Cytotoxic and Anti-HSV-1 Effects of Caulerpin Derivatives

Marine organisms represent a potential source of secondary metabolites with various therapeutic properties. However, the pharmaceutical industry still needs to explore the algological resource. The species Caulerpa lamouroux Forssk presents confirmed biological activities associated with its major compound caulerpin, such as antinociceptive, spasmolytic, antiviral, antimicrobial, insecticidal, and cytotoxic. Considering that caulerpin is still limited, such as low solubility or chemical instability, it was subjected to a structural modifications test to establish which molecular regions could accept structural modification and to elucidate the cytotoxic bioactive structure in Vero cells (African green monkey kidney cells, Cercopithecus aethiops; ATCC, Manassas, VA, USA) and antiviral to Herpes simplex virus type 1. Substitution reactions in the N-indolic position with mono- and di-substituted alkyl, benzyl, allyl, propargyl, and ethyl acetate groups were performed, in addition to conversion to their acidic derivatives. The obtained analogs were submitted to cytotoxicity and antiviral activity screening against Herpes simplex virus type 1 by the tetrazolium microculture method. From the semi-synthesis, 14 analogs were obtained, and 12 are new. The cytotoxicity assay showed that caulerpin acid and N-ethyl-substituted acid presented cytotoxic concentrations referring to 50% of the maximum effect of 1035.0 µM and 1004.0 µM, respectively, values significantly higher than caulerpin. The antiviral screening of the analogs revealed that the N-substituted acids with methyl and ethyl groups inhibited Herpes simplex virus type 1-induced cytotoxicity by levels similar to the positive control acyclovir.


Introduction
Drug discovery for various therapeutic areas, especially cancer, infectious diseases, vascular diseases, and multiple sclerosis, for example, has expanded in recent years in proportion to investigations of natural products and their semisynthetic derivatives, which arguably play a key role for sources of new candidates for these drugs [1].A good action plan for preparing derivatives is to enhance selectivity and therapeutic action arising from the promotion of physicochemical and pharmacokinetic activities and create patentable compounds.Thus, causing increased lipophilicity and promoting the insertion of atoms or groups of atoms into the chemical structures of natural products are excellent examples of changes that improve their biological activity [2].
In recent years, the number of organic compounds isolated from marine sources has been surprising due to industry research aiming to develop new drugs with therapeutic properties from the sea.In particular, seaweeds, whose commercial production has increased rapidly in recent decades, either by harvesting natural resources or by cultivation, and whose application is considered environmentally friendly, healthy, and sustainable for humans because of their many compounds that can be used as foods, cosmetics, medicines, and pharmaceuticals, can be applied in aquaculture and agriculture [3].
Alkaloids form a special class of secondary metabolites, grouped into heterocyclic and non-heterocyclic compounds based on the nitrogen atom position in their chemical structure.Most alkaloids are pharmacologically active or poisonous in excessive doses; they exhibit multiple biological activities, such as antitumor, antimicrobial, anticholinergic, antihypertensive, antidepressant, anti-inflammatory, and anti-ulcer, among others [4].The literature mentions four alkaloid drugs of marine origin in clinical use, such as anticancer [ara-C (Cytarabine ® ) and trabectedin (Yondelis ® )], antiviral [ara-A (Vidarabine ® )], and neuropathic analgesic [ziconotide (Prialt ® )] [5].
In this context, studies with Caulerpa lamouroux species proved the anti-inflammatory activity of the methanolic extract of Caulerpa mexicana in vitro and in vivo [6,7] and the anti-inflammatory [8] and ulcerative colitis activities of the methanolic extract of Caulerpa racemosa [7].They described the antinociceptive [8], spasmolytic [9,10], and antiviral activities against the Herpes simplex virus type 1 (HVS-1) [11] of the bis-indolic alkaloid, caulerpin (1), the major component of these species.Other authors have described the antimicrobial, insecticidal, and cytotoxic activities of extracts of the genus Caulerpa [12,13].
Given the variety of pharmacological properties presented by caulerpin (1), Canché Chay et al. [14] reported the synthesis of this natural product, starting from solutions of indole molecules and with higher yields than observed in extraction and purification processes of natural products.
Thus, recognizing the importance of the genus Caulerpa in the production of chemical constituents of the most varied classes with pharmacological potential, this study proposed performing structural modifications on the major component of Caulerpa racemosa, caulerpin (1), aiming to establish which molecular regions will be able to tolerate structural modulation and to elucidate the bioactive conformation by evaluating its cytotoxic and antiviral effects on the HSV-1.

