Cytotoxic Cyclolignans Obtained by the Enlargement of the Cyclolignan Skeleton of Podophyllic Aldehyde, a Selective Podophyllotoxin-Derived Cyclolignan

Podophyllotoxin, a cyclolignan natural product, has been the object of extensive chemomodulation to obtain better chemotherapeutic agents. Among the obtained podophyllotoxin derivatives, podophyllic aldehyde showed very interesting potency and selectivity against several tumoral cell lines, so it became our lead compound for further modifications, as described in this work, oriented toward the enlargement of the cyclolignan skeleton. Thus, modifications performed at the aldehyde function included nucleophilic addition reactions and the incorporation of the aldehyde carbon into several five-membered rings, such as thiazolidinones and benzo-fused azoles. The synthesized derivatives were evaluated against several types of cancer cells, and although some compounds were cytotoxic at the nanomolar range, most of them were less potent and less selective than the parent compound podophyllic aldehyde, with the most potent being those having the lactone ring of podophyllotoxin. In silico ADME evaluation predicted good druggability for most of them. The results indicate that the γ-lactone ring is important for potency, while the α,β-unsaturated aldehyde is necessary to induce selectivity in these cyclolignans.


Introduction
Natural products constitute a good source of bioactive compounds that can be used directly as drugs or, most frequently, as hits for developing new drugs.Also, natural products without any activity reports have often served as starting materials for the preparation of bioactive derivatives [1][2][3].We usually refer to the concepts of improving and inducing bioactivity by chemical transformations as the chemomodulation and chemoinduction of bioactivity, respectively [4].For many years, our research group has been involved in both the chemomodulation and chemoinduction of the bioactivity of natural compounds, such as cyclolignans, isolated from their natural sources.
Cyclolignans belong to the lignan family of natural compounds, which are widely distributed in Nature.Many biological activities have been described for them, such as anthelmintic, antiviral, or anticancer effects [5].Among lignans, podophyllotoxin stands out for its use as an antiviral in the treatment of venereal warts and, principally, as the starting material for obtaining the clinically used anticancer drugs etoposide and etopophos (Figure 1) [6][7][8][9].It is well known that both the natural compound and its semisynthetic derivatives have different mechanisms of action: while podophyllotoxin inhibits tubulin polymerization, etoposide and its analogs are inhibitors of the DNA-topoisomerase II enzyme.This change in the mechanism of action has been related to some structural changes in the cyclolignan skeleton, such as epimerization and glycosylation at C7 and O-demethylation at C4 ′ [10].The numbering of the cyclolignan skeleton used in this work (Figure 1) is in accordance with IUPAC rules for lignans, which is based on the two phenylpropanoid biogenetic subunits whose condensation leads to lignans.Thus, one of the subunits is numbered from 1 to 9 and the other from 1 ′ to 9 ′ (Figure 1) [11].
Molecules 2024, 29, x FOR PEER REVIEW 2 of 20 out for its use as an antiviral in the treatment of venereal warts and, principally, as the starting material for obtaining the clinically used anticancer drugs etoposide and etopophos (Figure 1) [6][7][8][9].It is well known that both the natural compound and its semisynthetic derivatives have different mechanisms of action: while podophyllotoxin inhibits tubulin polymerization, etoposide and its analogs are inhibitors of the DNA-topoisomerase II enzyme.This change in the mechanism of action has been related to some structural changes in the cyclolignan skeleton, such as epimerization and glycosylation at C7 and Odemethylation at C4′ [10].The numbering of the cyclolignan skeleton used in this work (Figure 1) is in accordance with IUPAC rules for lignans, which is based on the two phenylpropanoid biogenetic subunits whose condensation leads to lignans.Thus, one of the subunits is numbered from 1 to 9 and the other from 1′ to 9′ (Figure 1) [11].During the last decades, extensive chemomodulation of podophyllotoxin, 1, has been performed by our research group, modifying nearly all the rings and functions of the cyclolignan skeleton [4], including hybridization with other biologically active natural compound derivatives, such as purines [12] or terpenylhydroquinones [13].Such transformations are summarized in Figure 2 and had a variable influence in terms of structureactivity relationship (SAR) analysis.Most of the derivatives retained cytotoxicity at the micromolar level, with several differences.Noteworthy was the opening of the A-ring and the formation of pyrazole-and oxazole-fused rings, affecting the C-and D-rings, which showed interesting immunosuppressive activity either in vitro or in vivo [14], although they were less cytotoxic than podophyllotoxin.Also, hybrids formed by the union with another bioactive fragment, through position C7, C9, or C9′, led to promising agents with dual mechanisms of action [13].
Among the large number of podophyllotoxin derivatives obtained by our group, special mention should be made of podophyllotoxin-derived cyclolignans lacking the lactone ring, such as podophyllic aldehyde (2, Figure 1) and its imine derivatives, which showed selective cytotoxicity against certain human tumor cells, so 2 became our lead compound for further modifications.The antitumor evaluation of podophyllic aldehyde derivatives indicated that they kept the same mechanism of action as podophyllotoxin, and the α,βunsaturated aldehyde function was important for selectivity, while the nature and size of the chain attached to the carboxylic acid at C9′ had variable effects on cytotoxicity and selectivity, with evidence of a possible third mechanism of action for podophyllotoxinrelated lignans [15,16].
As a continuation of our research related to the chemomodulation of the bioactivity of cyclolignans, and with the aim of emphasizing the importance of the bioactivity of an untouched α,β-unsaturated aldehyde function at C9, in this work, we describe further chemical modifications performed at this aldehyde function, oriented toward the enlargement of the cyclolignan skeleton and its influence on bioactivity.They include the formation of vinylogues, reactions with different nucleophiles, and the incorporation of the aldehyde carbon into heterocyclic rings at the C2 position of several five-membered rings, such as thiazolidin-4-one and benzo-fused azoles (benzoxazole, benzothiazole, During the last decades, extensive chemomodulation of podophyllotoxin, 1, has been performed by our research group, modifying nearly all the rings and functions of the cyclolignan skeleton [4], including hybridization with other biologically active natural compound derivatives, such as purines [12] or terpenylhydroquinones [13].Such transformations are summarized in Figure 2 and had a variable influence in terms of structure-activity relationship (SAR) analysis.Most of the derivatives retained cytotoxicity at the micromolar level, with several differences.Noteworthy was the opening of the A-ring and the formation of pyrazole-and oxazole-fused rings, affecting the C-and D-rings, which showed interesting immunosuppressive activity either in vitro or in vivo [14], although they were less cytotoxic than podophyllotoxin.Also, hybrids formed by the union with another bioactive fragment, through position C7, C9, or C9 ′ , led to promising agents with dual mechanisms of action [13].

