Microwave-Assisted Synthesis of Schiff Bases of Isoniazid and Evaluation of Their Anti-Proliferative and Antibacterial Activities
Round 1
Reviewer 1 Report
Referee report on the manuscript molbank-1080990 entitled "Microwave assisted synthesis of (E)-N'-(5-(2-nitrophenyl)-furan-2-yl)-methylene) / (E)-N'-(substituted-benzylidene)-isonicotinohydrazide and their anti-proliferative and antibacterial activities" submitted by Bayan Hiary, Ashok K. Shakya, Rajashri R. Naik and Sanna Bardaweel,
The authors present the results of an investigation concerning the synthesis and biological activity of three new Schiff bases derived from isoniazid. These three compounds were synthesized using both microwave assisted and conventional method of synthesis, then were investigated for antiproliferative and antimicrobial activity.
Concerning the descriptive aspect of the investigated problem the paper fulfills the goals proposed by the authors. Nevertheless, there are some questions which should be clarified.
- Even in the abstract authors stated that the structure of the compounds was proved by elemental analysis and spectral characterization, no data for elemental analysis is presented.
- Some statements from introduction part require proper literature citations.
- Also, in the introduction part there are some English grammar that should be improved (some of them are highlighted in the corrected manuscript).
- Please present the used parameters of the microwave instrument (such as power) and include this instrument in the Material and methods part!
- Regarding the NMR spectra of the compounds, I have several remarks. Please include in Scheme 1 the NMR position numbering of the compounds for an easier tracking of the NMR peaks. The chemical shifts from the manuscript (chemical section) does not correspond with chemical shifts from the NMR spectrum from supporting information (e.g., for the compound 1, the chemical shift of the NH proton is 12.45 ppm in the manuscript, but in the NMR spectrum from supporting information is 12.04 ppm). Please check all the NMR signal to correspond both manuscript and supporting information. Please provide the spectra of all compounds at a better resolution and only for the interesting chemical shift interval (0-12.5 ppm in the case of first compound). Please provide both 1H- and 13C-NMR for al three compounds. Please provide the NMR signals in the same order (ascending or descending) for all the compounds.
- Please attribute the vibrational wave numbers to the corresponding functional group.
- Who is n from the legend of the Table 1?
- Please discus the role of vincristine sulfate from Table 1, and correlate the antiproliferative activity of the three compounds with the antiproliferative activity of the vincristine sulfate.
- In antibacterial screening part, authors stated that the second compound showed better antibacterial activity than the other two compounds. From the table 1, the diameters of inhibition zone for the compound 2 are the smallest, thus his antimicrobial activity is the smallest both under coli and Staphylococcus aureus. In fact, al the compounds have almost the same antimicrobial activity but half as the antimicrobial activity of amoxicillin.
- In General part, please specify for which nuclei are those operating frequencies?
- In conclusion part, the author stated that “The synthesized compound exhibit significant anti-proliferative and antibacterial activities”, but in my opinion “The synthesized compound exhibit very low anti-proliferative (ten thousand times lower than vincristine sulfate) and moderate to good antibacterial activities”.
Having in view this consideration I recommended the publication of this article in Molbank after major revision.
Comments for author File: 
Comments.pdf
Author Response
Thank you for commented and suggestions
the manuscript is updated as per the comments.
Author Response File: Author Response.pdf
Reviewer 2 Report
The authors describe in this article (ID: molbank‐1080990) the synthesis and the characterization of three new Schiff bases of isoniazid using conventional and microwaveassisted synthetic methods. In addition, they present the results of the evaluation of their antiproliferative and antibacterial activities. The manuscript is interesting and reasonably well written. I consider that the manuscript can be published in Molbank after a major revision (as
suggested below).
1. I suggest that the authors name their article “Microwave assisted synthesis of three new Schiff bases of isoniazid and evaluation of their anti‐proliferative and antibacterial activities”.
