Journal of Molecular Structure 1237 (2021) 130367

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Clerodane and 5 10-Seco-Clerodane-type diterpenoids from Salvia

involucrata
Celia Bustos-Brito a, Diana Pérez-Juanchi a, José Rivera-Chávez a,
Alejandro David Hernández-Herrera b, Brenda Y. Bedolla-García c, Sergio Zamudio d,
Teresa Ramírez-Apan a, Leovigildo Quijano a,∗, Baldomero Esquivel a,∗
a
Departamento de Productos Naturales, Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Ciudad de
México, 04510, México
b
Departamento de Bioquímica, Centro de Investigación Biomédica de la Facultad de Medicina, Unidad Torreón, Universidad Autónoma de Coahuila, Torreón,
México
c
Instituto de Ecología, A.C., Centro Regional del Bajío, Apartado Postal 386, 61600 Pátzcuaro, Michoacán, México
d
Apartado Postal 392, 61600 Pátzcuaro, Michoacán, México

a r t i c l e i n f o a b s t r a c t

Article history: From the leaves of Salvia involucrata Cav., eight clerodane diterpenoids were isolated, of which 1−3
Received 15 January 2021 have not been previously described. Their structures were established by spectroscopic and spectro-
Revised 15 March 2021
metric means and named as involucratin A (1), involucratin B (2), and involucratin C (3). The absolute
Accepted 22 March 2021
configuration of compounds 1 and 3 was established using a combination of experimental and theoret-
Available online 26 March 2021
ical ECD data, whereas the one for compound 2 was determined by NOESY experiments together with
Keywords: ECD and DP4+ statistical analysis. Furthermore, the known clerodane compounds (5R,7R,8S,9R,10R,12R)-
Salvia involucrata 7-hydroxycleroda-1,3,13(16),14-tetraene-17,12;18,19-diolide (4), (–)-hardwickiic acid (5), 1-deoxybacrispine
clerodane diterpenoids (6), 7α -hydroxybacchotricuneatin A (7), and kingidiol (8), along with the flavonoid salvigenin, and a mix-
DP4+ statistical analysis ture of ursolic and oleanolic acids were also isolated. The antiproliferative activity of compounds 1–3, 5,
cytotoxic activity and 7, 8 was evaluated against a panel of cancer cell-lines (six strains) using the sulforhodamine B assay.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction
In Mexico, Lamiaceae is one of the largest and most diverse
plant families, with 591 species and accounting for approximately
8.11 % of the family. It is distributed in 32 native or naturalized
genera in the country, with an endemism of 65.8 % [1]. The most
abundant genus of Lamiaceae in Mexico is Salvia L., with over
295 natives species belonging to subgenus Calosphace [2] and nine
species to subgenus Audibertia [3].
Previous phytochemical studies on Salvia species demonstrated
that diterpenoids are the most diversified secondary metabo-
lites, with neo-clerodane and abietane skeletons isolated most
frequently, labdane, pimarane, kaurene, and totarane-type diter-
penoids have also been isolated. Several rearranged diterpenoids
derived from neo-clerodane and abietane scaffolds have also been
described, highlighting the chemical features of Salvia spp. The
distribution of these type of compounds in the different subgen-
era proposed for this genus, originally by Bentham (Salvia, Leonia,
∗
Corresponding authors.
E-mail addresses: quijano@unam.mx (L. Quijano), baldo@unam.mx (B. Esquivel).
Sclarea, Calosphace) [4] and recently by Drew et al. (Audibertia, Do-
rystaechas, Meriandra, Perovskia, Rosmarinus and Zhumeria) [5], are
of chemosystematic relevance [6] and could explain the medicinal
uses of these plants across the world.
As part of a systematic study of Mexican Salvia spp, looking
for biologically active diterpenoids, we have recently described the
chemical constituents and biological evaluation of S. clinopodioides
Kunth [7] and S. cinnabarina M. Martens & Galeotti [8]. In continu-
ation of our studies, we herein describe the chemical composition
of two populations of Salvia involucrata Cav. (Section Holwaya), har-
vested from Xilitla, State of San Luis Potosí (Mexico). S. involucrata
is a native species of Mexico, distributed in the central-southern
portion of the Sierra Madre Oriental at 1100 to 2600 m of alti-
tude, in the States of Nuevo León, México, Tamaulipas, San Luis
Potosí, Guanajuato, Querétaro, Hidalgo, Puebla and Veracruz [9, 10].
It blooms and fructifies from July to January [11]. In Mexico, S. in-
volucrata is also known as “mirto real” and is part of the medicinal
complex known as “mirto” together with S. fulgens Cav., S. coccinea
Buc’hoz ex Etl., S. microphylla Kunth and S. elegans Vahl, and is
used in the treatment of the folk illness “aire” [12]. Although there
are no reports of the chemical constituents of this species, the

https://doi.org/10.1016/j.molstruc.2021.130367
0022-2860/© 2021 Elsevier B.V. All rights reserved.
C. Bustos-Brito, D. Pérez-Juanchi, J. Rivera-Chávez et al.
aqueous-ethanolic extract displaced nicotine and scopolamine from
nicotinic and muscarinic receptors in homogenates of human cere-
bral cortical cell membranes, an activity tested in medicinal plants
used to improve failing memory and cognitive functions [13].
2. Materials and methods
2.1. General Experimental Procedures
Melting points (uncorrected) were determined on a Fisher-Johns
apparatus. The optical rotations were measured on a Perkin-Elmer
323 polarimeter. The ECD spectra were recorded on a Jasco J-1500
spectropolarimeter in MeOH at 450 ppm for compounds 1 and 3
and 10 0 0 ppm for compound 2. The UV spectra were recorded on
a Shimadzu UV 160U spectrophotometer. The IR spectra were ob-
tained on a Bruker Tensor 27 spectrometer; 1D and 2D NMR exper-
iments were performed on Bruker Advance III HD spectrometers at
700 MHz for 1 H and 175 MHz for 13 C and 500 MHz for 1 H and
125 MHz for 13 C as indicated. Chemical shifts were referenced to
CDCl3 (δ H = 7.26, δ C = 77.16). The HRDARTMS spectra were per-
formed on a Jeol, AccuTOF JMS-T100LC mass spectrometer; silica
gel 230−400 mesh (Macherey-Nagel) was used for column chro-
matography. Analytical and semipreparative HPLC were performed
on Gemini-NX 5 μm particle size C18 columns (4.6 × 250 mm,
and 10.0 × 250 mm for analytical and semipreparative runs, re-
spectively; Phenomenex, Torrance, CA, USA). Percentage of purity,
of isolated compounds, was determined by NMR.