Chemistry
Podophyllic aldehyde, 2, was obtained from podophyllotoxin, 1, isolated from the rhizome resin of Podophyllum emodi [17], following the procedure previously described by Among the large number of podophyllotoxin derivatives obtained by our group, special mention should be made of podophyllotoxin-derived cyclolignans lacking the lactone ring, such as podophyllic aldehyde (2, Figure 1) and its imine derivatives, which showed selective cytotoxicity against certain human tumor cells, so 2 became our lead compound for further modifications.The antitumor evaluation of podophyllic aldehyde derivatives indicated that they kept the same mechanism of action as podophyllotoxin, and the α,β-unsaturated aldehyde function was important for selectivity, while the nature and size of the chain attached to the carboxylic acid at C9 ′ had variable effects on cytotoxicity and selectivity, with evidence of a possible third mechanism of action for podophyllotoxinrelated lignans [15,16].
As a continuation of our research related to the chemomodulation of the bioactivity of cyclolignans, and with the aim of emphasizing the importance of the bioactivity of an untouched α,β-unsaturated aldehyde function at C9, in this work, we describe further chemical modifications performed at this aldehyde function, oriented toward the enlargement of the cyclolignan skeleton and its influence on bioactivity.They include the formation of vinylogues, reactions with different nucleophiles, and the incorporation of the aldehyde carbon into heterocyclic rings at the C2 position of several five-membered rings, such as thiazolidin-4-one and benzo-fused azoles (benzoxazole, benzothiazole, benzimidazole), purine, and 1-deazapurine.Subsequent cytotoxicity assays of the obtained derivatives were performed on three types of human cancer cells.