2. Lines 55‐56: “Since various derivatives of isoniazid are well reported for their antitubercular and antibacterial properties…” . This should be justified by an example from the literature.
3. In section 2 relating to results and discussion, the general procedure for the synthesis of Isoniazidʹs Schiff bases according to the two protocols, the microwaveassisted synthesis (Lines 64‐69) and the conventional method (Line 70 to Line 75, just before “The synthesized compounds were characterized...”), should be moved to section 3 corresponding to Materials and Methods. Authors are requested to present (without the details of the reaction protocol) in the chemistry section, the two methods used, to discuss the results obtained, and to specify the advantage of using microwave chemistry, a technique more and more used in laboratories and in industry because it meets the criteria of green chemistry. Simple to implement, fast and selective, it has many interests and allows to synthesize many compounds. All this can be justified by bibliographic references.
4. The physico‐chemical characteristics of the three compounds (Lines: 86-115) should also be moved to section 3 corresponding to the materials and methods.
5. The analysis and the skinning of the 1H NMR Spectrum of compound (1) in the experimental part (Lines: 87‐90) is incorrect. Below, the analysis we have done.
1H‐NMR (300 MHz, DMSO‐d6): δ : 12.04 (1H; s; NH‐N=); 8.76‐8.74 (2H, br/m, H2’ and H6’ of pyr); 8.35 (1H, s; N=CH‐); 7.91‐7.94 (2H, br/m; H3’’ and H6’’ of ArH); 7.72‐7.85 (3H; m; H3’, H5’ of pyr and H5’’ of ArH); 7.58‐7.63 (1H, H4’’ of ArH); 7.01‐7.05 (1H, d, H4 of furan, J = 12Hz), 6.97‐7.01 (1H, d, H3 of furan, J = 12Hz).
6. For the skinning of the 13C NMR Spectrum of product (2), there are just two corrections to be made : Line 100 : 122.02 (C‐3’ and C‐5’, pyr) and Line 101 : 146.15 (C”).
7. The authors specify in the abstract of their paper the characterization of their products by elementary analysis, whereas the experimental and theoretical results of this analysis are absent in the experimental part.
8. How can the authors explain that the compound (E)‐Nʹ‐(4‐fluoro‐3‐
nitrobenzylidene)isonicotinohydrazide (2) showed better antibacterial activity than the compounds (1) and (3), while the product (1) had the highest zone of inhibition diameters? Have the authors carried out a statistical study to determine the mean of the inhibition diameters as well as the standard deviations?
9. In order to better evaluate the antibacterial activity, a further quantitative study may be carried out by the authors to determine the minimum inhibitory concentrations (MICs) or Bactericides (CMBs) of the Three new Schiff bases of isoniazid against the selected strains of bacteria.
10. The pixel resolution of some spectra in the supplemenatry data is extremely low. The authors should improve their resolution for better visibility.
11. Table 1 should be created according to the New Template of 2021.
12. The font size of the references should also be changed to Palatino Linotype, 9.
13. Changes can be made such as :
Line 31: …. lifetime[2]. ……………. lifetime [2]. . …
Line 79: …. (Scheme 1). ……………. (Scheme 1). …
Line 138: ….than compound 1 and 3……..than compounds (1) and (3)
Line 145: …. (d6DMSO). ……………. (DMSO‐d6). …
Comments for author File: Comments.pdf
Author Response
Thank you for comments.
The manuscript is updated as per comments. Please refer Answer to reviewer.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript entitled "Microwave assisted synthesis of (E) -N '- (5- (2-nitrophenyl) -furan-2-yl) -methylene) / (E) -N' - (substituted-benzylidene) -isonicotinohydrazide and their anti-proliferative and antibacterial activities ” presents the synthesis and biological activity of 3 compounds.
Compounds 1 and 3 are known, only 2 is a new compound: it is in line with the requests of the journal, but the authors should justify the choice to present these compounds at least from the point of view of SAR.
English is to be reviewed throughout the manuscript, then there are grammatical errors and disconnected sentences.
