Synthesis and antimycobacterial evaluation of natural oridonin and its enmein-type derivatives

Article abstract of DOI:10.1016/j.fitote.2014.10.005

A series of enmein-type derivatives were synthesized and assayed for their antimycobacterial effects. The structures of the synthesized compounds were established by ¹H NMR, ¹³C NMR and mass spectral analysis. All the compounds were screened for their antimycobacterial properties against Mycobacterium phlei, Mycobacterium smegmatis and Mycobacterium marinum. Compounds 2, 6g and 6i were found to exhibit potent antimycobacterial activity against M. phlei at a concentration of 0.5 μg/mL, which was comparable to that of positive drug streptomycin. Furthermore, five compounds were tested against Mycobacterium tuberculosis H₃₇Rv based on the promising preliminary screening results. Among them, compound 10 showed potent activity with IC₅₀ value of 17.1 μg/mL against M. tuberculosis H₃₇Rv strain. Thus, compound 10 could emerge as a promising lead for further research work.

Full text of DOI:10.1016/j.fitote.2014.10.005

Fitoterapia 99 (2014) 300–306
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Synthesis and antimycobacterial evaluation of natural oridonin
and its enmein-type derivatives
Shengtao Xu a,b, Lingling Pei a,b, Dahong Li a,b,c, Hong Yao a,b, Hao Cai a,b, Hequan Yao a,b
,
Xiaoming Wu a,b, Jinyi Xu a,b,
⁎
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China
b
c
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education and School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
103 Wen Hua Road, Shenyang 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 August 2014
Accepted in revised form 3 October 2014
Available online 12 October 2014
A series of enmein-type derivatives were synthesized and assayed for their antimycobacterial
effects. The structures of the synthesized compounds were established by 1H NMR, 13C NMR
and mass spectral analysis. All the compounds were screened for their antimycobacterial
properties against Mycobacterium phlei, Mycobacterium smegmatis and Mycobacterium marinum.
Compounds 2, 6g and 6i were found to exhibit potent antimycobacterial activity against M. phlei at
a concentration of 0.5 μg/mL, which was comparable to that of positive drug streptomycin.
Furthermore, five compounds were tested against Mycobacterium tuberculosis H37Rv based on the
promising preliminary screening results. Among them, compound 10 showed potent activity with
IC50 value of 17.1 μg/mL against M. tuberculosis H37Rv strain. Thus, compound 10 could emerge as a
promising lead for further research work.
Keywords:
Oridonin
Tuberculosis
Antimycobacterial activity
Enmein-type
Natural product
© 2014 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article:
Oridonin (PubChem CID: 34378)
Enmein (PubChem CID: 352542)
Lasiokaurin (PubChem CID: 99618)
1. Introduction
[3]. Natural products currently play an important role in the
chemotherapy of tuberculosis, for example, streptomycin, cap-
Tuberculosis is an infection caused by Mycobacterium
tuberculosis and is the leading cause of infectious disease mor-
tality in the world [1]. It is estimated that one-third part of
the world population is infected with the tubercle bacillus.
Each year there are approximately eight million new cases and
two million deaths [2]. The goals of tuberculosis control are
focused on curing active disease, preventing relapse, reducing
transmission and averting the emergence of drug-resistance
reomycin, cycloserine, and semisynthetic rifamycin analogs are
used as either front-line or second line drugs [4]. These natural
products and their semisynthetic analogs have verified that
their inhibitory activity against M. tuberculosis is widespread in
nature.
Recently, there is a re-emerging interest in natural prod-
ucts as being able to provide novel structures for the drug
discovery and being particularly effective as antibacterial
leads [5,6]. Isodon (formally Rabdosia) is a cosmopolitan
and important genus of the Labiatae family, and Isodon
diterpenoids have attracted considerable attention as anti-
bacterial, anti-inflammatory and anti-tumor agents [7]. As
an ent-kaurane diterpenoid isolated from the herb Isodon
⁎
Corresponding author at: Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China.
Tel.: +86 25 83271299; fax: +86 25 83302827.
E-mail address: jinyixu@china.com (J. Xu).
http://dx.doi.org/10.1016/j.fitote.2014.10.005
0367-326X/© 2014 Elsevier B.V. All rights reserved.

S. Xu et al. / Fitoterapia 99 (2014) 300–306
301
rubescens, oridonin (1) has been attracting a rising attention
in recent years due to its extensive biological activities [8].