Chemistry
Podophyllic aldehyde, 2, was obtained from podophyllotoxin, 1, isolated from the rhizome resin of Podophyllum emodi [17], following the procedure previously described by us [15].To achieve our objective of producing new bioactive podophyllic aldehyde derivatives, several carbon and heteroatom nucleophiles were used, taking advantage of the electrophilic character of the aldehyde function and the easiness of nucleophilic addition reactions.

Addition of Carbon Nucleophiles
Several vinylogous-like derivatives of 2 were prepared by a reaction with nitromethane and through Wittig reactions to obtain the extended derivatives 3-8 (Scheme 1).
Molecules 2024, 29, x FOR PEER REVIEW 4 of 20 conditions, while in compound 8, both ester groups were reduced, without the alteration of the double bond in any of them, as deduced from their NMR data.Grignard and Reformatsky reagents were also used as C-nucleophiles (Scheme 1).Thus, the reaction of 2 with methylmagnesium iodide led to a complex reaction product, in whose 1 H and 13 C NMR spectra were signals assignable to the olefinic proton and carbon at position C7 (6.34 and 119 ppm approx., respectively), while signals for the aldehyde function were absent, as were those of the methyl ester at C9′ (around 3.60 and 55 ppm).Instead, two doublets at 1.63 and 1.54 ppm were present.All of these data indicated the addition of the organomagnesium compound to the aldehyde with relactonization [20] to give a mixture of isomers, 9. Chromatography of the reaction product led to the isolation of small amounts of compounds 9a-c.Compounds 9a and 9b were epimers at C9, in which the migration of the Δ 7 double bond toward Δ 8(8′) occurred during chromatography, while in 9c, the aromatization of the C-ring took place.The configuration of C9 in 9a and 9b was defined through nOe (nuclear Overhauser effect) NMR experiments.Thus, a positive nOe effect for compound 9a between the methyl at C9 and the aromatic protons of the pendant trimethoxyphenyl ring indicated an S configuration for carbon C9.The absence of such an nOe effect for 9b allowed us to assign the R configuration to 9b.Scheme 1.Reactions of podophyllic aldehyde, 2, with C-nucleophiles.
When 2 was treated with ethyl bromodifluoroacetate in the presence of activated zinc dust in THF under reflux, compound 10 was isolated from the reaction product as a single epimer, in which not only did condensation with the aldehyde group take place, but relactonization also occurred again.The structure and configuration of 10 were determined after the analysis of its NMR spectra, 2D NMR experiments, and nOe effects.In this reaction, only epimer 10 was detected, probably because the additional coordination of zinc The nitro derivative 3 was obtained by a reaction with nitromethane in the presence of ammonium acetate.When the reaction was performed in glacial acetic acid as a solvent under reflux [18], the unreacted aldehyde 2 was recovered.However, when nitromethane was used as a reagent and as a solvent [19], the nitro derivative 3 was obtained in low yield (7%), also recovering the unreacted aldehyde 2 (39%).HMBC and HMQC NMR experiments confirmed the structure of 3 (Figures S3 and S4 in Supplementary Materials).
Analogs 4-6 were obtained through the Wittig reaction using triphenyl phosphonium ylides stabilized by carbonyl groups at the α-position.When (thiphenylphosphoranylidene)acetaldehyde was used, an irresolvable mixture of the starting aldehyde and vinylogue 4 was obtained.A careful analysis of the NMR spectra of the mixture allowed us to identify the signals corresponding to the extended aldehyde 4.
The reduction of the diester 6 with lithium aluminum hydride at a low temperature led to a mixture of 7 and 8.For compound 7, only the ester at C9 ′ was reduced under such conditions, while in compound 8, both ester groups were reduced, without the alteration of the double bond in any of them, as deduced from their NMR data.
Grignard and Reformatsky reagents were also used as C-nucleophiles (Scheme 1).Thus, the reaction of 2 with methylmagnesium iodide led to a complex reaction product, in whose 1 H and 13 C NMR spectra were signals assignable to the olefinic proton and carbon at position C7 (6.34 and 119 ppm approx., respectively), while signals for the aldehyde function were absent, as were those of the methyl ester at C9 ′ (around 3.60 and 55 ppm).Instead, two doublets at 1.63 and 1.54 ppm were present.All of these data indicated the addition of the organomagnesium compound to the aldehyde with relactonization [20] to give a mixture of isomers, 9. Chromatography of the reaction product led to the isolation of small amounts of compounds 9a-c.Compounds 9a and 9b were epimers at C9, in which the migration of the ∆ 7 double bond toward ∆ 8(8 ′ ) occurred during chromatography, while in 9c, the aromatization of the C-ring took place.The configuration of C9 in 9a and 9b was defined through nOe (nuclear Overhauser effect) NMR experiments.Thus, a positive nOe effect for compound 9a between the methyl at C9 and the aromatic protons of the pendant trimethoxyphenyl ring indicated an S configuration for carbon C9.The absence of such an nOe effect for 9b allowed us to assign the R configuration to 9b.
When 2 was treated with ethyl bromodifluoroacetate in the presence of activated zinc dust in THF under reflux, compound 10 was isolated from the reaction product as a single epimer, in which not only did condensation with the aldehyde group take place, but relactonization also occurred again.The structure and configuration of 10 were determined after the analysis of its NMR spectra, 2D NMR experiments, and nOe effects.In this reaction, only epimer 10 was detected, probably because the additional coordination of zinc with the C9 ′ -β-ester made the nucleophile approach the zinc-chelated aldehyde intermediate via the less hindered side only.
The presence of two electronegative fluorine atoms in the reagent was important for the reaction progress.When the same reaction was performed with ethyl bromoacetate under the same reaction conditions, mainly unreacted aldehyde was recovered, and only a small amount of dehydrodeoxypodophyllotoxin [21], resulting from the reduction of the aldehyde with the subsequent relactonization and aromatization of the C-ring, was detected.