Chemical Studies
Caulerpin (1) was isolated from the extract of the green alga C. racemosa with a yield corresponding to 7% of the crude extract.The structure elucidation of the natural product was based mainly on the analysis of its IR, NMR 1 H, and 13 C spectra and in comparison with the literature data.The IR spectrum revealed bands at ν max 3382 (N-H), 1687 (referring to C=O stretching of ester conjugated).The 1 H NMR spectral data (Table 1) revealed four signals characteristic of ortho-disubstituted benzene: two doublets (δ 7.43, 1H and δ 7.30, 1H) and two triplets (δ 7.18, 1H and δ 7.09, 1H).The singlet observed at δ 9.21 suggests the presence of an indole nucleus, characteristic of the alkaloid class [15,16].The presence of two singlets at δ 8.06 (1H) and δ 3.90 (3H) suggests the presence of cyclooctatetraene and methyl ester groups, respectively [17].The 13 C-APT NMR data (Table 1) confirm the presence of the benzene ring (δc 111.7, 118.4,120.8, 123.5, hydrogenated aromatic carbons and δc 128.3, 137.8, non-hydrogenated aromatic carbons) and cyclooctatetraene (δc 142.9, hydrogenated aromatic carbon and δc 112.0, 133.0, 125.6, positive phase) in addition to the methyl ester group (δc 52.7, non-hydrogenated aromatic carbons) and the carbonyl group (δc 166.8) [18].Given the results, it was possible to identify the compound from the C. racemosa extract as the majority alkaloid caulerpin (Figure 1), already isolated previously in several Caulerpa species [17].tatetraene (δc 142.9, hydrogenated aromatic carbon and δc 112.0, 133.0, 125.6, positive phase) in addition to the methyl ester group (δc 52.7, non-hydrogenated aromatic carbons) and the carbonyl group (δc 166.8) [18].Given the results, it was possible to identify the compound from the C. racemosa extract as the majority alkaloid caulerpin (Figure 1), already isolated previously in several Caulerpa species [17].The natural product caulerpin (1) comes from a family of bis-indolic alkaloids and has an extra eight-membered ring between two indolic rings directly incorporated with the carbonyl group.This alkaloid has several important biological activities already described in the literature.Macedo et al. [11] described 1 as an alternative drug to acyclovir ® during the treatment of HSV-1 infections by inhibiting the alpha and beta phases of the viral replication cycle.
Due to the promising biological activities of 1, several analogs were proposed for elaboration, evaluation of their biological activities, and consequent study of structure-activity relationships.
The literature reports several examples of modified natural products with recognized pharmacological activity, such as nicotine (pyridine alkaloid); adrenaline, mescaline, morphine, and tubocurarine (tyrosine alkaloids); ephedrine and pseudoephedrine (phenylalanine alkaloids); vitamins B1, B2, and B5; some tropane alkaloids such as co- The natural product caulerpin (1) comes from a family of bis-indolic alkaloids and has an extra eight-membered ring between two indolic rings directly incorporated with the carbonyl group.This alkaloid has several important biological activities already described in the literature.Macedo et al. [11] described 1 as an alternative drug to acyclovir ® during the treatment of HSV-1 infections by inhibiting the alpha and beta phases of the viral replication cycle.
Due to the promising biological activities of 1, several analogs were proposed for elaboration, evaluation of their biological activities, and consequent study of structureactivity relationships.
The literature reports several examples of modified natural products with recognized pharmacological activity, such as nicotine (pyridine alkaloid); adrenaline, mescaline, morphine, and tubocurarine (tyrosine alkaloids); ephedrine and pseudoephedrine (phenylalanine alkaloids); vitamins B1, B2, and B5; some tropane alkaloids such as cocaine and atropine; and more complex products such as paclitaxel, testosterone, and progesterone [20].The introduction of methyl (2) and ethyl (3) alkyl groups into the bis-indolic core of 1 increases lipophilicity, impacting permeability across biological membranes, which causes potentiation of its activity.In parallel, the insertion of unsaturated allyl (4) and propargyl (5) groups favors the adjustment with the respective receptors.In contrast, the insertion of aromatic rings (6) enables the enlargement of molecular dimensions, a property useful for receptor sites where there is a hydrophobic cavity liable to be occupied by the ring [21].The structures of the analogs with substituents on the indolic nuclei are shown in Scheme 1.