2.2. Plant Materials
Salvia involucrata was collected from the Municipality of Xilitla,
State of San Luis Potosí, Mexico in May 2017 and a second collec-
tion was carried out in the same area in October 2018. A voucher
specimen from the first collection was identified by Dr. J. García-
Pérez and deposited at the Herbarium Isidro Palacios of the In-
stituto de Investigaciones Desérticas de San Luis Potosí (Voucher
SLPM-51136). Plant material from the second collection was iden-
tified by Dr. Brenda Y. Bedolla-Garcia and Dr. S. Zamudio and de-
posited in the herbarium of the Instituto de Ecología, A. C., Centro
Regional del Bajío (Voucher IEB-266885).
2.3. Extraction and Isolation
The phytochemical analysis of both harvests of S. involucrata
was carried out and the chemical profile were essentially the same.
Therefore, we only describe the extraction and isolation of com-
pounds from the first harvest. The dried and powdered aerial
parts of S. involucrata (1390 g) were extracted by percolation with
CH2 Cl2 (1.2 L). The CH2 Cl2 extract was concentrated to yield 38
g of residue. The extract was subjected to CC on silica gel us-
ing gradient elution with petrol:EtOAc (100:0−0:100) to obtain 96
eluates (300 mL each), which were combined into 32 major frac-
tions (A-Z). Compound 5 (350 mg, 97 % purity) crystallized from
fraction C. Fraction P (389 mg) was purified by CC on silica gel,
eluting with petrol:EtOAc (9:1 v/v) as mobile phase to obtain 22
eluates (PA1-PA22). Fractions PA15-PA18 (75 mg) were combined
and purified by TLC on silica gel, eluting with EtOAc:MeOH:H2 O
(200:16:7) as the mobile phase to give 1-deoxybacrispine (6, 23.2
mg, 94 % purity) and salvigenin (6.5 mg). Fraction S (600 mg) was
subjected to “flash” column on silica gel, with an isocratic mobile
phase of petrol:acetone (8:2) to obtain 68 eluates (SA1-SA68), 25
mL each. Fraction SA8 (60.0 mg) was subjected to TLC eluting with
CH2 Cl2 :MeOH (100:2.5) to yield compound 8 (25.3 mg, 96 % pu-
rity). Fractions SA12-SA14 (63.7 mg) were combined and subjected
2
Journal of Molecular Structure 1237 (2021) 130367
to TLC eluting with petrol:EtOAc (1:1) to yield compound 3 (23.5
mg, 95 % purity). Fraction V (1.00 g) was subjected to CC on silica
gel using CH2 Cl2 :acetone (9:1) to obtain 23 eluates (VA1-VA23), 25
mL each. Compound 2 (12.7 mg, 91 % purity) was identified from
fraction VA15. Fraction X (803 mg) was subjected to CC eluting
with petrol:EtOAc (1:1) to obtain 23 eluates (XA1-XA23). Fractions
XA14-XA21 were combined and further purified by CC eluting with
petrol:EtOAc (1:1) to yield compounds 1 and 4 as a mixture (10
mg); which was then subjected to semipreparative reverse phase
chromatography on a GeminiNX C18 column using a gradient sol-
vent system of CH3 CN-H2 O (0.1 % formic acid) 20:80 − 100:0 over
30 min at a 4.6 mL/min flow rate, to afford compound 1 (5.0 mg,
91 % purity) and 4 (2.0 mg, 86 % purity). Compound 7 (125 mg,
97 % purity) crystalized from fraction Y. Ursolic and oleanolic acids
were identified as mixture from fraction D.
Involucratin A (1). White powder; mp 158-162 °C; [α ]25 D +15.0
(c 0.38, MeOH); UV (MeOH) λmax nm (log ε ): 207 (3.21), 299
(3.40); IR (CHCl3 ) ν max ; 1756, 1602, 1590, 1504, 1365, 1208, 1187,
1027, 919 and 876 cm−1 ; 1 H and 13 C NMR, see Table 1; HRDARTMS
m/z 399.14588 (calcd for C22 H23 O7 , 399.14438).
Involucratin B (2). White powder; mp 194−200 °C; [α ]25 D +76
(c 0.20, MeOH); UV (CH3 CN) λmax nm (log ε ): 285 (3.22), 297
(3.21); IR (CHCl3 ) ν max 2920, 1737, 1565, 1372, 1231, 1073, 1020,
732, 601 cm−1 ; 1 H and 13 C NMR, see Table 1; HRDARTMS m/z
339.12263 (calcd for C20 H19 O5 , 339.12325).
Involucratin C (3). White powder; mp 122-126 °C; [α ]25 D – 44 (c
0.72, CHCl3 ); UV (MeOH) λmax nm (log ε ): 212 (3.66), 291 (3.42);
IR (CHCl3 ) ν max 2934, 2866, 1748, 1645, 1616, 1505, 1441, 1323,
1059, 1035, 983, 954, 932, 876 cm−1 ; 1 H and 13 C NMR, see Table 2;
HRDARTMS m/z 399.14479 (calcd for C22 H23 O7 , 399.14438).
(-)-Hardwickiic acid (5): White powder; mp 112-116 °C (reported
[14] 104-105 °C); [α ]25 D – 101.0 (c 0.51, MeOH) (reported – 114.8
(c 1.0, MeOH) [14]) 1 H NMR (CDCl3 , 700 MHz) δ 7.35 (1H, s, H-15),
7.21 (1H, s, H-16), 6.88–6.85 (1H, m, H-3), 6.26 (1H, s, H-14), 2.44
(1H, dt, J=13.0, 3.2 Hz, H-1a), 2.32-2.35 (1H, m, H-2a), 2.32 (1H,
dd, J=14.0, 4.7 Hz. H-12a), 2.19-2.22 (1H, m, H-2b) 2.18 (1H, ddd,
J=14.6, 10.2, 4.4 Hz, H-12b), 1.70 (1H, dd, J=13.7, 7.4 Hz, H-6a), 1.66
(1H, dd, J=13.1, 5.1 Hz, H-11a), 1.57 (1H, dd, J=12.6, 4.3 Hz, H-8),
1.53-1.59 (1H, m, H-11b), 1.47-1.55 (2H, m, H-6b, H-7a), 1.43 (1H,
dd, J=13.9, 3.7 Hz, H-7b), 1.39 (1H, d, J=12.1 Hz, H-10), 1.26 (3H, s,
CH3 -19), 1.18 (1H, td, J=13.1, 3.9 Hz, H-1b), 0.84 (3H, d, J=6.7 Hz,
CH3 -17), 0.77 (3H, s, CH3 -20). 13 C NMR (CDCl3 , 175 MHz) δ 173.0
(C, C-18), 142.9 (CH, C-15), 141.7 (C, C-4), 140.5 (CH, C-3), 138.5
(CH, C-16), 125.7 (C, C-13), 111.1 (CH,C-14), 46.8 (CH, C-10), 39.0
(C, C-9), 38.8 (CH2 , C-11), 37.7 (C, C-5), 36.4 (CH, C-8), 36.0 (CH2 ,
C-1), 27.7 (CH2 , C-2), 27.4 (CH2 , C-7), 20.7(CH3 , C-19), 18.4 (CH3 , C-
20), 18.3 (CH2 , C-12), 17.6 (CH2 , C-6), 16.1 (CH3 , C-17). HRDARTMS
m/z 317.21205 (calcd for C20 H29 O3 , 317.21167).
