Structure Activity Relationships of Multitarget Coumarins on Inhibitory Aggregation of Platelets: An Integrated In Vitro and In Silico Study
, Eva Florencia Lejarazo Gómez 2, Ulises MartÃnez-Ortega 3, Mirthala Flores-GarcÃa 4, Ana MarÃa MejÃa-DomÃnguez 5, Aurora de la Peña-DÃaz 1,6 and Fausto Alejandro Jiménez-Orozco 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsCoumarins constitute the core scaffold of numerous natural and synthetic compounds and are well-known oral anticoagulants widely used in clinical practice. However, only a limited subset of coumarins exhibits anticoagulant activity, which requires specific substitutions at the 3- and 4-positions of the basic coumarin structure. Beyond their antithrombotic properties, coumarins have attracted increasing attention due to their diverse biological activities, including anti-inflammatory, antioxidant, cardioprotective, anticancer, and neuroprotective effects. In this context, the authors aimed to develop safer and more effective antiplatelet agents by synthesizing three nitrogen-substituted coumarin derivatives and evaluating their inhibitory effects on platelet aggregation induced by epinephrine, collagen, and ADP.
Major concerns:
- In the present study, the concentrations of ADP and epinephrine appear to correspond to doses that induce maximal platelet aggregation, whereas the concentration of collagen used is generally not sufficient to elicit maximal aggregation. The authors should provide a clear justification for the selection of these agonist concentrations. Alternatively, the study would be strengthened by including results obtained using low, intermediate, and high concentrations of each agonist to better support the conclusions.
- Platelet aggregation assays were performed using blood samples obtained from only five human donors. Given the well-known inter-individual variability in platelet responsiveness, this sample size is highly limited and may not adequately control for donor-dependent variability. In this regard, the use of experimental animal models could have provided a more controlled system for mechanistic validation. The authors should therefore explicitly discuss this limitation in the manuscript and/or provide additional supporting experiments to strengthen the robustness of the findings.
Minor concerns:
- The term epinephrine is inconsistently spelled as epinefrine in several parts of the manuscript and should be corrected throughout.
- Errors in the figures and their references should be corrected.
- The formatting and placement of subheadings require correction, including spacing and consistency (e.g., “3.3. α2-Adrenergic receptor”).
- Lines 113-192a. The introduction section is repeated twice. Authors should please delete the duplicated statements (from lines 113 to 192a).
Author Response
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Response to Reviewer 1 Comments
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1. Summary |
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Article Structure–Activity Relationships of Multitarget Coumarins on Inhibitory Aggregation of Platelets: An Integrated In Vitro and In Silico Study Ixchel Ramírez-Camacho 1, Fernando León Cedeño 2, José Germán Vázquez Cuevas 2, Eva Florencia Lejarazo Gómez 2, Ulises Martínez-Ortega 3, Mirthala Flores-García 4, Ana María Mejía-Domínguez 6, Aurora de la Peña-Díaz 1,5 and Fausto Alejandro Jiménez-Orozco 1 *.
Coumarins constitute the core scaffold of numerous natural and synthetic compounds and are well-known oral anticoagulants widely used in clinical practice. However, only a limited subset of coumarins exhibits anticoagulant activity, which requires specific substitutions at the 3- and 4-positions of the basic coumarin structure. Beyond their antithrombotic properties, coumarins have attracted increasing attention due to their diverse biological activities, including anti-inflammatory, antioxidant, cardioprotective, anticancer, and neuroprotective effects. In this context, the authors aimed to develop safer and more effective antiplatelet agents by synthesizing three nitrogen-substituted coumarin derivatives and evaluating their inhibitory effects on platelet aggregation induced by epinephrine, collagen, and ADP.
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
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Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
[Please give your response if necessary. Or you can also give your corresponding response in the point-by-point response letter. The same as below] |
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Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
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Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: A) In the present study, the concentrations of ADP and epinephrine appear to correspond to doses that induce maximal platelet aggregation, whereas the concentration of collagen used is generally not sufficient to elicit maximal aggregation. The authors should provide a clear justification for the selection of these agonist concentrations
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A) We thank the reviewer for this insightful comment regarding the agonist concentrations used in our platelet aggregation experiments.
The primary objective of this study was to evaluate the inhibitory activity of the tested compounds on platelet aggregation. For this reason, agonist concentrations were selected to elicit robust, reproducible aggregation responses in control samples, thereby enabling clear detection of inhibition.