Addition of Nitrogen Nucleophiles
In our previous research, several N-nucleophiles, namely, substituted aromatic and aliphatic hydrazines, hydroxylamines, and amines, were used to obtain the corresponding hydrazones, oximes, and imines, for which very good results in potency and selectivity were obtained, particularly for the imine derivatives [22,23].In those works, some of the aromatic amines used to form previous imines contained another heteroatom (N, O, S), located at the ortho position of the amine group, that can also act as a second nucleophile and add to the imine double bond.We describe here this kind of transformation to achieve the corresponding benzo-fused azoles that incorporate the C9 carbon of the cyclolignan skeleton into the additional five-membered heterocycle formed.
Thus, 2 was made to react with 1,2-phenylendiamines to obtain benzimidazoles 11-15, with moderate to good yields.When the reaction with 1,2-phenylendiamine itself was performed in refluxing ethanol and acidic conditions, a transesterification reaction occurred, and the ethylester 12 was obtained in moderate yield.To prevent transesterification, acidic conditions were avoided, and instead, an oxidant was added to facilitate the final aromatization of benzimidazoles 11-15, which were then obtained with better yields.When o-aminophenols or o-aminothiophenols were used as nucleophiles, the corresponding benzoxazoles 16-18 and benzothiazoles 19-20 were obtained in moderate yields (Scheme 2).
aliphatic hydrazines, hydroxylamines, and amines, were used to obtain the corresponding hydrazones, oximes, and imines, for which very good results in potency and selectivity were obtained, particularly for the imine derivatives [22,23].In those works, some of the aromatic amines used to form previous imines contained another heteroatom (N, O, S), located at the ortho position of the amine group, that can also act as a second nucleophile and add to the imine double bond.We describe here this kind of transformation to achieve the corresponding benzo-fused azoles that incorporate the C9 carbon of the cyclolignan skeleton into the additional five-membered heterocycle formed.
Thus, 2 was made to react with 1,2-phenylendiamines to obtain benzimidazoles 11-15, with moderate to good yields.When the reaction with 1,2-phenylendiamine itself was performed in refluxing ethanol and acidic conditions, a transesterification reaction occurred, and the ethylester 12 was obtained in moderate yield.To prevent transesterification, acidic conditions were avoided, and instead, an oxidant was added to facilitate the final aromatization of benzimidazoles 11-15, which were then obtained with better yields.When o-aminophenols or o-aminothiophenols were used as nucleophiles, the corresponding benzoxazoles 16-18 and benzothiazoles 19-20 were obtained in moderate yields (Scheme 2).
Diaminopyridine and diaminopyrimidine were also used as nucleophiles, leading, in these cases, to the 1-deazapurinyl and purinyl derivatives 21 and 22, respectively (Scheme 2).With the aim to enlarge the type of heterocycles attached to C8, several 1,3-thiazolidin-4-one derivatives, substituted at the nitrogen atom, were also obtained from the condensation of 2 with several amines and the subsequent reaction of the formed imines 23-27 with thioglycolic acid to yield thiazolidinones 28-32 as mixtures of epimers at C9 (Scheme 3), as deduced from the presence of several duplicated signals in their NMR spectra (Figures S38 to S44 in Supplementary Materials).Diaminopyridine and diaminopyrimidine were also used as nucleophiles, leading, in these cases, to the 1-deazapurinyl and purinyl derivatives 21 and 22, respectively (Scheme 2).
With the aim to enlarge the type of heterocycles attached to C8, several 1,3-thiazolidin-4-one derivatives, substituted at the nitrogen atom, were also obtained from the condensation of 2 with several amines and the subsequent reaction of the formed imines 23-27 with thioglycolic acid to yield thiazolidinones 28-32 as mixtures of epimers at C9 (Scheme 3), as deduced from the presence of several duplicated signals in their NMR spectra (Figures S38-S44 in Supplementary Materials).The formation of thiazolidinones 28-32 occurs in two steps: first the formation of the corresponding imines 23-27, followed by condensation with thioglycolic acid.So, it can be carried out either in one pot, with the addition of thioglycolic acid to the reaction flask after the imine is formed, or with the previous isolation of the corresponding imine and further reaction with thioglycolic acid.For the latter, the corresponding imines 23-27 were prepared following the procedure described by us [23].Thus, thiazolidinone 29 was obtained by both procedures with similar yields, while thiazolidinone 30 was only obtained The formation of thiazolidinones 28-32 occurs in two steps: first the formation of the corresponding imines 23-27, followed by condensation with thioglycolic acid.So, it can be carried out either in one pot, with the addition of thioglycolic acid to the reaction flask after the imine is formed, or with the previous isolation of the corresponding imine and further reaction with thioglycolic acid.For the latter, the corresponding imines 23-27 were prepared following the procedure described by us [23].Thus, thiazolidinone 29 was obtained by both procedures with similar yields, while thiazolidinone 30 was only obtained in one pot, and thiazolidinones 28, 31, and 32 were obtained by the two-step procedure, as specified in the Section 3.1.