We have reported in a preliminary communication about
the synthesis and antimycobacterial evaluation of various
oridonin derivatives [9]. The results showed that some
semisynthetic analogs of oridonin especially its enmein-
type derivatives exhibited good antimycobacterial activity
which may be regarded as interesting leads for the further
design and synthesis of M. tuberculosis inhibitors.
126.0, 119.7, 113.9, 113.5, 95.8, 76.1, 72.9, 62.9, 61.4, 59.4, 54.9,
54.1, 40.9, 40.8, 38.2, 33.3, 32.0, 30.0, 29.5, 21.1, 19.2; MS (ESI)
m/z: 525.3 [M + H]+; HR-MS (ESI) m/z: calcd for C30H37O8
[M + H]+ 525.2483, found 525.2493.
ent-6β-hydroxy-(14α-O-cyclopentoyl)-1,7-epoxy-7,15-dioxo-
6,20-hemiketal-6,7-seco-16-kaurene (Compound 4a)
46% as white solid, mp. 206–208 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 6.23 (s, 1H), 5.69 (s, 1H), 5.54 (s, 1H),
5.34 (s, 1H), 5.32 (m, 1H), 4.56 (m, 1H), 4.05, 3.96 (dd, JA
=
In continuation of our ongoing research program, directed
toward design and synthesis of enmein-type derivatives as
antitubercular drug candidates, herein, we would like to de-
scribe our further research results involved in the synthesis and
in vitro antimycobacterial activity evaluation of enmein-type
derivatives of oridonin.
JB = 9.3 Hz, each 1H), 3.12 (d, J = 9.9 Hz, 1H), 2.68 (m, 2H),
2.52 (m, 1H), 2.33 (m, 1H), 1.98 (m, 3H), 1.75 (br, 10H), 1.50
(m, 2H), 1.02 (s, 3H), 0.96 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ
(ppm) 198.2, 175.2, 167.2, 150.7, 118.9, 100.4, 76.2, 73.8, 71.3,
61.0, 54.8, 49.9, 47.4, 44.2, 43.1, 37.2, 32.3, 30.1, 30.0, 29.8, 25.1,
23.4, 22.9, 18.8; MS (ESI) m/z: 459.2 [M + H]+, 476.2 [M +
NH4]+; HR-MS (ESI) m/z: calcd for C26H35O7 [M + H]+
459.2377, found 459.2370.
2. Experimental
ent-6β-hydroxy-(14α-O-benzoyl)-1,7-epoxy-7,15-dioxo-6,20-
hemiketal-6,7-seco-16-kaurene (Compound 4b)
2.1. Chemistry
81% as white solid, mp. 152–154 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 7.95 (d, J = 7.2 Hz, 2H), 7.53 (t, J =
7.5 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H), 6.25 (s, 1H), 5.87 (s, 1H),
5.56 (s, 1H), 5.34 (d, J = 3.0 Hz, 1H), 5.30 (d, J = 3.0 Hz, 1H),
4.61 (m, 1H), 3.99, 4.09 (dd, JA = JB = 9.3 Hz, each 1H), 3.38
(d, J = 9.3 Hz, 1H), 2.50 (m, 2H), 1.72 (m, 2H), 1.70 (s, 1H), 1.67
(m, 2H), 1.36 (m, 2H), 1.27 (m, 1H), 1.03 (s, 3H), 0.97 (s, 3H);
13C NMR (DMSO-d6): δ (ppm) 198.5, 166.8, 166.2, 147.9, 133.7,
130.3, 129.3, 128.6, 120.7, 102.0, 74.8, 74.7, 60.3, 54.1, 50.4,
48.8, 41.2, 37.3, 33.1, 31.3, 29.9, 23.5, 23.3, 20.0; MS (ESI) m/z:
467.1 [M + H]+, 484.1 [M + NH4]+, 501.3 [M + Cl]-; HR-MS
(ESI) m/z: calcd for C27H31O7 [M + H]+ 467.2064, found
467.2071.
ent-6β-hydroxy-(14α-O-p-flurobenzoyl)-1,7-epoxy-7,15-
dioxo-6,20-hemiketal-6,7-seco-16-kaurene (Compound 4c)
37% as white solid, mp.151–153 °C; 1H NMR (CDCl3,
500 MHz): δ (ppm) 7.97 (m, 2H), 7.05 (t, J = 8.2 Hz, 2H),
6.25 (s, 1H), 5.86 (s, 1H), 5.56 (s, 1H), 5.35 (s, 1H), 4.60
(m, 1H), 4.10, 4.00 (dd, JA = JB = 9.0 Hz, each 1H), 3.32
(d, J = 9.0 Hz, 1H), 2.82 (m, 1H), 2.63 (m, 1H), 2.04 (m, 1H),
1.98 (m, 2H), 1.81 (m, 1H), 1.73 (m, 2H), 1.54 (m, 2H), 1.25
(s, 1H), 1.03 (s, 3H), 0.97 (s, 3H); 13C NMR (DMSO-d6,
125 MHz): δ (ppm) 199.1, 167.0, 163.8, 148.2, 132.1, 132.0,
120.3, 116.0, 115.8, 100.6, 75.6, 75.0, 72.6, 59.5, 53.6, 49.4,
48.3, 36.1, 32.5, 30.5, 29.5, 22.9, 22.6, 18.8; MS (ESI) m/z:
485.2 [M + H]+, 502.2 [M + NH4]+, 507.2 [M + Na]+; HR-
MS (ESI) m/z: calcd for C27H30FO7 [M + H]+ 485.1970, found
485.1966.
The reagents were purchased from commercial sources
and used without further purification. Analytical thin layer
chromatography (TLC) was HSGF 254 (0.15–0.2 mm thick-
ness). Column chromatography was carried out on Merck
silica gel 60 (200–300 mesh). Proton and carbon magnetic
resonance spectrums (1H NMR and 13C NMR) were recorded
using tetramethylsilane (TMS) in the solvent of CDCl3 as the
internal standard (1H NMR: TMS at 0.00 ppm; CHCl3 at
7.26 ppm, 13C NMR: CDCl3 at 77.0 ppm). Chemical shifts
were reported in parts per million (ppm, δ) downfield from
tetramethylsilane. Proton coupling patterns are described as
singlet (s), doublet (d), triplet (t), quartet (q), double doublet
(dd), multipet (m) and broad (br). Low-and high-resolution
mass spectrums (LRMS and HRMS) were measured on Agilent
QTOF 6520.
The synthetic method and physicochemical date of the
compounds 3, 5, 7, 8, 9 were disclosed in our previous report
[10] and also were described in the supporting information.
2.1.1. Synthesis of target compound
Compound 1 (72 mg, 0.2 mmol) was mixed with 4-
methoxycinnamic acid (0.22 mmol), EDCI (0.25 mmol) and
DMAP (0.01 mmol) in 15 mL of dry dichloromethane and
stirred at room temperature for 18 h. The mixture was poured
into 15 mL of 10% HCl, and extracted with dichloromethane
(10 mL × 3). The organic layer was combined, washed with
water and saturated NaCl solutions sequentially, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude
product was purified by column chromatography (MeOH/
CH2Cl2 1:150 v/v) to give the compound 2.
ent-6β-hydroxy-(14α-O-o-pyrazinoyl)-1,7-epoxy-7,15-dioxo-
6,20-hemiketal-6,7-seco-16-kaurene (Compound 4d)
45% as white solid, mp.178–180 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 9.17 (s, 1H), 8.73 (d, J = 2.1 Hz, 1H), 8.70
(d, J = 2.1 Hz, 1H), 6.28 (s, 1H), 6.02 (s, 1H), 5.63 (s, 1H), 5.35
(s, 1H), 4.65 (m, 1H), 4.08, 3.96 (dd, JA = JB = 10.2 Hz, each
1H), 3.39 (d, J = 9.3 Hz, 1H), 3.16 (m, 1H), 2.83 (m, 1H), 2.71
(m, 1H), 2.04 (m, 1H), 1.80 (m, 1H), 1.71 (m, 5H), 1.36 (m, 1H),
1.11 (s, 3H), 1.02 (s, 3H); 13C NMR (DMSO-d6, 75 MHz):
δ (ppm) 195.4, 167.1, 163.3, 148.6, 134.9, 132.7, 120.7, 113.8,
109.9, 101.9, 76.3, 75.8, 74.4, 59.8, 53.3, 49.8, 48.7, 40.7, 37.2,
33.0, 30.5, 29.6, 23.3, 22.9, 16.6; MS (ESI) m/z: 469.2 [M + H]+;
HR-MS (ESI) m/z: calcd for C25H29N2O7 [M + H]+ 469.1969,
found 469.1958.