1-Deoxybacrispine (6): White powder; mp 200-202 °C (reported
[15] 198-202 °C); [α ]25 D – 53 (c 0.13, MeOH) (reported [15] – 55.9
(c 1.07, MeOH)); 1 H NMR (CDCl3 , 700 MHz) δ 7.37 (1H, t, J = 1.6
Hz, H-15), 7.22 (1H, s, H-16), 6.73 (1H, dd, J = 7.4, 2.0 Hz, H-3), 6.27
(1H, s, H-14), 5.30 (1H, d, J = 7.6 Hz, H-19a), 4.11 (1H, q, J = 3.4
Hz, H-7), 3.92 (1H, dd, J = 7.6, 1.6 Hz, H-19b), 2.36-2.41 (1H, m,
H-2a), 2.36 (1H, dd, J = 14.3, 2.4 Hz, H-6a), 2.31-2.36 (1H, m, H-
12a), 2.13-2.20 (2H, m, H-2b, H-12b), 1.80 (1H, d, J = 12.1 Hz, H-
10), 1.77 (1H, dd, J = 7.1, 3.7 Hz, H-8), 1.72-1.74 (1H, m, H-1a), 1.68
(1H, td, J = 13.8, 4.6 Hz, H-11a), 1.54-1.59 (1H, m, H-11b), 1.40-
1.43 (1H, m, H-6b), 1.16 (1H, qd, J = 12.5, 3.9 Hz, H-1b), 1.07 (3H,
d, J = 7.2 Hz, CH3 -17), 0.87 (3H, s, CH3 -20). 13 C NMR (CDCl3 , 175
MHz) δ 170.1(C, C-18), 143.1(CH, C-15), 139.3(C, C-4), 138.6(CH, C-
16), 135.3(CH, C-3), 125.0(C, C-13), 111.0(CH, C-14), 72.9(CH2 , C-19),
72.8(CH, C-7), 48.5(CH, C-10), 45.1(C, C-9), 40.7(CH, C-8), 40.6(CH2 ,
C-6), 38.8(C, C-5), 38.7(CH2 , C-11), 27.8(CH2 , C-2), 19.5(CH2 , C-1),
19.4(CH3 , C-20), 18.4(CH2 , C-12), 12.1(CH3 , C-17). HRDARTMS m/z
331.18969 (calcd for C20 H27 O4 , 331.19093).
C. Bustos-Brito, D. Pérez-Juanchi, J. Rivera-Chávez et al. Journal of Molecular Structure 1237 (2021) 130367

Table 1
NMR Data (1 H 700 MHz and 13
C 175 MHz, CDCl3 ) of 1 and 2.

1 2

Position δC type δ H (J in Hz) HMBC δC type δ H (J in Hz) HMBC

1 131.7 CH 5.94, dd (9.8, 2.6) 3, 5, 9, 10 133.6 CH 6.26, dd (9.8, 2.8) 3, 5, 9, 10

2 122.8 CH 6.22, ddd (9.7, 5.4, 3.0) 4, 10 125.2 CH 6.34, m 3, 10
3 128.8 CH 6.86, d (5.4) 1, 2, 6∗ 10∗ 18, 19 129.4 CH 7.01, d (5.1) 1, 2, 5, 18
4 128.0 C 131.0 C
5 39.0 C 40.4 C
6a 32.0 CH2 2.30, dd (14.7, 9.4) 4, 5, 7, 8, 10, 19 27.8 CH2 2.34, d (17.8) 5, 8, 10, 19
6b 1.80, ddd (14.7, 7.2, 2.2) 4, 5, 7, 10, 19 2.15, dd (17.8, 6.8) 5, 8, 10, 19
7 63.2 CH 5.76, ddd (9.4, 7.2, 4.8) 8, 9, 1’ 134.9 CH 6.78, dd (6.8, 1.8) 5, 6, 8, 9, 17
8 46.0 CH 2.46, d (4.8) 6, 7, 9, 10, 11, 17, 20 137.2 C
9 34.2 C 34.8 C
10 52.2 CH 2.92, brs 1, 2, 3∗ 5, 6, 11, 20 48.8 CH 2.79, brs 1, 2, 5, 6, 9, 11, 19
11a 47.6 CH2 2.25, dd (14.6, 4.1) 8, 9, 10, 12, 13, 20 45.3 CH2 2.18, dd (14.1, 10.9) 8, 9, 10, 12, 13, 20
11b 2.11, dd (14.6, 10.5) 8, 9, 10, 12, 13, 20 2.07, dd (14.1, 5.5) 8, 9, 10, 12, 13, 20
12 70.6 CH 5.33, dd (10.5, 4.0) 9, 1, 13, 14, 16 71.0 CH 5.06, dd (10.9, 5.5) 9, 11, 13, 14, 16, 17
13 123.7 C 123.8 C
14 108.4 CH 6.42, dd (1.7, 1.0) 12, 13, 15, 16 108.7 CH 6.43, brs 12, 13, 15, 16
15 144.1 CH 7.44, t (1.7) 13, 14, 16 144.1 CH 7.44, brs 13, 14, 16
16 139.9 CH 7.48, brs 13, 14, 15 140.0 CH 7.48, brs 13, 14, 15
17 168.6 C 168.7 C
18 168.5 C 3, 4, 5, 19 168.9 C
19pro-R 78.7 CH2 4.44, d (8.3) 4, 5, 6, 18 77.2 CH2 4.31, d (8.3) 3, 5, 6, 18
19pro-S 4.00, dd (8.3, 2.2) 5, 6, 10 4.15, dd (8.3, 1.5) 5, 6, 18
20 27.6 CH3 1.35, s 8, 9, 10 29.1 CH3 1.44, s 8, 9, 10, 11
1’ 170.2 C
2’ 21.2 CH3 2.05, s 12, 13, 11
∗
Through four-bonds interaction.

Table 2 2.5 Hz, H-6a), 2.45 (1H, dddd, J=18.1, 7.4, 4.1, 1.8 Hz, H-2a), 2.15
NMR data (1 H 700 MHz, 13
C 175 MHz, CDCl3 ) of 3.