Bioactivity
The cytotoxicity of most compounds was evaluated in vitro, using the colorimetric sulforhodamine B (SRB) method, to determine their in vitro antiproliferative activity against a panel of three human tumor cell lines: A549 (non-small-cell lung carcinoma), HT-29 (colon adenocarcinoma), and MEL-28 (malignant melanoma).The results are shown in Figure 3 and in Table S1 (GI 50 values in µM).The natural compound podophyllotoxin, 1, and podophyllic aldehyde, 2, were included as references.In Figure 3, the 0 value on the y-axis coincides with the GI 50 value of 1 µM (values are expressed as -log GI 50 ) so that positive values correspond to cytotoxicity values below the micromolar level (higher cytotoxicity, GI 50 < 1 µM) and negative values to lower cytotoxicity (GI 50 > 1 µM).Each cell line is represented separately, with the figure itself indicating the different groups of compounds studied, arranged as follows: precursors (A: 1 and 2), C9 vinylogous derivatives (B: 3-8), lactone derivatives (C: 9-10), benzoheteroazoles (D: 11-20), and thiazolidinones (E: 28-32).
From these results, several considerations of the structure-activity relationship can be deduced.It can be stated that many of them were cytotoxic, but in general, the selectivity toward a certain cell line disappeared, with a few exceptions.
Among the vinylogous derivatives, compounds 3, 4, and 5 retained cytotoxicity under the µM level, in the same range as their parent compound 2, but with the loss of selectivity against HT-29 cells, while derivative 6 showed a certain selectivity, being fivefold more potent against HT-29 than against the other two cell lines tested, but with a lower potency, which was partially recovered in A549 and MEL-28 when the ester groups were reduced to the corresponding alcohols (7 and 8 vs. 6).
The best cytotoxicity results were observed for the 9-methyl lactones 9, 9a, and 9b.These compounds showed GI 50 values in the same range as that of 1 on the three cell lines tested and similar to that of 2 on HT-29 cells, but without the selectivity observed for podophyllic aldehyde between this cell line and the other two.However, the presence of a bulkier substituent at that position led to a considerable decrease in potency (10 vs. 9).It can also be observed that the mixture of epimers 9, with a ∆ 7 double bond, was nearly three times less potent than the single isomers 9a and 9b, which had a ∆ 8(8 ′ ) double bond, while no difference was observed between epimers (9 vs. 9a and 9b).
Several differences can be observed among the evaluated benzazoles, which incorporated the C9 carbon into heterocyclic rings.Thus, benzimidazoles 11-15 retained cytotoxicity at the µM level, while benzoxazoles 16-18, benzothiazoles 19-20, and the purinyl derivative 22 were nearly inactive; only the deazapurinyl analog 21 kept the GI 50 values at the µM level, with a slight selectivity against HT-29.Among the thiazolidinones, they also retained cytotoxicity at the µM level without selectivity toward any cell line, except in the case of those with aromatic substituents, 31 and 32, which were tenfold more potent on HT-29 than on A549 cells.From this, it can be said that this skeleton enlargement did not improve the cytotoxicity of the molecules.In fact, the benzazole group includes the least potent compounds of all the series prepared.
In order to correlate the cytotoxic activity obtained with structural features, we conducted an in silico analysis focused on drug-likeness and ADME parameters.The structures were submitted to the freely accessible Swissadme platform [24] to predict their pharmacokinetic properties and possible adverse effects.Attending to their chemical features, compounds were organized into the same groups, A to E, indicated above, and a color code was assigned in the figures to each group to observe trends.Most of the compounds showed good druggability since they agreed with Lipinski's rule of five.Groups A, B, and C (except compound 10) fulfilled the five rules, and groups D and E only presented one violation.All the compounds also showed good non-rotatable bond (n-ROTB) values (<10) and acceptable H-bond acceptor values (7-10) (Table S2).Further correlations through in silico values were found when plotting TPSA and log S (referring to solubility) and log P (lipophilicity) in relation to cytotoxicity values.Figure 4 represents the values obtained for the A549 cell line (similar results were found for HT-29 cells and are shown in Figure S1).In both cases, a certain correlation among the groups is observed, which also follows a trend in terms of their cytotoxicity.At first glance, it can be seen that higher TPSA and log P values, relative to the precursors, correlate with lower cytotoxicity.Specifically, the most interesting in silico correlation was observed between log P and cytotoxicity.In this case, it can be seen that the values of groups B and E cluster together.Some components of these groups showed good cytotoxicity at the micromolar level.On the other hand, group D, which is generally less cytotoxic, clusters with higher log P values.Similarly, the more cytotoxic compounds (3-5 and 9-10) seem to maintain this feature.In the case of metabolism, various isoforms of cytochrome P450 (CYP) were evaluated, with the results showing different patterns among the groups and the isoform considered (Table S3).In summary, these results seem to indicate that the γ-lactone ring is important for the cytotoxic potency of these cyclolignans derived from podophyllotoxin, as has been previously considered [26].In this sense, it is also worth mentioning that the majority of the podophyllotoxin derivatives described in the literature had this lactone ring [27,28] unsubstituted at C9, so little or nothing was known till now about the influence of that substitution on bioactivity.According to our results, it seems that a small substituent is tolerable at C9 but not a bulky one; in any case, it will be necessary to consider more examples in future works.Also, in accordance with some of our previous works, the α,β-unsaturated aldehyde seems necessary for the selectivity of the podophyllic aldehyde analogs [15,29] since the absence of the electrophilic character at C9 and the enlargement of the cyclolignan skeleton by its incorporation into different heterocycles led to less potent analogs or, in some cases, nearly inactive derivatives.These results warrant further studies aimed at understanding their mechanisms of action at the molecular level in different types of tumor cells.