The introduction should be revised, it is not very fluent, many concepts are repeated. The authors should focus more on why they chose to synthesize these compounds, and why they decided to test the antitumor and antibacterial activity ... it all seems a bit loose.
Section 2.1.1: Just as the authors report all the steps and reaction times for b) Conventional method, so it should also be for method a). Alternatively, and I believe it is the best choice, all the synthetic steps, as well as the characterization of the compounds, should be reported in an appropriate section in Materials and methods. In this way, in Results and discussion, I would suggest to the authors to focus on the comparison between the two methods, in terms of yield, timing, costs, etc .... And the reasons that led them to try the two ways.
For these reasons I suggest MINOR REVISIONS.
Author Response
Thank you for comments the manuscript is revised as per the comments.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comment 01:
Please be more carefully when you present the NMR peaks. If you choose to present the NMR signals in descending order, then you have to give all the chemical shifts in descending order, even the chemical shift range of the same signal (e.g. 12.04 (1H, s, NH-N=); 8.76-8.74 (2H, br/m, H2’ and H6’ of pyr); 8.35 (1H, s, N=CH-); 7.91-7.94 7.94-7.91 (2H, br/m; H3’’ and H6’’ of ArH);). Also, all signals that are not singlet must be provided as a range chemical shift and not a number (e.g., for compound 2: 8.81(2H; d, d; J = 5.0Hz, H-2’ and H-6’, pyr)). Also, on the previous example the signal from 8.81 is presented as d, d and not dd, as was indicated in the general part.
Comment 02:
Regarding the operating frequencies of the NMR instrument, I understand that the authors used two NMR instrument (300 and 500 MHz) to record the NMR spectra of the compounds. Please provide the name and the parameters of both instruments.
Comment 03:
Regarding the antiproliferative activity of the compounds, even in the results and discussion part the authors say that „ the synthesized compounds showed low to moderate activity, compared to standard”, in the conclusion the same authors claim that „ The synthesized compound exhibited moderate anti-proliferative and antibacterial activities;”. Please correct the conclusion part to be in accordance with the results and discussion.
Having in view this consideration I recommended the publication of this article in Molbank after these corrections has been made.
Comments for author File: Comments.pdf
Author Response
Comment 01: IT IS CORRECTED, and for the doublet the single value (and J in Hz are mentioned) as:
%, 15 min. (b) 95%, 6h, yellow crystalline), m.p. 180-182 °C (decomposed). 1H-NMR (300 MHz, DMSO-d6): δ : 12.04 (s, 1H, NH‐N=); 8.76‐8.74 (br/m, 2H, H2’ and H6’ of pyr); 8.35 (s, 1H, N=CH‐); 7.94-7.91(br/m, 2H, H3’’ and H6’’ of ArH); 7.72‐7.85 (m, 3H, H3’, H5’ of pyr and H5’’ of ArH); 7.58‐7.63 (br, 1H, H4’’ of ArH); 7.03 (d, 1H, J = 12Hz, H4 of furan), 6.99 (d, 1H, J = 12Hz, H3 of furan). LCMS/MS for C17H12N4O4 (ESI+ion) : m/z = 337.6 [M+1], 336.6[M+], 319.2, 216, 148, 121, 104.8, 80, 79. IR (KBr, cm-1): 3442.9 (N-H), 3209.6 (C-H, arom.), 1660.7 (C=O), 1616.4 (C=C), 1533.4 (N-H), 1410.0 (C-N) and 690.5 (C-H, arom.). Elemental Analysis C17H12N4O4, Calculated C, 60.71; H, 3.60; N, 16.66; Found C, 60.49; H, 3.59; N 16.62. Rf value = 0.69 (chloroform: methanol : acetic acid:90:10:0.5 v/v/v).