caine and atropine; and more complex products such as paclitaxel, testosterone, and progesterone [20].The introduction of methyl (2) and ethyl (3) alkyl groups into the bis-indolic core of 1 increases lipophilicity, impacting permeability across biological membranes, which causes potentiation of its activity.In parallel, the insertion of unsaturated allyl (4) and propargyl (5) groups favors the adjustment with the respective receptors.In contrast, the insertion of aromatic rings (6) enables the enlargement of molecular dimensions, a property useful for receptor sites where there is a hydrophobic cavity liable to be occupied by the ring [21].The structures of the analogs with substituents on the indolic nuclei are shown in Scheme 1. Insertion of 3,4,5-trihydroxybenzyl groups into the indole nitrogen of caulerpin 1 using 3,4,5-trihydroxybenzyl chloride in DMF, according to the methodology of Zhao et al. [22], did not occur.Analyses of 1 H and 13 C NMR spectral data, including two-dimensional data, suggested that the reaction product would have the presence of two carbonyls, δC 168.4 (C-10), and 166.8 (C-10′), which correlate with H-9 and H-9′, respectively.However, only C-10′ demonstrates 3 JCH coupling with 3H-11′, suggesting the monoacid analog 7, the result of a monohydrolysis of caulerpin 1 (Scheme 2).Furthermore, this monoindolic insertion behavior also occurred in the reaction of caulerpin 1 with ethyl bromoacetate [22], yielding analog 8 (Scheme 2).Prototype molecules with the introduction of acid groups in the chemical structure produce analogs with higher water solubility due to the ability of acids to form salts in vitro as the acidity in the structure increases.Usually, the most explored acid groups are carboxylic acid and sulfonic acid [21].Aiming to obtain products with different polarities Insertion of 3,4,5-trihydroxybenzyl groups into the indole nitrogen of caulerpin 1 using 3,4,5-trihydroxybenzyl chloride in DMF, according to the methodology of Zhao et al. [22], did not occur.Analyses of 1 H and 13 C NMR spectral data, including two-dimensional data, suggested that the reaction product would have the presence of two carbonyls, δC 168.4 (C-10), and 166.8 (C-10 ′ ), which correlate with H-9 and H-9 ′ , respectively.However, only C-10 ′ demonstrates 3 JCH coupling with 3H-11 ′ , suggesting the monoacid analog 7, the result of a monohydrolysis of caulerpin 1 (Scheme 2).Furthermore, this monoindolic insertion behavior also occurred in the reaction of caulerpin 1 with ethyl bromoacetate [22], yielding analog 8 (Scheme 2).caine and atropine; and more complex products such as paclitaxel, testosterone, and progesterone [20].The introduction of methyl (2) and ethyl (3) alkyl groups into the bis-indolic core of 1 increases lipophilicity, impacting permeability across biological membranes, which causes potentiation of its activity.In parallel, the insertion of unsaturated allyl (4) and propargyl ( 5) groups favors the adjustment with the respective receptors.In contrast, the insertion of aromatic rings (6) enables the enlargement of molecular dimensions, a property useful for receptor sites where there is a hydrophobic cavity liable to be occupied by the ring [21].The structures of the analogs with substituents on the indolic nuclei are shown in Scheme 1. Insertion of 3,4,5-trihydroxybenzyl groups into the indole nitrogen of caulerpin 1 using 3,4,5-trihydroxybenzyl chloride in DMF, according to the methodology of Zhao et al. [22], did not occur.Analyses of 1 H and 13 C NMR spectral data, including two-dimensional data, suggested that the reaction product would have the presence of two carbonyls, δC 168.4 (C-10), and 166.8 (C-10′), which correlate with H-9 and H-9′, respectively.However, only C-10′ demonstrates 3 JCH coupling with 3H-11′, suggesting the monoacid analog 7, the result of a monohydrolysis of caulerpin 1 (Scheme 2).Furthermore, this monoindolic insertion behavior also occurred in the reaction of caulerpin 1 with ethyl bromoacetate [22], yielding analog 8 (Scheme 2).Prototype molecules with the introduction of acid groups in the chemical structure produce analogs with higher water solubility due to the ability of acids to form salts in vitro as the acidity in the structure increases.Usually, the most explored acid groups are carboxylic acid and sulfonic acid [21].Aiming to obtain products with different polarities Prototype molecules with the introduction of acid groups in the chemical structure produce analogs with higher water solubility due to the ability of acids to form salts in vitro as the acidity in the structure increases.Usually, the most explored acid groups are carboxylic acid and sulfonic acid [21].Aiming to obtain products with different polarities and, consequently, different pharmacological potentials, we proceeded with the production of analog 9 from the natural product 1; and analogs 10, 11, 12, and 13, from the corresponding N-substituted 2, 3, 4, and 6 (Scheme 3).All products were obtained by hydrolysis of the ester groups in a basic medium, with a nucleophilic addition-elimination reaction occurring at the ester carbonyl [23,24].Two other analogs (14 and 15) are shown in Scheme 4, and were obtained to exchange the methyl groups of the esters for ethyl (1 and 2).The transesterification was carried out in order to amplify the lipophilic character [25].Of the and, consequently, different pharmacological potentials, we proceeded with the production of analog 9 from the natural product 1; and analogs 10, 11, 12, and 13, from the corresponding N-substituted 2, 3, 4, and 6 (Scheme 3).All products were obtained by hydrolysis of the ester groups in a basic medium, with a nucleophilic addition-elimination reaction occurring at the ester carbonyl [23,24].Two other analogs (14 and 15) are shown in Scheme 4, and were obtained to exchange the methyl groups of the esters for ethyl (1 and 2).The transesterification was carried out in order to amplify the lipophilic character [25].Of the 15 molecules presented in this study, the analogs 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, and 15 are reported for the first time in the literature.