– 2.29 (2H, m, H-2b, H-11a), 1.90 – 1.99 (2H, m, H-11b, H-10),
3 1.77 – 1.80 (1H, dd, J=12.9, 4.8 Hz, H-1a), 1.41 (1H, ddt, J=14.3,
Position δC type δ H (J in Hz) HMBC 4.1, 2.2 Hz, H-6b), 1.33 (1H, qd, J=12.6, 4.1 Hz, H-1b), 1.13 (3H,
s, CH3 -20); 13 C NMR (CDCl3 , 126 MHz) δ 174.3(C, C-17), 169.3(C,
1 123.8 CH 6.06, d (17.5) 2, 9, 10
2 133.4 CH 6.35, d (11.4) 4, 10
C-18), 144.3(CH, C-16), 139.9(CH, C-15), 138.6(C, C-4), 135.2(CH,
3 121.6 CH 6.12, d (11.4) 1, 2, 5, 10∗ 18 C-3), 124.2(C, C-13), 108.6(CH, C-14), 72.4(CH2 , C-19), 70.9(CH, C-
4 124.9 C 12), 66.1(CH, C-7), 54.6(CH, C-10), 51.3(CH, C-8), 44.9(CH2 , C-11),
5 151.9 C 44.5(C, C-5), 38.1(CH2 , C-6), 37.0(C, C-9), 27.8(CH2 , C-2), 21.7(CH3 ,
6a 33.1 CH2 3.57, t (12.7) 4 ,5, 7, 19
C-20), 20.0(CH2 , C-1); DARTMS m/z 359 (calcd for C20 H23 O6 , 359).
6b 2.64, dd (12.7, 6.0) 4, 5 ,7, 8, 19
7 70.9 CH 5.66, ddd (12.0, 6.0, 2.0) 5, 6, 8, 9, 17, 1’
8 46.4 CH 2.68, brs 9, 10, 17, 20
2.4. ECD and chemical shifts calculations
9 39.1 C
10 140.6 CH 6.04, d (17.5) 1, 2, 8, 9, 20
11a 45.0 CH2 2.14, d (14.2) 9, 10, 12 Compounds 1–3 were built and geometry optimized using a
11b 1.95, dd (14.6, 12.0) 9, 10, 12, 13, 20 semiempirical method (PM3), as implemented in Spartan’10. Con-
12 70.2 CH 5.34, dd (12.0, 2.8) 9, 11, 13, 14, 16, 17 formational analysis was performed by means of the same soft-
13 123.5 C
ware and force field. All conformers were filtered and checked for
14 108.4 CH 6.38, brs 12,13,15,16
15 143.9 CH 7.39, t (1.68) 13,14,16 redundancy. Then, the conformers were minimized, optimized, and
16 140.0 CH 7.42, brs 13,14,15 thermochemical properties, including IR and vibrational analyses,
17 170.9 C were obtained with Gaussian 09 using a DFT force field at the
18 174.6 C
B3LYP/DGDZVP level of theory for ECD or B3LYP/6-31G(d) for NMR
19a 71.8 CH2 4.86, d (18.1) 2∗∗ 3∗ 4, 5, 6, 7, 18
19b 4.74, d (18.1) 2, 3, 4, 5, 6, 7, 18
calculations.
20 23.5 CH3 1.53, s 8, 9, 10, 11 ECD calculations in MeOH solution were carried out employing
1’ 170.1 C a TD-SCF force field at the B3LYP/ DGDZVP theory level, with the
2’ 21.2 CH3 2.09, s 7∗ 1’ default solvent model. The calculated excitation energy (nm) and
∗
Through four-bonds interaction. rotatory strength (R) in dipole velocity (Rvel ) form was simulated
∗∗
Through five-bonds interaction. into an ECD curve using Eq. (1) as implemented in the SpecDis
software [17,18]. Where Ek and Rk are the transition energy and
rotatory strength of the kt h electronic transition, respectively, and
7α -hydroxybacchotricuneatin A (7): Colorless crystal; mp 248- σ is the exponential half-width.
253 °C (reported [16] 242-243 °C); [α ]25 D – 53 (c 0.13, CHCl3 ) (re-   
1 1  E − E0k 2
ported – 154.6 (c 0.24, CHCl3 [16])); 1 H NMR (CDCl3 , 500 MHz) δ ε ( λ ) = × √ E0k R0k e −
7.46–7.51 (1H, m, H-15), 7.45 (1H, t, J=1.7 Hz, H-16), 6.73 (1H, dd, 2.296 × 10−39 σ π k
σ
J=7.4, 2.0 Hz, H-3), 6.44 (1H, dd, J=1.8, 0.8 Hz, H-14), 5.39 (1H, dd, (1)
J=10.3, 7.3 Hz, H-12), 5.32 (1H, d, J=7.5 Hz, H-19a), 4.63 (1H, p,
J=2.7 Hz, H-7), 4.00 (1H, t, J=2.0 Hz, OH-7), 3.93 (1H, dd, J=7.5, The NMR shielding constants were calculated with the Gauge-
2.2 Hz, H-19b), 2.67 (1H, d, J=3.0 Hz, H-8), 2.53 (1H, dd, J=14.3, Independent Atomic Orbital (GIAO) method at the B3LYP/6-31G (d)

3
C. Bustos-Brito, D. Pérez-Juanchi, J. Rivera-Chávez et al.
level of theory, using the PCM solvent model in CHCl3 . The experi-
mental and calculated data were analyzed by the DP4+ probability
method.
All calculations were performed on the HP Cluster Platform
30 0 0SL “Miztli,” a parallel supercomputer with a Linux operating
system, containing 25,312 cores and a total of 15,0 0 0 GB of RAM
[19].
2.5. Cytotoxicity assay
The natural products were screened in vitro against the fol-
lowing human cancer cell lines: human mammary adenocarci-
noma (MCF-7), human chronic myelogenous leukemia (K562), hu-
man glioblastoma (U251), human lung adenocarcinoma (SKLU-1),
human colon cancer (HCT-15), human prostate cancer (PC-3) and
healthy gingival human fibroblasts (FGH) cell lines which were
supplied by the National Cancer Institute (USA), and ATTC. The hu-
man tumor cytotoxicity was determined using the protein-binding
dye sulforhodamine B (SRB) in microculture assay to measure cell
growth, as described in the protocol established by the NCI [20].
The cell lines were cultured in RPMI-1640 medium supplemented
with 10 % fetal bovine serum, 2 mM L-glutamine, 10 0 0 0 units/mL
penicillin G sodium, 10 0 0 0 μg/mL streptomycin sulfate, 25 μg/mL
amphotericin B (Gibco), and 1 % non-essential amino acids (Gibco).
