Synthesis and in vitro anticancer activities of substituted N-(4′-nitrophenyl)-l-prolinamides

Prolinamides are present in secondary metabolites and have wide-ranging biological properties as well as antimicrobial and cytotoxic activities. N-(4′-substituted phenyl)-l-prolinamides 4a–4w were synthesized in two steps, starting from the condensation of p-fluoronitrobenzene 1a–1b with l-proline 2a–2b, under aqueous–alcoholic basic conditions to afford N-aryl-l-prolines 3a–3c, which underwent amidation via a two-stage, one-pot reaction involving SOCl2 and amines, to furnish l-prolinamides in 20–80% yield. The cytotoxicities of 4a–4w against four human carcinoma cell lines (SGC7901, HCT-116, HepG2 and A549) were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; with good tumour inhibitory activities (79.50 ± 1.24%–50.04 ± 1.45%) against HepG2. 4a exhibited the best anti-tumour activity against A549 with percentage cell inhibition of 95.41 ± 0.67% at 100 µM. Likewise, 4s (70.13 ± 3.41%) and 4u (83.36 ± 1.70%) displayed stronger antineoplastic potencies against A549 than the standard, 5-fluorouracil (64.29 ± 2.09%), whereas 4a (93.33 ± 1.36%) and 4u (81.29 ± 2.32%) outperformed the reference (81.20 ± 0.08%) against HCT-116. SGC7901 showed lower percentage cell viabilities with 4u (8.02 ± 1.54%) and 4w (27.27 ± 2.38%). These results underscore the antiproliferative efficacies of l-prolinamides while exposing 4a and 4u as promising broad-spectrum anti-cancer agents. Structure-activity relationship studies are discussed.


Comments to the Author(s)
In this manuscript Profs. Bao and Familoni describe the synthesis and evaluation of a series of Narylated prolinamides as in vitro anticancer agents. The work is scientifically sound, and identified antiproliferative agents that could lead to further refinement and follow-up studies. I am therefore recommending publication of the present manuscript in Royal Society Open Science, after the following comments have been addressed by the authors: 1.
Please include additional information on the rationale of your structural design. The tested compounds have novel structures, and are quite different from the structures in figure 1. (those are either N-acyl or free amino versus N-aryl substitutions in the present study), and therefore there is no indication as to why the authors thought these molecules might have applications as anticancer agents. 2.
Related to the first point, please indicate what was the criteria to select the cell lines tested. Is any information about the potential mechanism of action or molecular target available? If so, please include it in your discussion. 3.
The activity observed ranges from modest to good, but the potential selectivity could be equally important or even more relevant. Please state whether any selectivity study using cell line models for "healthy cells" was conducted, or state whether that would be part of a planned follow-up study.

4.
For analog 4m, indicate in the structure the stereochemistry of the hydroxyl group at position 4 of the proline.
P4L16 -'peptides are typically favored over small organic molecules in drug development' This is not true (at least in the way the authors are describing) peptide or peptidomimetic compounds represent a very small % of approved drugs. Small molecules are preferred for the reasons discussed by the authors.
The authors screen their compounds at 1, 10 and 100 uM concentrations but why not add another concentration and determine an IC50 value? An IC50 is much more robust and allows better comparison between derivatives.
The discussion of SAR wherein longer chains provide more active compounds is a common observation in medicinal chemistry. The greater lipophilicity afforded by the longer chains enable greater cell uptake. See Wang et. al. ChemMedChem. 2017, 12, 1033. Consider adding this reference.
No selectivity data in a non-cancerous cell line is presented, despite the introduction discussing non-selective toxicity as a real problem. This should be determined at least for the most active compounds. This is especially important with compound 4u which is toxic against all the cells tested. Is this just a toxic compound? P12L21. '4k and 4m suspected of encouraging proliferation' This seems unlikely, is this not just general growth? A negative control in the experiment would determine levels of growth in vehicle alone.
P12L30; Sulphonamides alone are not a pharmacophore, it is their substituents so this sentence is incorrect.
P12:35; 4l has no inhibition activity or it just wasn't determined as shown in Table 2? It is unlikely that the physical state will be relevant to activity.

Minor issues:
The abstract is very long and switches between discussing cell inhibition % and cell survival % which are not the same thing.
The prolinamide structure in the nucleoside compounds in figure 1 is a little misleading as these are ligands of a protide structure that will be lost in the cell, it has no contribution to the pharmacophore.
Scheme 1 shows 3a-d but the text only refers to 3a-c.
A line to separate cell types in table 2 would be useful for clarity of presentation.