Cytotoxic Effect on MTT
Cytotoxic analysis showed low activity for caulerpin (1), with a CC50 value of 687.9 ± 35.2 µM.In another study, caulerpin extracted from C. peltata also showed low cytotoxicity, showing 12% inhibition of cancer cell growth [26].For analogs 2, 4, 5, 12, and 13, lower CC50 values were observed than analog 1, demonstrating greater cell growth inhibition (Table 2).and, consequently, different pharmacological potentials, we proceeded with the production of analog 9 from the natural product 1; and analogs 10, 11, 12, and 13, from the corresponding N-substituted 2, 3, 4, and 6 (Scheme 3).All products were obtained by hydrolysis of the ester groups in a basic medium, with a nucleophilic addition-elimination reaction occurring at the ester carbonyl [23,24].Two other analogs (14 and 15) are shown in Scheme 4, and were obtained to exchange the methyl groups of the esters for ethyl (1 and 2).The transesterification was carried out in order to amplify the lipophilic character [25].Of the 15 molecules presented in this study, the analogs 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, and 15 are reported for the first time in the literature.
In another study that investigated the cytotoxicity of 1 against Vero cells, a CC50 of 1176 µM was verified, demonstrating that this compound has potential as a promising drug in human cells [11].The difference in values obtained is related to the statistical methods used in the different works.The cytotoxicity of 1 was also evaluated in different colorectal cancer cells, showing an inhibitory effect on cell growth in the tested strains after 48 h of exposure, and the IC 50 values ranged from 20 to 31 µM [27].The effect of 1 in cancer cells may be associated with increased enzyme activity or decreased oxygen consumption by the cell [27].Higher CC50 values were observed compared to analogs 9 and 11 and, consequently, lower cytotoxicity than for 1.These results may be related to the hydrophilicity characteristics of the molecules, demonstrating that the insertion of lipophilic groups with different numbers of carbons, double bonds, and aromatic groups does not relate to the cytotoxic potency observed in Vero cells.
In a cytotoxicity study performed with tri(1-alkyl-indol-3yl) methylene salts on human colon carcinoma HCT116 and leukemia K526 cells, it was observed that in both cell lines, the average CC 50 decreases progressively with the increasing number of carbons up to the radical with five carbons, suggesting that the number of substituent carbons in the indolic nitrogen is related to higher cytotoxic potency [28].
Regarding the E máx values of the analogs, it was evidenced that acids 12 and 13 presented a significantly lower effect (p < 0.05) than 1, suggesting that the simultaneous presence of the acid group and the bulkier groups (allyl and benzyl) as substituents in the indole unit act to attenuate the cytotoxic efficiency.Differently, analogs 2, 4, 5, 9, 10, and 11 provided E máx values significantly higher than 1 (p < 0.05).
By analyzing the cell viability curves for each analog (Figure 2), it was possible to demonstrate that analogs 2 and 4 presented a maximum percentage of cell viability lower than 75%, showing high cytotoxicity compared to the other molecules.In general, the analogous compounds showed higher cytotoxicity than 1.In most of them, cell growth inhibition was less than 50%, demonstrating viability for studying antiviral and antifungal activities with these compounds.