They were maintained at 37 °C in a humidified atmosphere with 5
% CO2 . The viability of the cells used in the experiments exceeded
95 % as determined with trypan blue.
The cells were removed from the tissue culture flasks by treat-
ment with trypsin and diluted with fresh media. Of these cell sus-
pensions, 100 μl containing 5000-10,000 cells per well, were pipet-
ted into 96 well microtiter plates (Costar) and the material was in-
cubated at 37 °C for 24 h in a 5 % CO2 atmosphere. Subsequently,
100 μL of a solution of the compound, obtained by diluting the
stocks, were added to each well. The cultures were exposed to the
compound at 25 μM concentrations for 48 h. After the incubation
period, cells were fixed to the plastic substratum by the addition of
50 μL of cold 50% aqueous trichloroacetic acid. The plates were in-
cubated at 4 °C for 1 h, washed with tap water, and air-dried. The
trichloroacetic acid-fixed cells were stained by the addition of 0.4
% SRB. The free SRB solution was then removed by washing with
1 % aqueous acetic acid. The plates were air-dried, and the bound
dye was dissolved by the addition of 10 mM unbuffered Tris base
(100 μL). The plates were placed on a shaker for 10 min, and the
absorption was determined at 515 nm using an ELISA plate reader
(BioTek Instruments).
2.6. Scavenging Activity on Free Radical
2,2-Diphenyl-1-Picrylhydrazyl (DPPH)
Free radical scavenging activity was measured using an adapted
method of Mellors and Tappel [21]. The test was carried out in 96-
well microplates. A 50 μL aliquot of the solution of the test com-
pound was mixed with 150 μL of an ethanol solution of DPPH (fi-
nal concentration 100 μM). The mixture was incubated at 37 °C for
30 min, and the absorbance was then measured at 515 nm using a
BioTek microplate reader SYNERGY HT. The % inhibition was deter-
mined by comparison with a 100 μM DPPH ethanol blank solution.
3. Results and discussion
The phytochemical profile of both analyzed collections of S.
involucrata was essentially the same. From the dichloromethane
extract of both populations, three undescribed clerodane lac-
tones 1−3, and the known clerodanes, (5R,7R,8S,9R,10R,12R)-
7-hydroxycleroda-1,3,13(16),14-tetraene-17,12;18,19-diolide (4) [22],
4
Journal of Molecular Structure 1237 (2021) 130367
(-)-hardwickiic acid (5) [23], 1-deoxybacrispine (6) [15], 7α -
hydroxybacchotricuneatin A (7) [16], and kingidiol (8) [24] were
isolated, together with salvigenin, and the common mixture of ur-
solic and oleanolic acids (Chart 1). Compound 4 was previously iso-
lated from S. chamaedryoides Cav. [22], while (-)-hardwickiic acid
(5) was described originally as a constituent of the resin part of
Hardwickia pinnata Roxb. ex DC. [23], 1-deoxybacrispine (6) was
first isolated from Baccharis crispa Spreng [15] and later described
as the oxidation product of portulide C from Salvia melissodora
Lag. [25], 7α -hydroxybacchotricuneatin A (7) was described pre-
viously from Baccharis incarum (Wedd.) Perkins [16], and kingidiol
(8) from Baccharis kingie Cuatrec [24]. All known compounds were
identified by spectroscopic methods, mainly high field (700 MHz)
NMR, and their structures confirmed by comparison with literature
data.
Compounds 1 and 2 were characterized as furane-clerodane-
diterpenes, and diterpene 3 as a 5,10-seco-clerodane derivative
based on their spectroscopic features and the following discussion.
Compound 1 was isolated as a white powder and the mass spec-
trum showed a pseudo molecular ion [M + H]+ according with the
molecular formula of C22 H23 O7 (HRDART m/z 399.14588 [M + H]+ ,
calcd 399.14438), indicating 12 degrees of unsaturation. Its IR spec-
trum showed a strong stretching frequency band for C=O at 1756
cm−1 and characteristic bands for a β -substituted furan ring at
1504, 919 and 876 cm−1 . Typical UV absorptions for this chro-
mophore [λmax nm (log ε ); 207 (3.21)] [26], together with the ab-
sorption for a homoannular diene system at 298 nm (log ε = 3.40),
supported these assignments [27].
Analysis of the 1 H and 13 C NMR spectra of compound 1
(Table 1) indicated the presence of a monosubstituted furan ring
(δ H 7.48, brs, H-16/ δ C 139.9; δ H 7.44, t, J = 1.7 Hz, H-15/ δ C 144.1;
and δ H 6.42, dd, J = 1.7, 1.0 Hz, H-14/ δ C 108.4); a δ -lactone (δ C
168.6, C-17; δ H 5.33, dd, J = 10.5, 4.0 Hz, H-12/ δ C 70.6); a γ -
lactone (δ C 168.5, C-18; δ H 4.44, d, J = 8.3 Hz, H-19pro–R , 4.00, dd,
J = 8.3, 2.2 Hz, H-19pro–S / δ C 78.7), the pro-S diastereotopic pro-
ton of this group is typically W-coupled with the C-6β proton, in-
dicating an axial orientation for C-19 and the absence of a C-6β
substituent in a clerodane type skeleton [28].
The 13 C NMR spectrum (Table 1) of 1 showed 22 signals.
Four vinylic carbon signals were attributed to one non-protonated
carbon (δ C 128.0/C-4) and three methines (δ C /δ H , 128.8/6.86, d
(J = 5.4 Hz), 122.8/6.22, ddd (J = 9.7, 5.4, 3.0 Hz), and 131.7/5.94,
dd (J = 9.8, 2.6 Hz)) which were assigned by DEPT, HSQC, COSY and
HMBC experiments to carbons C-3, C-2, and C-1 respectively. The
carbon signal at δ C 168.5, ascribed to the carbonyl of a γ -lactone,
correlated in the HMBC spectra with the doublet at δ 6.86 (J = 5.4
Hz) which was ascribed to the olefinic proton H-3. The COSY ex-
periment indicated that H-3 is part of a spin system including H-2
and H-1. H-2 was observed as a ddd at δ 6.22 (J = 9.7, 5.4, 3.0 Hz),
and H-1 as a dd at δ 5.94 (J = 9.8, 2.6 Hz). This facts led us to con-
clude that in compound 1, a homoannular diene system is present
in A ring which is fused to a γ -lactone.