Decision letter (RSOS-200906.R0)
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However, the reviewers raise a number of valid concerns that should be addressed by the authors. In particular, addressing the concern over compound is a necessity. Also, I am inclined to agree that some toxicity data in healthy cells for the most active compounds is required.
I ask the authors to carefully consider the attached reviewer comments and submit a revised manuscript. Given, the present global circumstances, I ask the authors to do their best at addressing these concerns. There may be some concerns that require additional experimentation. If this is not possible, please justify why we should consider a revised manuscript without these experiments.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) In this manuscript Profs. Bao and Familoni describe the synthesis and evaluation of a series of Narylated prolinamides as in vitro anticancer agents. The work is scientifically sound, and identified antiproliferative agents that could lead to further refinement and follow-up studies. I am therefore recommending publication of the present manuscript in Royal Society Open Science, after the following comments have been addressed by the authors: 1. Please include additional information on the rationale of your structural design. The tested compounds have novel structures, and are quite different from the structures in figure 1. (those are either N-acyl or free amino versus N-aryl substitutions in the present study), and therefore there is no indication as to why the authors thought these molecules might have applications as anticancer agents. 2. Related to the first point, please indicate what was the criteria to select the cell lines tested. Is any information about the potential mechanism of action or molecular target available? If so, please include it in your discussion. 3. The activity observed ranges from modest to good, but the potential selectivity could be equally important or even more relevant. Please state whether any selectivity study using cell line models for "healthy cells" was conducted, or state whether that would be part of a planned follow-up study. 4. For analog 4m, indicate in the structure the stereochemistry of the hydroxyl group at position 4 of the proline.

Reviewer: 2
Comments to the Author(s) The authors describe the synthesis and anticancer activity of a series of N-substituted(4'nitrophenyl)-L-prolinamides. The compounds are interesting with a number (notably 4a and 4u) possessing greater activity than 5-FU control). However, a number of issues need attention before I can support publication: Major issues: No information on the purity of the compounds is provided. The compounds need HPLC or elemental analysis determined purity levels to ensure that an impurity is not responsible for the assigned activity.
P4L16 -'peptides are typically favored over small organic molecules in drug development' This is not true (at least in the way the authors are describing) peptide or peptidomimetic compounds represent a very small % of approved drugs. Small molecules are preferred for the reasons discussed by the authors.
The authors screen their compounds at 1, 10 and 100 uM concentrations but why not add another concentration and determine an IC50 value? An IC50 is much more robust and allows better comparison between derivatives.
The discussion of SAR wherein longer chains provide more active compounds is a common observation in medicinal chemistry. The greater lipophilicity afforded by the longer chains enable greater cell uptake. See Wang et. al. ChemMedChem. 2017, 12, 1033. Consider adding this reference.
No selectivity data in a non-cancerous cell line is presented, despite the introduction discussing non-selective toxicity as a real problem. This should be determined at least for the most active compounds. This is especially important with compound 4u which is toxic against all the cells tested. Is this just a toxic compound? P12L21. '4k and 4m suspected of encouraging proliferation' This seems unlikely, is this not just general growth? A negative control in the experiment would determine levels of growth in vehicle alone.
P12L30; Sulphonamides alone are not a pharmacophore, it is their substituents so this sentence is incorrect.
P12:35; 4l has no inhibition activity or it just wasn't determined as shown in Table 2? It is unlikely that the physical state will be relevant to activity.

Minor issues:
The abstract is very long and switches between discussing cell inhibition % and cell survival % which are not the same thing.
The prolinamide structure in the nucleoside compounds in figure 1 is a little misleading as these are ligands of a protide structure that will be lost in the cell, it has no contribution to the pharmacophore.
Scheme 1 shows 3a-d but the text only refers to 3a-c.
A line to separate cell types in table 2 would be useful for clarity of presentation.

Decision letter (RSOS-200906.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Reviewer 1's Comment
In this manuscript Profs. Bao and Familoni describe the synthesis and evaluation of a series of Narylated prolinamides as in vitro anticancer agents. The work is scientifically sound, and identified antiproliferative agents that could lead to further refinement and follow-up studies. I am therefore recommending publication of the present manuscript in Royal Society Open Science, after the following comments have been addressed by the authors: