(22E,24S)-24-Propylcholest-5en-3α-acetate: A New Steroid from the Stembark Aglaia angustifolia (Miq.) (Meliaceae)

Round 1

Reviewer 1 Report

Major Revisions Required

Major Item #1: The structure in Figure 1 may not be correctly assigned. The authors need to check their assignments correctly and revise this manuscript. If correct the authors are proposing a rather new structure of C20-C29 as numbered in Fig. 1. As shown in the attached PDF file it is more likely that they are looking at a 24-propylcholesterol derivative such as (24R)-24-propylcholest-5-en-3β-ol clearly defined synthetically by Griner, see:

Giner, José-Luis; Li, Xiaoyong Stereospecific synthesis of 24-propylcholesterol isolated from the Texas brown tide. [Tetrahedron, 2000, vol. 56, # 49, p. 9575 - 9580]

Giner, Jose-Luis; Li, Xiaoyong; Boyer, Gregory L Sterol composition of Aureoumbra lagunensis, the Texas brown tide alga. [Phytochemistry, 2001, vol. 57, # 5, p. 787 - 789]

It is possible that what the authors are looking at is the acetate of (24R or 24S)-24-propylcholest-5-en-3α-ol. What is proposed is an unprecedented, often erroneously assigned, 3 carbon linkage (CH₂CH₂CH₂) between C21 and C24 when in reality this is typically 2 (CH₂CH₂).

Figure 3 is missing the C21 methyl group? The authors need to very carefully check their NOESY assigments here.

If this assignment is revised and is correct the authors have a very strong finding as C30-sterols with n-propyl subtitution at C-24 appear are very limited to a small class of marine chromophyte algae as first reported by Fenical and Djerassi, see:

Rohmer, Michael; Kokke, W. C. M. C.; Fenical, W.; Djerassi, C. Isolation of two new C30 sterols, (24E)-24-N-propylidenecholesterol and 24ξ-N-propylcholesterol, from a cultured marine chrysophyte. Steroids, 1980, vol. 35, # 2, p. 219 – 231.

Major Item #2:

The compounds are not sufficiently pure and will need additional chromatographic purification prior to publication. The authors clearly ignore the impurity peaks in their NMR spectra including clear deletion of minor peaks in their 13C spectrum. Examples include the two peaks between the assigned peaks at 139.8 and 122.8. These two olefinic peaks appear in all of the 1D and 2D spectra presented and could be attributed to a (24E)-24-N-propylidenecholesterol congener of their material. That said, the purity of these compounds is not acceptable for publication and the authors will need to return and deliver a pure sample of their analog with a repeated 1D and 2D NMR dataset. As shown, this work cannot be published. What is rather concerning for this author is their well run DEPT-135 clearly shows these two olefinic peaks (as well as the others) but the authors do not report or discuss them. That said, this manuscript is close to publication, and highly interesting if really a rare sterol, and the authors did an excellent job writing up their manuscript.

Major Item #3

For clear evaluation, the authors need to provide a detailed table of each 1H, 13C, gCOSY, and NOESY assignment as required by most publications including this Journal and the Journal of Natural Products.

Major Item #4:

The spectral set provide in the Supporting Information needs serious improvement. For instance, The COSY was not run with sufficient transients or processed with software in a sufficient manner for this author to clearly evaluate the correlations that would be required to assign C24-C30. The SI figures need to allow the reader to directly see each correlation mentioned in the Table (Major Item #3 above) as well as discussed in the manuscript. Clearly no one can see the correlations in the COSY in the window of 1-2 PPM versus 1-2 PPM in Fig. S6. Expansions and detailed assignments of each correlation would really help this manuscript. Inexpensive software such as mNova would make such assignments rather easy.

As this is a novel sterol not found in plant materials the authors will need to do a detailed and careful assignment of their finding. This finding must be put in proper context with the published literature. As it stands without said effort this is an rather ‘unpublishable’ half-finished work.

Minor:

Line 3, should read “and the Pacific Islands”

Line 50, define what brand and type of silica gel that was used

Line 84 and general within the manuscript you do not have to write out ‘thirty two’ rather just put the number 32 as it is easier to read. The same goes for other spots within this paragraph and manuscript where ‘one’, ‘two’, ‘six’ can be easily described as 1, 2 and 6. All of these should be edited so the reader can better address the data description.

The structure in Figures 1-2 could use some editing as the bond orientations from C20-C25 are poorly drawn.

C20 in the structure in Figure 1 is given with double stereochemistry. It is best to show the proton with stereochemistry at C17 and no stereochemistry on the bond C17-C20 and then provide the dashed bond at C20.