A singlet at δ H 2.05 was attributed to the methyl of an ace-
toxy group at C-7, whose geminal proton (H-7) was responsible
for a doublet of doublet of doublets signal at δ 5.76 (1H, ddd,
J = 9.4, 7.2, 4.8 Hz), indicating the acetoxy group must be flanked
by a methylene and a methine. The coupling constants displayed
by H-7 (9.4, 7.2 Hz, axial-axial, axial-equatorial) suggest that the
acetoxy group must be oriented equatorially. Inspection of a Drei-
ding model and DFT-B3LYP/DGDZVP minimum energy conformer
of compound 1 indicated that the B-ring could adopt a boat-like
conformation due to a cis junction of the A-B rings displaying di-
hedral angles of H6α −C6−C7−H7β (28°) and H6β −C6−C7−H7β
(144°), thus explaining the coupling constants of 9.4 and 7.2 Hz
respectively. It has been documented that in cis-clerodanes, C-20
resonates lower in the field (δ C 2l-29) than trans-clerodanes (δ C
C. Bustos-Brito, D. Pérez-Juanchi, J. Rivera-Chávez et al.
Chart 1.
Fig. 1. Key NOESY correlations of compound 1.
17-19) [14]. The 13 C chemical shift of CH3 -20 (δ C 27.6), confirmed
the cis ring junction of rings A and B in compound 1.
The NOESY spectrum (Table 1, Fig. 1) supported the cofacial β
orientation of H-7, H-8, H-10, H-12, and H-19 and is thereby con-
5
Journal of Molecular Structure 1237 (2021) 130367
sistent with the α -orientation of CH3 -20, A/B cis and B/C trans ring
junctions.
Two related compounds have been identified from S.
chamaedryoides [22] and S. gesneriiflora [27]. The first one
was identified as the deacetyl derivative of compound 1,
(5R,7R,8S,9R,10R,12R)-7-hydroxycleroda-1,3,13(16),14-tetraene-
17,12;18,19-diolide (4) [22]. The second one was identified as
7α -acetoxy-7,8α -dihydrogensnerofolin B (9) [27], which could
be considered the 5S,8R,10S stereoisomer of 1. The observed 1 H
NMR spectroscopic changes at CH2 -19 (δ H 4.44/4.00 in 1 and
4.86/4.15 in 9) and CH-7 (δ H 5.76, ddd, J = 9.4, 7.2, 4.8 Hz in 1
and δ H 5.68, dt, J = 5.7, 3.0 Hz in 9) are attributed to the boat-like
conformation of ring B in 1. The specific rotation values were also
different in sign and magnitude, showing [α ]25 D +15.0 for 1 and
[α ]25 D –94.8 for 9.
To establish the absolute configuration of compound 1, the ex-
citation energy (nm) and rotatory strength (R) in dipole veloc-
ity (Rvel ) for the 5R, 7R, 8S, 9R, 10R, 12R isomer and its enan-
tiomer were calculated using TD-DFT and simulated into ECD
curves (Fig. 2). The experimental ECD spectrum for 1 displayed
two positive Cotton effects at 217 and 307 nm, one negative at
263 nm, and was in good agreement with the calculated data
for the 5R, 7R, 8S, 9R, 10R, 12R diastereoisomer. Additionally,
C. Bustos-Brito, D. Pérez-Juanchi, J. Rivera-Chávez et al.
Fig. 2. Comparison between the experimental (black line) and calculated ECD spec-
tra for enantiomers (5R, 7R, 8S, 9R, 10R, 12R)-1 (red dashed line) and (5S, 7S, 8R,
9S, 10S, 12S)-1 (blue dashed line).
Fig. 3. Minimum energy conformation for compound 2 and gensnerofolin B.
the experimental ECD curve shape for 1 matches the experimen-
tal curve of (5R,7R,8S,9R,10R,12R)-7-hydroxycleroda-1,3,13(16),14-
tetraene-17,12;18,19-diolide (4) [22]. Based on this evidence, the
absolute configuration of compound 1 was established as 5R, 7R,
8S, 9R, 10R, 12R.
Therefore, it follows that 1 is a new cis-clerodane derivative
identified as (5R,7R,8S,9R,10R,12R)-7-acetoxycleroda-1,3,13(16),14-
tetraene-17,12;18,19-diolide, which we have given the trivial name
involucratin A.
Compound 2 displayed the [M + H]+ ion at m/z 339.12263 in
the HRDART mass spectrum, which supports the molecular formula
C20 H19 O5 (calcd 339.12325). The 1 H and 13 C NMR spectra (Table 1)
of 2 displayed features similar to those of 1, except for the pres-
ence of a doublet of doublets signal at δ 6.78 (J = 6.8, 1.8 Hz, H-7)
in the 1 H NMR spectrum and two carbon resonances at δ C 134.9
(CH-7) and 137.2 (C-8) in the 13 C spectrum, associated with the
presence of a 7−8 unsaturation instead of the acetoxy group.
The proposed structure for compound 2 is closely related to
gensnerofolin B (10), a diterpenoid previously isolated from S. ges-
neriiflora [27]. When comparing the 1 H and 13 C NMR data of com-
pound 2 with the reported data for 10, a close similarity is ob-
served in almost all the signals except H-7; a triplet (J = 3.5 Hz)
was reported for 10, while in 2 it is observed as a doublet of dou-
blets (J = 6.8 and 1.8 Hz). Although both compounds have an A/B
cis junction, when H-10 and C-19 have a β orientation, the adopted
conformation by the B ring results in H-7 and H-6a being almost
in the same plane (Fig. 3), consequently, a strong coupling (J = 6.8
Hz) is observed between these two hydrogens.
The relative configuration of 2 was established on the basis
of NOESY correlations and was predicted using the DP4+ analy-
sis, a sophisticated approach for the stereochemical assignment of
6
Journal of Molecular Structure 1237 (2021) 130367
Fig. 4. Graph of the DP4+ (B3LYP/6-31G∗ ) probability obtained by correlating the
experimental NMR of compound 2 with the calculated data for 2a-2c. The proba-
bility for the correct assignment is shown in blue.
Fig. 5. Comparison between the experimental (black line) and calculated ECD spec-
tra for enantiomers 5R9R10R12R-2 (red dashed line) and 5S9S10S12S-2 (blue dashed
line).
organic molecules [29]. In this regard, the NMR isotropic shield-
ing tensors for diastereoisomers 5R, 9R, 10R, 12R (2a), 5S, 9R, 10S,
12S (2b), and 5R, 9R, 10R, 12S (2c) (Fig. 4) were calculated using
the GIAO method at the B3LYP/6-31G∗ level of theory. The results
from this prediction indicated that the diastereoisomer 5R, 9R, 10R,
12R (2a) is the most likely structure, with a probability of 97.3 %
(Fig. 4).