2 µg/mL collagen was used to trigger platelet aggregation, which is a widely employed concentration in optical aggregometry, the gold standard for determining platelet function. This concentration reliably induces strong platelet activation in platelet-rich plasma and is commonly used in pharmacological studies evaluating antiplatelet compounds. In our laboratory, collagen at 2 µg/mL consistently elicits aggregation responses within the validated internal reference range (83.1 ± 8.6% maximal aggregation), which is within the expected range reported for healthy individuals (approximately 70–94% aggregation). Its maximum response is comparable to that observed with 10 µM ADP and 10 µM epinephrine.
Importantly, several pharmacological studies investigating platelet inhibitors have used collagen concentrations in the 1–2 µg/mL range to evaluate the inhibitory effects of bioactive molecules. For example:
1. Hsia CW, Lin KC, Lee TY, Hsia CH, Chou DS, Jayakumar T, Velusamy M, Chang CC, Sheu JR. Esculetin, a Coumarin Derivative, Prevents Thrombosis: Inhibitory Signaling on PLCγ2-PKC-AKT Activation in Human Platelets. Int J Mol Sci. 2019; 20(11):2731. doi: 10.3390/ijms20112731. 2. Rodríguez L, Montecino-Garrido HL, Lagos F, Carrasco B, Palomo I, Ormazabal P, Trostchansky A, Fuentes E. Enhanced Antiplatelet Activity of Nitrated Fatty Acid Extracts from Phaseolus vulgaris L. Molecules. 2026; 31(3):488. doi: 10.3390/molecules31030488. 3. Branchford BR, Stalker TJ, Law L, Acevedo G, Sather S, Brzezinski C, Wilson KM, Minson K, Lee-Sherick AB, Davizon-Castillo P, Ng C, Zhang W, Neeves KB, Lentz SR, Wang X, Frye SV, Shelton Earp H 3rd, DeRyckere D, Brass LF, Graham DK, Di Paola JA. The small-molecule MERTK inhibitor UNC2025 decreases platelet activation and prevents thrombosis. J Thromb Haemost. 2018;16(2):352-363. doi: 10.1111/jth.13875. 4. Hutachok N, Angkasith P, Chumpun C, Fucharoen S, Mackie IJ, Porter JB, Srichairatanakool S. Anti-Platelet Aggregation and Anti-Cyclooxygenase Activities for a Range of Coffee Extracts (Coffea arabica). Molecules. 2020 Dec 22;26(1):10. doi: 10.3390/molecules26010010.
These studies of platelet function, among others, show that collagen at 2 µg/mL is commonly used to assess pharmacological inhibition of platelet aggregation, because it produces a strong and reproducible activation signal, like obtained with 10µM ADP and 10µM epinephrine. For these reasons, we consider that the concentration of agonists used in this study is appropriate for the experimental objective. |
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B) Alternatively, the study would be strengthened by including results obtained using low, intermediate, and high concentrations of each agonist to better support the conclusions. |
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Response: We thank the reviewer for this valuable observation. We agree that full concentration–response curves for each agonist provide important mechanistic insight. However, experiments with multiple agonist concentrations are useful for characterizing platelet hyperreactivity or agonist sensitivity; our goal was not to construct dose-response curves for platelet activation but rather to assess the capacity of the new compounds to inhibit platelet aggregation under conditions of robust stimulation. Under threshold stimulation conditions, aggregation responses tend to be more variable, and partial activation may obscure/hide the inhibitory effects.
Furthermore, in platelet aggregation inhibition assays, agonists should be used at concentrations that elicit a strong and consistent aggregation response. Commonly, 10 µM ADP, 10 µM epinephrine, and 2 µg/mL collagen are used as physiological agonists to evaluate the inhibitory activity of pharmacological compounds. |
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C) Platelet aggregation assays were performed using blood samples obtained from only five human donors. Given the well-known inter-individual variability in platelet responsiveness, this sample size is highly limited and may not adequately control for donor-dependent variability. In this regard, the use of experimental animal models could have provided a more controlled system for mechanistic validation. The authors should therefore explicitly discuss this limitation in the manuscript and/or provide additional supporting experiments to strengthen the robustness of the findings.