Comments for author File: Comments.pdf

Author Response

We had already revised our manuscript in line with the reviewer's comments and suggestions

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript described the isolation and structure elucidation of a new 23a-homostigmastane-type steroid from the stem bark of Aglaia angustifolia. Two questions should be answered before accepting publication.

The "new" compound is probably artifacts formed during the isolation process. The proof that it actually exists in the plant should be presented. As shown in Figure s1- s8, the purity of compound 1 is not so good. The proton signals of H20-H28 are overlapped so that the side chain of the compound needs to be carefully re-checked. In its HR-TOS-MS spectrum, the ion at 493.4 is too low. The ions at 935 [2M+Na]⁺ and 397 [M-CH3COO]⁺ suggest that this compound may be acetyl-β-sitosterol. Authors should verify this possibility.

Author Response

We had already revised in line with reviewer's comments

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Page 1, line 25 and Page 2, line 42, the capital P in propyl should be in small case
Page 1, line 35, define the ‘some parts’ such as roots, leaves, or bark?
Page 1, line 40, should read ‘phytochemically’
Page 2, line 48, ‘room temperature’ is commonly abbreviated ‘rt’
Page 2, lines 50-52, the abbreviations VLC and CC appear not used more than once and can be deleted
Page 2, Figure 1, the C20 stereochemistry as drawn is ambiguous remove the bold dash line from C17-C20 and the structure is correctly drawn. The Journal of Natural Products provides a nice guideline as to why this is an issue
Page 2, Figure 1 caption. The bolding should be removed as Structure or Compound are not proper names.
Page 3, line 78, split ‘thus requiring hydrogen deficiency index of six’ into its own sentence and explain how this was derived not everyone will know or just delete this as it is described in line 85. However if deleted you will need to explain how you got six just before line 85.
Page 3, line 83, its methyl groups not methyls and methane carbons not methines. The same is trie for methylene
Page 3, line 92 why is that proton in [ ]. Remove this notation completely throughout the text and write the sentence as a sentence. You have 2 perhaps three sentences cramped into one and writing it out would clearly help the reader.
Page 3, line 99, again you can remove the capital P in propyl, check this throughout the manuscript
Page 3, line 100, it should read the Texas brown tide and why is the letter A capitalized. The authors need to carefully check capitalization throughout the text. Only proper nouns like Texas are capitalized compound names are also not unless they are a commercial trade name.
Page 4, line 121 it is a 1H-1H NOESY please state
Page 4, Figure 3, one does not use BOLD DASHED bonds in this type of structural depiction use straight lines only. The positions of the carbons for the side chain are again rather clumsily drawn.
Page 5, line 134, why is the word ‘Compound’ used and in the next line why is ‘Cisplatin’ capitalized
Page 4, line 155, the actual name of the herbarium should be provided.
Page 4, line 160, 50 µL/50 mL is the same as 1 µL/mL
Page 4, line 169, it should read ‘Microsoft Excel’
Page 4, line 173, check if ‘A. angustifolia (Miq.) Miq’ is correct

Author Response

We had already revised in line with reviewer's suggestion:

Author Response File: Author Response.docx

Reviewer 2 Report

no

Author Response

New cytotoxic pregnane-type steroid from the Stem Bark of Aglaia elliptica (Meliaceae), Rec. Nat. Prod.,

Rohmer, M., Kokke, W. C. M. C., Fenical, W. Djerassi, C., Isolation of Two New C₃₀ Sterols, (24E)-24-N-Propylidenecholesterol and 24ε-N-Propylcholesterol, from a cultured Marine Chrysophyte. Steroids, 1980, 35(2), 219-231. DOI: https://doi.org/10.1016/0039-128X(80)90104-X.

Giner, J.-L., Li, Xiaoyong., Boyer, G. L. Sterol Composition of Aureumbra lagunensis, The Texas Brown Tide Alga. Phytochemistry, 2001, 57(5), 787-789.DOI: https://doi.org/10.1016/S0031-9422(01)00135-2.

Giner, J.-L., Li, Xiaoyong. Stereospecific synthesis of 24-Propylcholesterol Isolated from The Texas Brown Tide. Tetrahedron, 2000, 56(49), 9575-9580. DOI: https://doi.org/10.1016/S0040-4020(00)00931-5.

Nugroho, B. W., Edrada, R.A., Wray, V., Witte L., Bringmann, G., Gehling, M., and Proksch, P. An insectisidal rocaglamide derivates and related compounds from Aglaia odorata (Meliaceae). Phytochemistry  1999, 51, 367-376. DOI: https://doi.org/10.1016/S0031-9422(98)00751-1.

Author Response File: Author Response.docx