In order to establish the absolute configuration of 2, its ECD
spectrum was recorded and compared with that calculated for di-
astereoisomer 2a and its enantiomer. The theoretical spectrum of
2a (red dashed line) matched the shape of the experimental curve
of 2 (Fig. 5), which displayed a positive Cotton effect at 300 nm,
and a negative effect at 222 nm. Based on these results, the abso-
lute configuration of 2 was determined to be 5R, 9R, 10R, 12R.
Compound 3 was isolated as white powder with the molecu-
lar formula C22 H22 O7 based on its HRDARTMS data ([M + H]+ at
m/z 399.14479, calcd 399.14438), together with 1 H and 13 C NMR
analyses, indicating that 3 has 12 degrees of unsaturation. Its IR
spectrum was consistent with the presence of a furan ring (1505,
876 cm−1 ), olefinic double bonds (1645 cm−1 ), and the presence
of carbonyl groups (1748 cm−1 ). The UV spectrum supports these
assignments, since it showed typical absorptions for a furan group
[λmax MeOH nm (log ε )]: 212 (3.66) together with the presence of an
absorption for a homoannular conjugated triene system at 291 nm
C. Bustos-Brito, D. Pérez-Juanchi, J. Rivera-Chávez et al.
(log ε = 3.42). Significant similarities in the 1 H and 13 C NMR data
of 3 and 1 were observed. The 13 C NMR spectrum of 3 (Table 2)
exhibited 22 carbon resonances comprising of one acetate methyl
group, three methylenes (one oxygenated), ten methines (two oxy-
genated and eight olefinic and aromatic), seven quaternary carbons
(three carbonyls, three olefinic and one aliphatic), and is consistent
with an acetylated diterpene skeleton. The 1 H NMR spectrum of
3 (Table 2) revealed a typical resonance pattern due to the pres-
ence of a β -substituted furan ring, as was shown in compounds
1 and 2. The 1 H-1 H COSY spectrum displayed three spin sys-
tems: −CH=CH−CH=CH− (for C10−C1−C2−C3), −CH2 −CH− (for
C11−C12), and CH2 −CH−CH (for C6−C7−C8). The first spin sys-
tem showed proton signals at δ H 6.12 (brd, J = 11.4 Hz, H-3),
6.35 (brd, J = 11.4, H-2), 6.06 (d, J = 17.5 Hz, H-1), and 6.04 (d,
J = 16.0 Hz, H-10) which were part of the homoannular conju-
gated triene system suggested by the UV spectrum and also in-
volve the C4-C5 double bond. This triene olefinic system can only
be accommodated in a 5,10-seco-clerodane skeleton as depicted in
3. The coupling constant observed for the protons of this triene
indicated that the C10-C1 double bond must have an E configura-
tion (J = 17.5 Hz) and the C2-C3 double bond must be Z (J = 11.4
Hz). The olefinic proton at δ H 6.12 (H-3) exhibited a strong cor-
relation with a γ -lactone carbonyl group (δ C 174.6) in the HMBC
spectrum, in its turn this carbonyl showed a correlation with an
AB system (δ H 4.86, 4.74, d, J = 18.1 Hz) for a methylene group,
therefore the triene system should be conjugated with a γ -lactone
carbonyl moiety. The magnitude of the coupling constant (18.1 Hz)
showed in compound 3 for this AB system is typical for a rigid γ -
lactone as in the clerodanes salvileucanthsin B and salvileucanthsin
C isolated from S. leucantha Cav. [30], cardiophyllidin from S. car-
diophylla Benth [31], and a 5,10-seco-clerodane from Baccharis fla-
bellate Hook. & Arn. [32]. The second spin system, observed in the
1 H-1 H COSY spectrum of 3, showed proton signals at δ 5.34 (dd,
H
J = 12.0, 2.8, H-12), 2.14 (d, J = 14.2, H-11a) and 1.95 (dd, J = 14.6,
12.0, H-11b). This spin system is part of a C17, C12 δ -lactone based
on the NMR signals at δ C 170.9 (C-17) and 70.2 (C-12). The HMBC
correlations of C-17 with H-12 and H-7 (δ H 5.66, ddd, J = 12.0, 6.0,
2.0 Hz /δ C 70.9) confirmed the 17,12 δ -lactone and indicated that
the third spin system is constituted by C-6, C-7, and C-8 carbons.
A broad singlet at δ 2.68 was ascribed to H-8, the multiplicity of
this signal suggested a dihedral angle near to 90° between H-8 and
H-7. The signal for the C-6 methylene protons were observed at δ H
3.57 and 2.64 (Table 2).
The relative configuration of compound 3 was established
by considering the α -orientation of CH3 -20, by NOE effects be-
tween H-7, H-8 and H-12, and the lack of these effects with
CH3 -20, establishing a β -orientation of H-8 and an α -orientation
for the furan ring. The absolute configuration of compound 3
was established as 7R, 8S, 9R, 12R based on the results ob-
tained from comparison between the theoretical (blue and red
dashed lines) and experimental ECD curves (black line, Fig. 6),
and also by comparison of the experimental curve with that
reported for 7β -acetoxysalvimicrophyllin A, isolated from Salvia
decora Epling [19], and (7R,8S,9R,12R)-7-hydroxy-5,10-seco-neo-
cleroda-1(10),2,4,13(16),14-pentaene-17,12;18,19-diolide [22]. Based
on these data, compound 3 was given the trivial name involucratin
C.
Involucratin A (1) and involucratin C (3) could be related
through a conrotatory electrocyclic reaction catalyzed by light. The
possibility of this reaction was supported in a theoretical way by
quantum chemical calculations in a closely related compound iso-
lated from Salvia microphylla Kunth [33].
Based on the fact that clerodane diterpenes have showed sig-
nificant cytotoxic activity, including (–)-hardwickiic acid [34], com-
pounds 1−3, 5, and 7-8 were tested for antiproliferative activity
using six human cancer cell lines (U251, PC-3, K562, HCT-15, MCF-
7
Journal of Molecular Structure 1237 (2021) 130367
Fig. 6. Comparison between the experimental (black line) and calculated ECD spec-
tra for enantiomers (7R, 8S, 9R, 12R)-3 (red dashed line) and (7S, 8R, 9S, 12S)-3 (blue
dashed line).
7, and SKLU-1) and a normal monkey kidney (COS-7) at 25.0 μM.
Adriamycin at 0.5 μM, was used as the positive control. Results are
summarized in Table 3. Of all evaluated compounds, compounds
1 and 8 showed inhibitory effects in cell lines U251 and K562
respectively. The calculated IC50 for compound 1 on U251 (66.8 ±
5.7 μM) showed a weak effect. The calculated IC50 for compound 8
on K562 indicated a moderate cytotoxic effect (19.0 ± 1.8 μM).