Response: We thank the reviewer for raising this important point regarding donor variability in platelet aggregation studies. However, we would like to clarify that the experiments were not performed using samples from only five individual donors. In our experimental design, each independent experiment (n = 5) was conducted using platelet-rich plasma pooled from nine different healthy donors, resulting in a total of 45 donors included in the study. Pooling samples from multiple donors is a commonly used approach in platelet function studies to reduce inter-individual variability and obtain a more representative platelet response. We acknowledge that this methodological detail was not sufficiently described in the original manuscript, which may have led to this misunderstanding. Therefore, the Methods section has been revised to clearly specify that each experimental replicate consisted of pooled samples from nine donors. This clarification better reflects the robustness of the experimental design and the measures taken to minimize donor-dependent variability.
Comments 2: The term epinephrine is inconsistently spelled as epinefrine in several parts of the manuscript and should be corrected throughout. Response 2: We thank the reviewer for pointing out this typographical inconsistency. The spelling of the term “epinephrine” has now been carefully corrected throughout the entire manuscript and figure 3 to ensure consistency and accuracy.
4. Response to Comments on the Quality of English Language Response 1: The English was reviewed and improved in the manuscript and in the figures. |
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5. Additional clarifications |
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Errors in the figures and their references should be corrected.
Response: We thank the reviewer for pointing out these issues. All figures and their corresponding references in the text have been carefully reviewed and corrected to ensure accuracy and consistency throughout the manuscript. The figure numbering, captions, and in-text citations have been revised accordingly.
The formatting and placement of subheadings require correction, including spacing and consistency (e.g., “3.3. α2-Adrenergic receptor”).
Response: We thank the reviewer for pointing out these formatting inconsistencies. The formatting and placement of all subheadings have been carefully revised to ensure consistent spacing, numbering, and style throughout the manuscript. In particular, the formatting of section titles such as “3.3. α2-Adrenergic receptor” has been corrected to follow the journal’s formatting guidelines. These revisions have been implemented in the updated version of the manuscript.
Lines 113-192a. The introduction section is repeated twice. Authors should please delete the duplicated statements (from lines 113 to 192a).
Response: We thank the reviewer for identifying this duplication in the Introduction section. The repeated text between lines 113–192a has been carefully removed to eliminate redundancy and improve the clarity and structure of the manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis is an interesting manuscript concerning the multitarget activity of some coumarins, 3-substituted with N-heterocycles, on inhibitory aggregation of platelets. There are some remarks specified below:
- Introduction: The first nine paragraphs are double.
- Page 5, lines 192-199: They are double with lines 185-192.
- Page 6, 2.2.1: There is not any chemical equation for the general synthesis.
- Page 6, line 244: It is not possible to find the Supplementary Materials.
- Page 8, Table 1: There is not any control experiment with a reference compound.
- Page 10, There is not chapter 3.3.
- Page 11, line 404: Figure 3A, 3D, 3H profoundly are Figure 4A, 4D, 4H.
- Pages 10-11, Molecular Docking: The binder affinity for all cases is not so strong exept for B (-0.839) as P2Y12 receptor.
- Page 19, line 20: There is not the 13C-NMR.
- Page 21-22, References: Ref. [1], [2], [4], [6], [7], [9], [10], [12], [14], [15], [17], [21] must be corrected to include journal names etc.
- Page 22, ref. [20] is the same with ref. [22].
Author Response
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Response to Reviewer 2 Comments
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1. Summary |
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Article Structure–Activity Relationships of Multitarget Coumarins on Inhibitory Aggregation of Platelets: An Integrated In Vitro and In Silico Study Ixchel Ramírez-Camacho 1, Fernando León Cedeño 2, José Germán Vázquez Cuevas 2, Eva Florencia Lejarazo Gómez 2, Ulises Martínez-Ortega 3, Mirthala Flores-García 4, Ana María Mejía-Domínguez 6, Aurora de la Peña-Díaz 1,5 and Fausto Alejandro Jiménez-Orozco 1 *
This is an interesting manuscript concerning the multitarget activity of some coumarins, 3-substituted with N-heterocycles, on inhibitory aggregation of platelets. There are some remarks specified below
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
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Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
[Please give your response if necessary. Or you can also give your corresponding response in the point-by-point response letter. The same as below] |
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Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
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Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: The first nine paragraphs are double |
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Response 1: We thank the reviewer for identifying this duplication in the Introduction section. The repeated text has been carefully removed to eliminate redundancy and improve the clarity and structure of the manuscript.