Chemistry
NMR spectra were recorded on a Bruker (Billerica, MA, USA) WP 200 SY (200 and 50.3 MHz for 1 H and 13 C) or Bruker Avance 400DRX (400 and 100 MHz) spectrometer in CDCl3 using TMS as an internal reference.Chemical shift (δ) values are expressed in ppm and followed by multiplicity and coupling constants (J) in Hz.IR spectra were obtained on a Nicolet Impact 410 spectrophotometer in NaCl film.EM was run on a Hewlett-Packard (Palo Alto, CA, USA) 5890 Series II GC-MS with an electron impact ionization source.HRMS was run on a VG TS-250 spectrometer working at 70 eV and using electrospray Also, the Swissadme platform provided very interesting information on other drug parameters.As can be seen in Figure S2, using the BOILED-egg model provided by the platform [25], most of the compounds were predicted to show good gastrointestinal absorption, and only compounds 9, 9a, and 9b were predicted to passively permeate the blood-brain barrier.In terms of whether or not the compounds can be substrates of Pglycoprotein, the precursors appear to be substrates of this cellular efflux system.Similarly, the more cytotoxic compounds (3-5 and 9-10) seem to maintain this feature.In the case of metabolism, various isoforms of cytochrome P450 (CYP) were evaluated, with the results showing different patterns among the groups and the isoform considered (Table S3).
In summary, these results seem to indicate that the γ-lactone ring is important for the cytotoxic potency of these cyclolignans derived from podophyllotoxin, as has been previously considered [26].In this sense, it is also worth mentioning that the majority of the podophyllotoxin derivatives described in the literature had this lactone ring [27,28] unsubstituted at C9, so little or nothing was known till now about the influence of that substitution on bioactivity.According to our results, it seems that a small substituent is tolerable at C9 but not a bulky one; in any case, it will be necessary to consider more examples in future works.Also, in accordance with some of our previous works, the α,β-unsaturated aldehyde seems necessary for the selectivity of the podophyllic aldehyde analogs [15,29] since the absence of the electrophilic character at C9 and the enlargement of the cyclolignan skeleton by its incorporation into different heterocycles led to less potent analogs or, in some cases, nearly inactive derivatives.These results warrant further studies aimed at understanding their mechanisms of action at the molecular level in different types of tumor cells.