ent-1α,6β,7β-trihydroxy-(14β-O-p-methoxycinnamoyl)-
15-oxo-7,20-epoxy-16-kaurene
(Compound 2)
75% as white solid, mp. 175–177 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 7.59, 6.20 (dd, JA = JB = 15.9 Hz, each
1H), 7.44 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 6.21 (m,
2H), 5.93 (s, 1H), 5.49 (s, 1H), 4.42 (s, 1H), 4.09, 4.35 (dd, JA
=
JB = 9.6 Hz, each 1H), 3.86 (s, 3H), 3.79 (m, 1H), 3.53 (m, 1H),
3.27 (m, 1H), 2.63 (m, 1H), 2.38 (m, 1H), 2.04 (m, 1H), 1.80 (m,
2H), 1.71 (m, 5H), 1.54 (m, 1H), 1.11 (s, 6H); 13C NMR (CDCl3,
75 MHz): δ (ppm) 206.3, 165.0, 161.4, 149.4, 145.8, 129.6,

302
S. Xu et al. / Fitoterapia 99 (2014) 300–306
ent-6β-hydroxy-(14α-O-2-indoloyl)-1,7-epoxy-7,15-dioxo-6,
20-hemiketal-6,7-seco-16-kaurene (Compound 4e)
(d, J = 9.3 Hz, 1H), 2.65 (m, 1H), 2.26 (m, 2H), 2.11 (m, 1H),
1.80 (m, 11H), 1.38 (m, 4H), 1.21 (s, 3H), 1.06 (s, 3H); 13C NMR
(CDCl3, 75 MHz): δ (ppm) 197.3, 176.3, 175.2, 165.7, 147.1,
122.9, 121.0, 74.2, 72.8, 71.3, 59.4, 50.6, 47.4, 45.9, 43.4, 40.3,
36.2, 32.9, 32.1, 29.7, 25.8, 23.5, 22.9, 19.0; MS (ESI) m/z: 457.1
[M + H]+, 474.2 [M + NH4]+; HR-MS (ESI) m/z: calcd for
C26H33O7 [M + H]+ 457.2221, found 457.2213.
53% as white solid, mp. 112–114 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 7.36 (m, 5H), 6.26 (s, 1H), 5.82 (s, 1H),
5.59 (s, 1H), 5.33 (s, 1H), 4.57 (m, 1H), 4.11, 3.77 (dd, JA = JB =
10.2 Hz, each 1H), 3.39 (d, J = 9.3 Hz, 1H), 3.20 (m, 1H), 2.79
(m, 1H), 2.68 (m, 1H), 2.11 (m, 1H), 1.83 (m, 1H), 1.75 (m, 5H),
1.38 (m, 1H), 1.10 (s, 3H), 1.01 (s, 3H); 13C NMR (DMSO-d6,
75 MHz): δ (ppm) 197.2, 167.7, 162.8, 148.9, 132.4, 130.2,
120.8, 120.6, 119.9, 113.8, 112.4, 111.4, 101.8, 76.2, 75.7, 74.2,
59.9, 53.1, 49.3, 48.2, 40.9, 37.3, 33.1, 30.5, 29.7, 23.1, 22.8, 18.6;
MS (ESI) m/z: 506.2 [M + H]+; 523.2 [M + NH4]+; HR-MS
(ESI) m/z: calcd for C29H32NO7 [M + H]+ 506.2173, found
506.2170.
ent-(14α-O-benzoyl)-1,7-epoxy-6,7,15-trioxo-6,20-epoxy-6,7-
seco-16-kaurene (Compound 6b)
73% as white powder, mp. 298–299 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 7.94 (d, J = 7.2 Hz, 2H), 7.54 (t, J = 7.5 Hz,
1H), 7.39 (t, J = 7.5 Hz, 2H), 6.30 (s, 1H), 5.87 (s, 1H), 5.64
(s, 1H), 4.64 (m, 1H), 4.38, 4.09 (dd, JA = JB = 10.2 Hz, each
1H), 3.36 (d, J = 9.3 Hz, 1H), 2.60 (m, 1H), 2.30 (m, 2H), 2.06
(m, 1H), 1.88 (m, 1H), 1.61 (m, 4H), 1.37 (m, 1H), 1.22 (s, 3H),
1.07 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 197.3, 189.5,
176.7, 175.4, 172.7, 165.8, 146.9, 140.8, 133.6, 130.1, 128.8,
121.4, 77.3, 76.8, 71.4, 59.4, 50.9, 47.6, 40.5, 36.9, 33.0, 29.6,
23.8, 19.1; MS (ESI) m/z: 465.1 [M + H]+, 482.1 [M + NH4]+,
479.5 [M + Cl]-; HR-MS (ESI) m/z: calcd for C27H29O7 [M + H]+
465.1908, found 465.1910.