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Comments 2: Page 5, lines 192-199: They are double with lines 185-192 |
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Response 2: We thank the reviewer for identifying this duplication in the Introduction section. The repeated text between lines 113–192a has been carefully removed to eliminate redundancy and improve the manuscript´s clarity and structure
Comments 3: Page 6, 2.2.1: There is not any chemical equation for the general synthesis Response 3: We thank the reviewer for this helpful suggestion. To improve the clarity of the synthetic methodology, we have now included a general reaction scheme illustrating the synthesis of the coumarin derivatives used in this study. The corresponding chemical scheme (Scheme 1) has been added to the revised manuscript to provide a clearer representation of the synthetic route and the key reaction steps involved in the preparation of the compounds.
Comments 4: Page 6, line 244: It is not possible to find the Supplementary Materials. Response 4: We thank the reviewer for this comment. The supplementary material file was removed from the annexes and replaced by appendices A, B, and C in the text.
Comments 5: Page 8, Table 1: There is not any control experiment with a reference compound. Response 5: We thank the reviewer for this valuable observation. In our study, we used human platelet-rich plasma and stimulated it with epinephrine, collagen, or ADP, following a standard light-transmission aggregometry protocol. In this experimental framework, the reference condition corresponds to PRP stimulated with the agonist in the presence of the vehicle (0.4% DMSO), which we used to define the maximum aggregation response (100%). The main goal of our work was to study the structure–activity relationships (SARs) among three new coumarin derivatives under the same experimental conditions, not to compare their potencies with those of established antiplatelet drugs. Because of this, we designed our experiments to compare the inhibition of platelet activation by compounds A, B, and C via different pathways. This approach makes it easier to see how structural changes affect biological activity within this chemical series. All assays used the same PRP preparation, agonist concentrations, and aggregation protocol. This maintained consistency in our results and enabled us to reliably compare ICâ‚…â‚€ values for each compound. We appreciate the reviewer’s suggestion to include a pharmacological reference inhibitor for a better context when comparing potency. This is a helpful recommendation, and we will consider adding reference compounds in future studies to further improve the pharmacological characterization of these multitarget coumarin derivatives.
Comments 6: Page 10, There is not chapter 3.3. Response 6: Response: We thank the reviewer for pointing out this inconsistency in the section numbering. The manuscript has been carefully revised, and the numbering of the subsections has been corrected to ensure proper sequence and consistency throughout the document. The commentary to section 3.3 has been adjusted accordingly in the revised version of the manuscript.
Comments 7: Page 11, line 404: Figure 3A, 3D, 3H profoundly are Figure 4A, 4D, 4H. Response 7: Figure numbering has been corrected as suggested
Comments 8: Pages 10-11, Molecular Docking: The binder affinity for all cases is not so strong exept for B (-0.839) as P2Y12 receptor. Response 8: The Molecular Docking section has been revised to clarify that binding affinities are interpreted qualitatively and in conjunction with experimental data.
Comments 9: Page 19, line 20: There is not the 13C-NMR. Response 9: We thank the reviewer for this important observation. In response to this comment, the ¹³C-NMR spectra for the synthesized compounds have now been obtained and included in the revised manuscript to provide additional structural confirmation. The corresponding ¹³C-NMR data have been incorporated into the characterization section, and the spectra are now provided here and in Appendix section for completeness. These additions strengthen the structural validation of the synthesized coumarin derivatives.
Comments 10: Page 21-22, References: Ref. [1], [2], [4], [6], [7], [9], [10], [12], [14], [15], [17], [21] must be corrected to include journal names etc. Response 10: We thank the reviewer for pointing out these inconsistencies in the reference list. The cited references [1], [2], [4], [6], [7], [9], [10], [12], [14], [15], [17], and [21] have been carefully reviewed and corrected to include the complete bibliographic information, including journal names, volume, page numbers, and other relevant details in accordance with the journal’s formatting guidelines. Additionally, the entire reference list has been thoroughly checked to ensure accuracy, completeness, and consistency.
Comments 11: Page 22, ref. [20] is the same with ref. [22]. Response 11: We thank the reviewer for identifying this duplication in the reference list. References [20] and [22] corresponded to the same article. The duplicate reference has been removed, and the reference list has been corrected accordingly. The numbering of the subsequent references and their corresponding citations in the text have also been revised to ensure consistency.