Antiviral Assay Concomitant Treatment of Infection
This assay sought to evaluate the samples' ability to stop the infection at the same moment the cells were infected with the virus.As cell viability was less than 75%, analogs 2 and 4 were not tested in this assay.
The results observed for analogs 3, 5, 6, 11, and 12 suggested that the introduction of methylene groups in chemical structures of prototype molecules increases their dimensions, as well as their lipophilicity, allowing for an increase in the potency of the biological properties.Only Acyclovir (ACV) obtained an inhibition percentage greater than 50% (52.79%).

Antiviral Assay Concomitant Treatment of Infection
This assay sought to evaluate the samples' ability to stop the infection at the same moment the cells were infected with the virus.As cell viability was less than 75%, analogs 2 and 4 were not tested in this assay.
Treatment of cells with analogs 3, 5, 6, 11, and 12 resulted in a significant inhibition of HSV-1-induced cytotoxicity (p < 0.0001) when compared to the untreated cells (control) (Figure 3).Despite the numerical difference in the inhibition percentage values between acyclovir and molecules 11 and 12, statistical analysis indicates that these new compounds exhibit efficacy similar to the antiviral agent acyclovir when the treatment occurs concurrently with the infection.Considering that acyclovir is one of the most widely used anti-HSV-1 drugs in clinical applications, molecules 11 and 12 represent an effective and safe alternative for treating the high rates of patients infected with this virus.