ent-6β-hydroxy-(14α-O-o-quinoloyl)-1,7-epoxy-7,15-dioxo-
6,20-hemiketal-6,7-seco-16-kaurene (Compound 4f)
31% as white solid, mp.138–140 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 8.19 (t, J = 9.0 Hz, 2H), 7.97 (d, J =
8.4 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.70 (t, J = 7.2 Hz, 1H), 7.55
(t, J = 7.2 Hz, 1H), 6.18 (s, 1H), 5.95 (s, 1H), 5.51 (s, 1H), 5.30
(s, 1H), 4.60 (m, 1H), 4.03, 3.92 (dd, JA = JB = 10.2 Hz, each
1H), 3.37 (d, J = 9.3 Hz, 1H), 2.80 (m, 1H), 2.61 (m, 1H), 2.02
(m, 1H), 1.91 (m, 2H), 1.71 (m, 3H), 1.48 (m, 1H), 1.18 (m, 2H),
0.96 (s, 3H), 0.89 (s, 3H); 13C NMR (DMSO-d6, 75 MHz):
δ (ppm) 197.9, 166.0, 165.4, 147.0, 137.2, 130.1, 129.8, 128.2,
127.1, 121.2, 120.2, 101.2, 75.9, 75.2, 73.9, 59.5, 53.4, 49.7, 48.2,
40.3, 36.5, 32.4, 30.5, 29.2, 22.8, 22.5, 19.3; MS (ESI) m/z: 518.2
[M + H]+; HR-MS (ESI) m/z: calcd for C30H32NO7 [M + H]+
518.2173, found 518.2166.
ent-(14α-O-p-methxoylbenzoyl)-1,7-epoxy-6,7,15-trioxo-6,20-
epoxy-6,7-seco-16-kaurene (Compound 6c)
37% as white powder, mp. 257–259 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 7.89 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.7 Hz,
2H), 6.29 (s, 1H), 5.83 (s, 1H), 5.62 (s, 1H), 4.62 (m, 1H), 4.38,
4.10 (dd, JA = JB = 10.2 Hz, each 1H), 3.83 (s, 3H), 3.33 (d, J =
9.6 Hz, 1H), 2.60 (m, 1H), 2.31 (m, 2H), 2.03 (m, 1H), 1.84
(m, 1H), 1.62 (m, 4H), 1.38 (m, 1H), 1.22 (s, 3H), 1.07 (s, 3H);
13C NMR (CDCl3, 75 MHz): δ (ppm) 197.9, 175.7, 166.0, 164.4,
147.5, 132.5, 121.8, 114.4, 74.9, 74.0, 71.8, 59.8, 55.9, 51.2, 48.1,
46.4, 41.0, 36.7, 33.4, 32.6, 29.8, 24.0, 23.5, 19.5; MS (ESI) m/z:
495.2 [M + H]+; HR-MS (ESI) m/z: calcd for C28H31O8 [M +
H]+ 495.2013, found 495.2013.
ent-6β-hydroxy-(14α-O-2-chloro-4-pyridoyl)-1,7-epoxy-7,15-
dioxo-6,20-hemiketal-6,7-seco-16-kaurene (Compound 4g)
35% as white solid, mp. 236–238 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 8.19 (m, 1H), 8.09 (m, 1H), 7.21 (m, 1H),
6.18 (s, 1H), 5.90 (s, 1H), 5.53 (s, 1H), 5.28 (s, 1H), 4.56 (m, 1H),
4.01, 3.91 (dd, JA = JB = 10.2 Hz, each 1H), 3.26 (d, J = 9.3 Hz,
1H), 2.77 (m, 1H), 2.60 (m, 1H), 1.94 (m, 3H), 1.70 (m, 3H),
1.48 (m, 1H), 1.20 (m, 2H), 0.97 (s, 3H), 0.89 (s, 3H); 13C NMR
(CDCl3, 75 MHz): δ (ppm) 197.5, 174.2, 163.1, 162.1, 151.9,
149.8, 146.1, 146.0, 122.3, 121.5, 102.1, 74.7, 74.1, 72.6, 59.4,
52.3, 48.2, 47.2, 37.8, 32.6, 31.6, 29.0, 23.1, 22.1, 18.9; MS (ESI)
m/z: 502.2 [M + H]+, 540.6 [M + K]+; HR-MS (ESI) m/z: calcd
for C26H29ClNO7 [M + H]+ 502.1627, found 502.1631.
