 343–350.

[18] SHELX97 [Includes SHELXS97, SHELXL97, CIFTAB] - Programs for Crystal Structure Analysis (Release 97-2). G. M. Sheldrick, Institut für Anorganische Chemie der Universität, Tammanstrasse 4, D-3400 Göttingen, Germany, 1998.

[19] L. J. Farrugia, *J. Appl. Crystallogr.* **1999**, *32*, 837–838.

[20] INTERNATIONAL tables for X-Ray crystallography, 1974, Vol IV, Kynoch press, Birmingham, England.

<footnote>Manuscript received: December 6, 2023</footnote>

<footnote>Version of record online: ■■, ■■</footnote>