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5. Additional clarifications |
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Appendix C: Characterization section
- All reagents used in this study were purchased from Sigma-Aldrich and used as received and solvents employed in the reactions and purification procedures were distilled according to previously reported literature methods. (la Hoz, A et al., 2002)
- Column chromatography was carried out using silica gel 60 (0.063–0.200 mm) from Merck as the stationary phase.
- Thin-layer chromatography (TLC) analyses were performed on precoated plates with fluorescent indicator supplied by Sigma-Aldrich.
- Visualization of TLC plates was achieved using short- and long-wavelength UV light, iodine vapors, ninhydrin, p-anisaldehyde, and vanillin staining reagents, prepared according to literature procedures.
- Melting points were determined using a Fisher–Jones melting point apparatus and are uncorrected.
- Microwave-assisted reactions were performed using a CEM Discover SP microwave reactor.
- Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL Eclipse 300 MHz NMR spectrometer, a Bruker Avance III 400 MHz NMR spectrometer, and a Magritek Spinsolve 80 MHz NMR spectrometer.
- Mass spectra were obtained using a chromatographic system from SCIEX coupled to a mass spectrometer.
Appendixes C2. Demethylation of 2,4,5-Trimethoxybenzaldehyde.
The demethylation reaction of 2,4,5-trimethoxybenzaldehyde was carried out using boron tribromide (BBr₃), following previously reported procedures described by Y. H. Seo et al. and C. Moolman et al., to afford 2-hydroxy-4,5-dimethoxybenzaldehyde in 79% yield.
Appendix C3. 2-Hydroxy-4,5-dimethoxybenzaldehyde.
Slightly yellow crystalline solid (1.75 g, 92.4%); mp 102–103 °C.
¹H NMR (400 MHz, CDCl₃) δ 11.36 (s, 1H), 9.66 (d, J = 0.6 Hz, 1H), 6.87 (s, 1H), 6.43 (s, 1H), 3.90 (s, 3H), 3.84 (s, 3H).
¹³C NMR (100 MHz, CDCl₃) δ 194.12, 161.09, 157.36, 142.30, 113.25, 112.23, 104.01, 56.52, 56.42.
Appendix C4. Synthesis of N-(Cyanomethyl)imidazole
Imidazole (1.7 g, 25 mmol) was added to a suspension of NaH (25 mmol) in anhydrous THF. A solution of bromoacetonitrile (1.8 mL, 25 mmol) in anhydrous THF was then added dropwise. The reaction mixture was stirred at room temperature for 4 h.
After completion of the reaction, THF was removed under reduced pressure. Distilled water (10 mL) was added, and the product was isolated by liquid–liquid extraction with DCM (25 mL × 3). The combined organic layers were dried over anhydrous Naâ‚‚SOâ‚„, and the solvent was removed under reduced pressure.
The crude product was purified by column chromatography on silica gel (DCM–acetone 98:2) to afford a brownish-yellow liquid (1 g, 40.1%).
¹H NMR (80 MHz, CDCl₃) δ 7.55 (s, 1H), 7.07 (d, 2H), 4.77 (s, 2H).
Appendix C5. General Synthesis of 3-Pyrrolyl and 3-Imidazolyl Coumarins
A mixture of compound (2) (0.1 g, 0.5 mmol), tert-BuOK (0.13 g, 1.1 mmol), and compound (5) (0.5 mmol) was dissolved in DMF (1 mL). The reaction mixture was heated at 110 °C for 16 h. After cooling to room temperature, distilled water was added, and the mixture was stirred for an additional 0.3 h.
The crude product was extracted with EtOAc. The combined organic phases were dried over anhydrous Naâ‚‚SOâ‚„, and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel (DCM–MeOH 100:0–92:8).
3-(1-Imidazolyl)-6,7-dimethoxycoumarin (29). Beige solid (0.034 g, 25%).
¹H NMR (300 MHz, CDCl₃–CD₃OD) δ 8.04 (s, 1H), 7.67 (s, 1H), 7.38 (t, J = 1.4 Hz, 1H), 7.20 (s, 1H), 6.92 (s, 1H), 6.91 (s, 1H), 3.98 (s, 3H), 3.94 (s, 3H).
¹³C NMR (100 MHz, CDCl₃–CD₃OD) δ 157.71, 153.58, 148.63, 147.26, 136.94, 133.67, 129.06, 120.97, 119.28, 110.67, 108.06, 99.78, 56.52, 56.38.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have addressed most concerns raised by this reviewer.