ent-6β-hydroxy-(14α-O-2-chloro-3-pyridoyl)-1,7-epoxy-7,15-
dioxo-6,20-hemiketal-6,7-seco-16-kaurene (Compound 4h)
67% as white solid, mp. 194–196 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 8.72 (d, J = 8.4 Hz, 1H), 8.50 (d, J =
8.4 Hz, 1H), 7.92 (t, J = 8.1 Hz, 1H), 6.18 (s, 1H), 5.95 (s, 1H),
5.52 (s, 1H), 5.27 (s, 1H), 4.56 (m, 1H), 4.02, 3.92 (dd, JA = JB =
10.2 Hz, each 1H), 3.34 (d, J = 9.3 Hz, 1H), 2.78 (m, 1H), 2.61
(m, 1H), 1.90 (m, 3H), 1.70 (m, 3H), 1.44 (m, 1H), 1.21 (m, 2H),
0.99 (s, 3H), 0.90 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm)
197.5, 169.2, 163.1, 152.6, 151.9, 148.8, 146.0, 138.7, 129.4,
121.3, 101.8, 74.7, 74.0, 72.5, 58.9, 52.3, 48.2, 47.8, 37.6, 32.5,
32.0, 28.9, 23.2, 21.9, 18.8; MS (ESI) m/z: 502.2 [M + H]+, 540.5
[M + K]+; HR-MS (ESI) m/z: calcd for C26H29ClNO7 [M + H]+
502.1627, found 502.1636.
ent-(14α-O-p-flurobenzoyl)-1,7-epoxy-6,7,15-trioxo-6,20-
epoxy-6,7-seco-16-kaurene (Compound 6d)
56% as white solid, mp. 117–119 °C; 1H NMR (CDCl3,
500 MHz): δ (ppm) 8.12 (m, 2H), 7.14 (m, 2H), 6.30 (s, 1H),
5.86 (s, 1H), 5.64 (s, 1H), 4.61 (m, 1H), 4.38, 4.09 (dd, JA
=
JB = 10.0 Hz, each 1H), 3.34 (d, J = 10.5 Hz, 1H), 2.68 (m,
1H), 2.14 (m, 2H), 2.10 (m, 1H), 1.84 (m, 1H), 1.67 (m, 4H),
1.34 (m, 1H), 1.23 (s, 3H), 1.08 (s, 3H); 13C NMR (CDCl3,
75 MHz): δ (ppm) 197.1, 175.2, 165.6, 164.5, 146.8, 132.6,
121.6, 121.2, 115.8, 74.3, 74.0, 71.2, 59.3, 50.7, 47.6, 45.9,
40.4, 36.3, 32.4, 31.8, 29.4, 23.8, 23.0, 19.0; MS (ESI) m/z:
483.2 [M + H]+, 500.2 [M + NH4]+, 505.2 [M + Na]+, 521.1
[M + K]+; HR-MS (ESI) m/z: calcd for C27H28FO7 [M + H]+
483.1814, found 483.1814.
ent-(14α-O-o-pyrazinoyl)-1,7-epoxy-6,7,15-trioxo-6,20-
epoxy-6,7-seco-16-kaurene (Compound 6e)
48% as white solid, mp. 168–170 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 9.09 (s, 1H), 8.67 (d, J = 2.1 Hz, 1H), 8.63
(d, J = 2.1 Hz, 1H), 6.26 (s, 1H), 5.93 (s, 1H), 5.63 (s, 1H), 4.60
(m, 1H), 4.00, 4.31 (dd, JA = JB = 10.2 Hz, each 1H), 3.34 (d, J =
9.3 Hz, 1H), 2.64 (m, 1H), 2.23 (m, 2H), 2.04 (m, 1H), 1.80
(m, 1H), 1.71 (m, 4H), 1.36 (m, 1H), 1.15 (s, 3H), 1.01 (s, 3H);
13C NMR (CDCl3, 75 MHz): δ (ppm) 196.2, 174.7, 166.6, 150.4,
147.5, 146.1, 146.0, 144.1, 121.6, 74.7, 74.2, 70.8, 58.9, 50.3,
47.0, 45.6, 39.8, 35.9, 32.6, 31.7, 29.1, 23.1, 22.6, 18.7; MS (ESI)
m/z: 467.2 [M + H]+; HR-MS (ESI) m/z: calcd for C25H27N2O7
[M + H]+ 467.1813, found 467.1810.