Post-Infection Treatment Assay
In this assay, the ability of the compounds to block an HSV-1 infection already established in Vero cells was verified.The inhibition (%) of HSV-1-induced cytotoxicity after treatment with compound 1 and its analogs is shown in Figure 4.It was evidenced that treatment with 1 and analogs 3, 5, 6, 10, 11, and 12 resulted in higher percentages of cellular inhibition compared to the control group, suggesting that the introduction of alkyl groups may enhance anti-HSV-1 activity in post-infection treatment.Previous studies have demonstrated the antiviral activity of seaweed extracts rich in 1 at concentrations ranging from 2.22 to 4.20 µg/mL [29,30].The results observed for analogs 3, 5, 6, 11, and 12 suggested that the introduction of methylene groups in chemical structures of prototype molecules increases their dimensions, as well as their lipophilicity, allowing for an increase in the potency of the biological properties.Only Acyclovir (ACV) obtained an inhibition percentage greater than 50% (52.79%).
Despite the numerical difference in the inhibition percentage values between acyclovir and molecules 11 and 12, statistical analysis indicates that these new compounds exhibit efficacy similar to the antiviral agent acyclovir when the treatment occurs concurrently with the infection.Considering that acyclovir is one of the most widely used anti-HSV-1 drugs in clinical applications, molecules 11 and 12 represent an effective and safe alternative for treating the high rates of patients infected with this virus.

Post-Infection Treatment Assay
In this assay, the ability of the compounds to block an HSV-1 infection already established in Vero cells was verified.The inhibition (%) of HSV-1-induced cytotoxicity after treatment with compound 1 and its analogs is shown in Figure 4.It was evidenced that treatment with 1 and analogs 3, 5, 6, 10, 11, and 12 resulted in higher percentages of cellular inhibition compared to the control group, suggesting that the introduction of alkyl groups may enhance anti-HSV-1 activity in post-infection treatment.Previous studies have demonstrated the antiviral activity of seaweed extracts rich in 1 at concentrations ranging from 2.22 to 4.20 µg/mL [29,30].
The continuous use of available medications for HSV-1 treatment promotes the selection of resistant strains and their relative toxicities upon prolonged administrations.In this context, analogs 10 and 11 emerge as promising molecules in combating this virus by exhibiting antiviral effects statistically similar to acyclovir.

Cytotoxicity
The method of Cheng et al. [32] was followed with modifications to evaluate the cytotoxicity of caulerpin analogs 1, 2, 3, 4, 5, 7, 8, 9, 10, and 11 at concentrations ranging from 200 to 1800 µM.Vero cells (African green monkey kidney cells Cercopithecus aethiops; ATCC, Manassas, VA, USA) grown in Dulbecco's modified medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 0.1 µM HEPES, and 2.5 µg/mL gentamicin at 37 • C in 5% CO 2 were used.Each test sample was diluted in DMEM containing 2% fetal bovine serum at six different concentrations.Vero cells were plated at a concentration of 2 × 10 4 cells/well and incubated for 24-36 h.When the cells showed 90% confluence, the medium was removed from the wells, and the samples were added and incubated for 72 h.After this time, the medium was discarded and 20 µL of MTT solution was added to each well.The plates were incubated for four hours at 37 • C. Subsequently, the supernatant was removed, and 150 µL of DMSO was added to each well to dissolve the formazan crystals.The plates were shaken for 10 min, followed by absorbance reading in an ELISA microplate reader at a wavelength of 540 nm.

Figure 2 .
Figure 2. Graphs of the effect of 1 and its analogs on cell viability.

Figure 2 .
Figure 2. Graphs of the effect of 1 and its analogs on cell viability.

Figure 3 .
Figure 3. Percentage of HSV-1 inhibition by caulerpin analogs in Vero cells infected with HSV-1 (MOI 0.2).The cells were treated with the compound's CC 20 and further incubated at 37 • C in 5% CO 2 for 72 h.Cell viability was assessed using the MTT method.Statistical analyses were performed with ANOVA and Tukey's posttest (**** p < 0.0001 versus control).ACV-Acyclovir.Molecules 2024, 29, x FOR PEER REVIEW 9 of 16

Table 2 .
Absolute CC 50 values of 1 and its analogs against Vero cells (ANOVA p < 0.05).Different lowercase letters in the same column represent a significant difference between the analogs.Different capital letters in the same column represent a significant difference between the analogs.