Chemistry
NMR spectra were recorded on a Bruker (Billerica, MA, USA) WP 200 SY (200 and 50.3 MHz for 1 H and 13 C) or Bruker Avance 400DRX (400 and 100 MHz) spectrometer in CDCl 3 using TMS as an internal reference.Chemical shift (δ) values are expressed in ppm and followed by multiplicity and coupling constants (J) in Hz.IR spectra were obtained on a Nicolet Impact 410 spectrophotometer in NaCl film.EM was run on a Hewlett-Packard (Palo Alto, CA, USA) 5890 Series II GC-MS with an electron impact ionization source.HRMS was run on a VG TS-250 spectrometer working at 70 eV and using electrospray ionization (ESI, Edinburgh, UK) or fast atom bombardment (FAB, Tokyo, Japan).Optical rotations were recorded on a Perkin-Elmer 241 polarimeter in a CHCl 3 solution.UV spectra were obtained on a Hitachi (Tokyo, Japan) 100-60 spectrophotometer in an ethanol solution, and λ max values are given in nm.Column chromatography (CC) purifications were performed using silica gel 60 (40-63 mm, 230-400 mesh, Merck), and TLC was carried out on silica gel 60 F 245 (Merck, 0.25 mm thick).Solvents and reagents were purified by standard procedures as necessary.
Method A from imines 23-27.To a solution of the corresponding imine in dry benzene, MgSO 4 and thioglycolic acid were added, and the reaction was refluxed for the specified time in a flask equipped with a Dean-Stark trap under a nitrogen atmosphere.After cooling, the mixture was filtered, concentrated under reduced pressure, diluted with an aq sat NaHCO 3 solution, and extracted with EtOAc.The combined organic layers were washed with brine, dried over Na 2 SO 4 , and evaporated.Purification of the crude by silica gel CC (CH 2 Cl 2 /EtOAc) provided the corresponding thiazolidinones.
Method B from aldehyde 2. To a suspension of aldehyde 2 in dry benzene was added the appropriate amine, and the mixture was refluxed for the specified time in a flask equipped with a Dean-Stark trap under a nitrogen atmosphere.After cooling to room temperature, thioglycolic acid was added dropwise to the solution, and the resulting mixture was refluxed for additional time.It was then cooled and concentrated under reduced pressure.The obtained residue was diluted with an aq sat NaHCO 3 solution and extracted with EtOAc.The combined organic layers were washed with brine, dried over Na 2 SO 4 , and evaporated.Purification of the crude by silica gel CC (CH 2 Cl 2 /EtOAc) provided the corresponding thiazolidinones.Following method B from aldehyde 2 (52 mg, 0.12 mmol) and propylamine (13 µL, 0.16 mmol) in dry benzene (10 mL) for 6 h and thioglycolic acid (9 µL, 0.13 mmol) for an additional 5 h.Purification by silica gel CC (CH 2 Cl 2 /EtOAc 8:2) provided compound 29 (31 mg, 47%) as a 7:3 mixture of epimers.When the reaction was continuously refluxed for 20 h and an additional 8 h, compound 29 was obtained in 59% yield.