ent-(14α-O-cyclopentoyl)-1,7-epoxy-6,7,15-trioxo-6,20-epoxy-
6,7-seco-16-kaurene (Compound 6a)
65% as white solid, mp. 245–248 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 6.25 (s, 1H), 5,67 (s, 1H), 5.62 (s, 1H),
4.60 (m, 1H), 4.34, 4.05 (dd, JA = JB = 10.5 Hz, each 1H), 3.15

S. Xu et al. / Fitoterapia 99 (2014) 300–306
303
ent-(14α-O-m-pyridoyl)-1,7-epoxy-6,7,15-trioxo-6,20-epoxy-
6,7-seco-16-kaurene (Compound 6f)
(m, 3H), 1.12 (s, 3H), 1.10 (s, 3H); 13C NMR (CDCl3, 125 MHz):
δ (ppm) 206.0, 169.4, 167.2, 165.0, 152.1, 131.5, 119.6, 115.5,
96.9, 74.9, 73.3, 72.7, 62.8, 61.1, 59.9, 51.6, 42.7, 39.2, 37.7, 33.2,
32.2, 29.8, 24.7, 21.2, 21.1, 17.5; MS (ESI) m/z: 529.2 [M + H]+;
HR-MS (ESI) m/z: calcd for C29H34FO8 [M + H]+ 529.2232,
found 529.2228.
46% as white solid, mp. 150–152 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 9.12 (s, 1H), 8.77 (s, 1H), 8.21 (d, J =
7.8 Hz, 1H), 7.36 (m, 1H), 6.25 (s, 1H), 5.91 (s, 1H), 5.68 (s, 1H),
4.63 (m, 1H), 4.38, 4.11 (dd, JA = JB = 10.6 Hz, each 1H), 3.37
(d, J = 9.3 Hz, 1H), 2.70 (m, 1H), 2.26 (m, 2H), 2.10 (m, 1H),
1.82 (m, 1H), 1.71 (m, 4H), 1.38 (m, 1H), 1.22 (s, 3H), 1.07
(s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 196.9, 175.1, 165.6,
164.5, 153.9, 151.0, 146.7, 137.4, 124.9, 123.3, 121.7, 74.6, 74.3,
71.2, 59.4, 50.7, 47.5, 46.0, 40.4, 36.3, 33.0, 32.1, 29.5, 23.5, 23.0,
19.0; MS (ESI) m/z: 466.2 [M + H]+; HR-MS (ESI) m/z: calcd for
C26H28NO7 [M + H]+ 466.1860, found 466.1856.
2.2. Biology
2.2.1. Minimum inhibitory concentration determination
MIC assays were performed in 96-well round-bottom
plates (Costar no. 3799, Corning Inc., NY). Each compound
was suspended in DMSO at a final concentration of 12.8 mg/ml
and diluted 100-fold into 7H9-OADC liquid medium con-
taining 0.05% Tween-80. This 128 μg/ml stock was diluted
by 2-fold serial dilutions (10 dilutions total) in the same
medium, resulting in 50 μL total volume in each well. M. phlei,
M. smegmatis and M. marinum were grown to an optical density
at 600 nm of 0.8 and diluted to 0.01 before adding 50 μL to each
individual well of the 96-well plates. Upon incubation for 1 day
ent-(14α-O-o-indoloyl)-1,7-epoxy-6,7,15-trioxo-6,20-epoxy-
6,7-seco-16-kaurene (Compound 6g)
28% as white solid, mp. 177–179 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 8.92 (s, 1H), 7.65 (s, 1H), 7.28 (m, 4H),
6.32 (s, 1H), 5.83 (s, 1H), 5.66 (s, 1H), 4.62 (m, 1H), 4.38, 4.10
(dd, JA = JB = 9.6 Hz, each 1H), 3.43 (d, J = 9.3 Hz, 1H); 2.70
(m, 1H), 2.34 (m, 2H), 2.04 (m, 1H), 1.88 (m, 1H), 1.71 (m, 4H),
1.36 (m, 1H), 1.20 (s, 3H), 1.08 (s, 3H); 13C NMR (CDCl3,
75 MHz): δ (ppm) 196.7, 175.1, 165.8, 163.9, 152.1, 146.4,
131.4, 121.7, 120.6, 120.0, 119.4, 112.8, 74.5, 74.1, 70.9, 58.9,
50.6, 47.5, 45.8, 39.8, 36.2, 32.3, 31.0, 29.2, 23.1, 22.8, 18.9; MS
(ESI) m/z: 504.3 [M + H]+, 521.2 [M + NH4]+; HR-MS (ESI) m/z:
calcd for C29H30NO7 [M + H]+ 504.2017, found 504.2010.