Compounds 3 and 8 were also tested in the 2,2-diphenyl-1-
picrylhydrazyl (DPPH); however, both compounds were inactive.
In a recent paper, a thorough taxonomic treatment and detailed
description of Salvia involucrata Cav. (section Holwaya), as well as
the comparison with other members of the same section, led to
the conclusion that Salvia puberula Fernald must be considered a
synonym of S. involucrata [11].
Several years ago, the diterpenoid content of a population of
Salvia puberula Fernald (actually S. involucrata) collected in the mu-
nicipality of Rayón in the State of San Luis Potosí was published,
and the presence of benzonorcaradiene (salvipuberulin) and ben-
zocyclohepatriene (iso-salvipuberulin) diterpenoids of clerodanic
origin was reported [35]. Nevertheless, these compounds were not
found in the two collections of S. involucrata studied here, and
therefore, the diterpenoid content is significantly different between
the two populations described in this work and the previously
published. Although it has been well documented for some Mexi-
can Salvia species, that the diterpenoid content can vary depending
on the location, time of the year when the harvesting takes place,
and the local environment [36], it is worthy to note that both spec-
imens of S. involucrata reported in this manuscript have the same
chemical profile, despite being harvested at different times.
It is important to point out that while the two populations de-
scribed herein grow in the municipality of Xilitla (State of San
Luis Potosí), which is located in the eastern slope of the Sierra
Madre Oriental mountain range, the population of S. involucrata
analyzed under the binomial S. puberula, was harvested from the
municipality of Rayón (State of San Luis Potosí), but in the west-
ern slope of the Sierra Madre Oriental and about 75 kilometers
from Xilitla. This geographical isolation prevents genetic flow be-
tween the populations, probably resulting in the chemical differ-
ences observed between the Xilitla and Rayón collections of S. in-
volucrata. Geographical isolation is considered one of several fac-
tors that have triggered the great diversity and richness of Salvia
subgenus Calosphace in Mexico [2].
Further phytochemical analysis in different populations of Salvia
involucrata and its synonymous species S. puberula are necessary
to establish its variability in chemical composition and support
the previously described taxonomic conclusion. The content of
clerodane-type diterpenoids found for S. involucrata is consistent
with the fact that these types of secondary metabolites are the
most frequently isolated from Salvia species belonging to the sub-
C. Bustos-Brito, D. Pérez-Juanchi, J. Rivera-Chávez et al.
Table 3
Primary screening of compounds 1–3, 5, and 7–8 on antiproliferative activity at concentration of 25.0 μM.
Compound Antiproliferative activity (%)
U251 PC-3 K562
1 49.6 14.7 24.8
2 5.1 23.5 34.7
3 NC 11.0 19.4
5 22.4 1.8 45.5
7 3.8 12.8 20.2
8 22.4 13.0 51.6
Adriamycin 0.5 μM 96.0 85.2 100
Results are represented as the mean (n = 2); U251 = human glioblastoma; PC-3 = human prostate cancer; K562 = hu-
man chronic myelogenous leukemia; HCT-15 = human colon cancer; MCF-7 = human mammary adenocarcinoma;
SKLU-1 = human lung adenocarcinoma; COS-7 normal monkey kidney; NC = Not cytotoxic.
genus Calosphace, and chemically supports the divergence with
Salvia species grown in the Old World and China, which mainly
biosynthesize abietane-derived diterpenoids [37].
4. Conclusions
From the dichloromethane extract of two collections of Salvia
involucrata Cav., several natural products were isolated including
two unpublished cis-clerodane diterpenoids, named involucratin A
(1) and involucratin B (2), and one unreported 5,10-seco-clerodane
named involucratin C (3). Five known clerodane diterpenoids (4−8)
were also obtained and their structures were established by spec-
troscopic means and confirmed by comparison with literature data.
While the absolute configuration of compounds 1 and 3 were es-
tablished by using a combination of experimental and theoretical
ECD data, the relative and absolute configuration of compound 2
was addressed by DP4+ statistical analysis and ECD data. Some
of the diterpenoids were tested for antiproliferative activity and
only 1 and 8 showed moderate activity in U251 (human glioblas-
toma cancer) and K562 (human chronic myelogenous leukemia)
cell lines respectively with IC50 = 66.8 ± 5.7 (μM) for compound
1 and IC50 =19.0 ± 1.8 (μM) for compound 8. Diterpenoids 3 and
8 showed no activity in the DPPH assay. The content of clerodane-
type diterpenoids found for S. involucrata, is consistent with the
chemical profile most frequently found for Salvia species belonging
to the American subgenus Calosphace.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgments
The authors acknowledge H. Rios, B. Quiroz, E. Huerta, A. Peña,
R. Patiño, L. Velasco, C. García, and J. Pérez for collecting NMR, UV,
IR and MS data. The authors are indebted to Dr. J. Garcia Pérez
from the Instituto de Investigaciones Desérticas de San Luis Po-
tosi, for plant identification of the first collection. This study made
use of UNAMś NMR lab: LURMN at IQ-UNAM, which is funded by
CONACYT- Mexico (Project 0224747), and UNAM. J. R.-C. is indebted
to the Dirección General de Cómputo y de Tecnologiás de Informa-
ción y Comunicación (DGTIC), UNAM, for providing the resources
to carry out the computational calculations through the Miztli Sys-
tem, through the seed project LANCAD-UNAM-DGTIC-374. The au-
thors thank to Ms. Sc. Jacklyn Gallagher for English language revi-
sion.
8
Journal of Molecular Structure 1237 (2021) 130367
HCT-15 MCF-7 SKLU-1 COS-7
- NC 12.6 NC
11.8 0.5 36.7 21.6
9.7 NC 16.8 11.9
10.4 1.4 11.5 19.8
13.3 NC 33.0 14.2
15.5 0.8 22.9 19.7
86.9 99.1 90.0 53.4
Credit Author Statement
Celia Bustos-Brito, Diana Pérez-Juanchi, Alejandro David
Hernández, Baldomero Esquivel and Leovigildo Quijano, par-
ticipated in the isolation and structure elucidation. Brenda Y.
Bedolla-García, Sergio Zamudio participated in the harvest and
identification of plants. José Rivera-Chávez determined the ab-
solute configuration of the compounds and carried out all the
corresponding calculations. Teresa Ramírez-Apan participated in
the performance of the antiproliferative activity. Celia Bustos-Brito,
Baldomero Esquivel, Leovigildo Quijano, Brenda Y. Bedolla-García,
Sergio Zamudio and José Rivera-Chávez participated in the
redaction of the manuscript. All co-authors participated equally
and substantially to the paper preparation and revision of the
manuscript.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130367.
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