Biological Evaluation
A colorimetric type of assay using the sulforhodamine B (SRB) reaction was adapted for the quantitative measurement of cell growth and viability, following a previously described method [30].This assay employs 96-well cell culture microplates of 9 mm diameter.Cell lines derived from different human cancer types were obtained from the American Type Culture Collection (ATCC).Cells were maintained in RPMI 1640 10% Fetal Bovine Serum (FBS) supplemented with 0.1 g/L penicillin and 0.1 g/L streptomycin sulfate and then incubated at 37 • C, 5% CO 2 , and 98% humidity.For the experiments, cells were harvested from subconfluent cultures using trypsin and resuspended in fresh medium before plating.
Cells were seeded in 96-well microtiter plates at 5 × 10 3 cells per well in aliquots of 195 µL of RPMI medium, and they were allowed to attach to the plate surface by growing in a drug-free medium for 18 h.Afterward, samples were added to aliquots of 5 µL in a range from 10 to 10 −8 µg/mL, dissolved in DMSO:EtOH:Phosphate-Buffered Saline (PBS) (0.5:0.5:99).After 72 h of exposure, the antitumor effect was measured by the SRB methodology: cells were fixed by adding 50 µL of cold 50% (wt/vol) trichloroacetic acid (TCA) and incubating for 60 min at 4 • C. Plates were washed with deionized water and dried; 100 µL of SRB solution (0.4% wt/vol in 1% acetic acid) was added to each microtiter well and incubated for 10 min at room temperature.Unbound SRB was removed by washing with 1% acetic acid.Plates were air-dried, and bound stain was solubilized with tris(hydroxymethyl)aminomethane (Tris) buffer.Optical densities (ODs) were read on an automated spectrophotometric plate reader at a single wavelength of 490 nm.Data analyses were generated automatically by LIMS implementation.Using control OD values (C), test OD values (T), and time-zero OD values (T 0 ), the drug concentration that

Figure 1 .
Figure 1.Chemical structures of podophyllotoxin and several derivatives, with the IUPAC numbering system used in this work.

Figure 1 .
Figure 1.Chemical structures of podophyllotoxin and several derivatives, with the IUPAC numbering system used in this work.

Molecules 2024 ,
29, x FOR PEER REVIEW 3 of 20 benzimidazole), purine, and 1-deazapurine.Subsequent cytotoxicity assays of the obtained derivatives were performed on three types of human cancer cells.

Figure 2 .
Figure 2. A schematic representation of the chemomodulation of podophyllotoxin performed by our group.

Figure 2 .
Figure 2. A schematic representation of the chemomodulation of podophyllotoxin performed by our group.

Figure 4 .
Figure 4. Graphical representation of log P and TPSA values of the cyclolignans, calculated by Swissadme platform.Log P and TPSA values are plotted with the cytotoxicity values of the compounds in A549.Color code for the different groups of compounds: red for precursors (A: 1 and 2), blue for C9 vinylogous derivatives (B: 3-8), purple for lactone derivatives (C: 9-10), green for benzoheteroazoles (D: 11-20), and yellow for thiazolidines (E: 28-32).

Figure 4 .
Figure 4. Graphical representation of log P and TPSA values of the cyclolignans, calculated by Swissadme platform.Log P and TPSA values are plotted with the cytotoxicity values of the compounds in A549.Color code for the different groups of compounds: red for precursors (A: 1 and 2), blue for C9 vinylogous derivatives (B: 3-8), purple for lactone derivatives (C: 9-10), green for benzoheteroazoles (D: 11-20), and yellow for thiazolidines (E: 28-32).