(E)-N'-(4-fluoro-3-nitrobenzylidene)isonicotinohydrazide (2): (yield, time (a) 98%, 10 min. (b) 95%, 6h, brown crystals), m.p. 200-205 °C (decomposed). 1H- NMR (500 MHz, DMSO-d6): δ : 12.32 (1H; s; NH-N=); 8.81(d, 2H, J = 5.0Hz, H-2’ and H-6’, pyr); 8.54 (1H, s, N=C-H), 8.51 (d, 1H, J=6.8 Hz, H-2), 8.19 (d, 1H, J=7.0 Hz, H-6), 7.83 (d, 2H, J = 5.0Hz, H-3’ and H-5’, pyr), 7.69 (dd, 1H, 3JH-F =10.5 Hz, H-5); 13CNMR (125 MHz, DMSO-d6) : δ 162.38 (C=O), 155.77 (d, 1JC-F = 263 Hz, C-4), 150.84 (C-2’ and C-6’, pyr), 146.15(C”), 140.64 (C-4’), 137.78 (d, 2JC-F = 7.50 Hz, C-2), 134.95 (d, 2JC-F = 9.5 Hz, C-3), 131.98 (C-1), 124.80 (C-6), 122.02 (C-3’ and C-5’, pyr), 119.74 (d, 2JC-F = 21.50 Hz, C-5). LCMS/MS for C13H9FN4O3 (ESI+ion) : m/z = 289.6 [M+1], 288.6, 242.8, 123, 121, 104, 80, 79. IR (KBr, cm-1): 3446.8 (N-H), 3201.8, 3118.9, 3086.1 (C-H, arom.), 1674.2 (C=O), 1618.3 (C=C), 1577.8 (N-H), 1537.3 (C=C), 1411.9(C-N), 1159.2 (C-F), 752.2 and 694.4 (C-H, arom.). Elemental Analysis C13H9FN4O3, Calculated C, 54.17; H, 3.15; N, 19.44; Found C, 54.29; H, 3.16; N 19.51. Rf value = 0.52 (chloroform: methanol: acetic acid:90:10:0.5 v/v/v).
(E)-N'-(2-fluoro-5-nitrobenzylidene)-isonicotinohydrazide (3) : (yield, time (a) 98.5%, 20 min. (b) 95.0%, 8h, off white, fine crystals), m.p. 208-210 °C; 1H- NMR (300 MHz, DMSO-d6): δ: 12.38 (s, 1H, NH-N=C); 8.82(d, J = 5.1Hz, 2H, H-2’ and H-6’, pyr), 8.70 (s/br, N=C-H, overlapped with 1H, H-6), 8.35-8.37 (m/br, 1H, H-4), 7.85 (d, J = 5.1Hz, 2H, H-3’ and H-5’, pyr), 7.64 (dd, 1H, 3JH-F =10.5 Hz, H-3).
All Signals are presented in descending order,
(Please let me these are correct or range is required; I saw in recent pubication as single value, Molbank 2021, 2021(1), M1184; https://doi.org/10.3390/M1184).
Comment 02:
2 instruments are used and details are added in the text as
1H-NMR were recorded on Bruker DPX-300 instrument (300MHz) and Bruker 500 MHz-Avance III (500 MHz) spectrometer, while 13C‐NMR spectra were recorded on Bruker 500 MHz-Avance III (at 125 MHz) at room temperature using TMS as reference.
Comment 03:
It is updated as
- Conclusions
We have successfully developed an efficient protocol for the synthesis of a Schiff bases in excellent yields, under microwave irradiation and conventional conditions. The microwave assisted method presented here is rapid and economical All the synthesized compounds were thoroughly characterized using elemental analysis, NMR, IR, and LC-MS/MS spectrometric analyses. The synthesized compounds were then utilized for anti-proliferative and biological studies. The synthesized compound exhibited low to moderate anti-proliferative and weak antibacterial activities; more structural modifications is required to enhance the efficacy of synthesized compounds.
Reviewer 2 Report
The authors have revised the manuscript. They have taken the comments into account and have made all the requested changes to the revised manuscript. The manuscript is much improved and can therefore be published at molbank in the present form
Author Response
Thank you very much