ent-(14α-O-2-chloro-3-pyridoyl)-1,7-epoxy-6,7,15-trioxo-6,
20-epoxy-6,7-seco-16-kaurene (Compound 6h)
at 37 °C (M. phlei, M. smegmatis) or
7 day at 28 °C
(M. marinum), the MIC was determined to be the lowest
concentration that prevents visible growth.
The M. tuberculosis H37Rv (ATCC 27294) strain maintained
in 7H9 broth [Middlebrook 7H9 broth base (Difco)] medium
supplemented with 0.05% Tween 80 and 10% ADS supplement
was used for MIC determination. Compounds dissolved in
DMSO were 2-fold serial diluted in duplicates in 96-well
microtiter plates. A volume of 100 μL of M. tuberculosis H37Rv
suspension in 7H9 broth was added to each well and the assay
plates were incubated at 37 °C for 6 days. MTT was added, and
after 1 day of incubation, solubilization buffer was added to
each well. The optical densities were measured at 570 nm. The
MIC was determined as the lowest concentration of drug that
inhibited bacterial growth (absorbance from untreated bacilli
was taken as a control for growth). Reported MICs are an
average of three individual measurements.
77% as white solid, mp. 145–147 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 8.42 (m, 1H), 8.07 (dd, J = 7.8, 1.8 Hz,
1H), 7.21 (m, 1H), 6.22 (s, 1H), 5.88 (s, 1H), 5.59 (s, 1H), 4.56
(m, 1H), 4.00, 4.30 (dd, JA = JB = 10.2 Hz, each 1H), 3.26 (d, J =
9.3 Hz, 1H), 2.63 (m, 1H), 2.21 (m, 2H), 2.04 (m, 1H), 1.81 (m,
1H), 1.71 (m, 4H), 1.36 (m, 1H), 1.15 (s, 3H), 1.00 (s, 3H); 13C
NMR (CDCl3, 75 MHz): δ (ppm) 196.1, 174.6, 163.1, 151.9,
146.1, 146.0, 121.9, 121.5, 74.3, 74.1, 70.7, 58.9, 50.3, 47.0, 45.6,
39.8, 35.9, 32.6, 31.7, 29.0, 23.1, 22.5, 18.6; MS (ESI) m/z: 522.2
[M + Na]+; HR-MS (ESI) m/z: calcd for C26H27ClNO7 [M + H]+
500.1471, found 500.1463.
3. Results and discussion
ent-(14α-O-p-fluorophenylvinoyl)-1,7-epoxy-6,7,15-trioxo-6,
20-epoxy-6,7-seco-16-kaurene (Compound 6i)
3.1. Chemistry
64% as white solid, mp. 118–120 °C; 1H NMR (CDCl3,
300 MHz): δ (ppm) 7.54 (d, J = 15.9 Hz, 1H), 7.48 (m, 2H),
7.07 (t, J = 8.4 Hz, 2H), 6.29 (d, J = 15.9 Hz, 2H), 6.25 (s, 1H),
5.80 (s, 1H), 5.65 (s, 1H), 4.62 (m, 1H), 4.38, 4.07 (dd, JA = JB =
9.9 Hz, each 1H), 3.28 (d, J = 9.3 Hz, 1H), 2.60 (m, 1H), 2.33 (m,
2H), 2.09 (m, 1H), 1.86 (m, 1H), 1.60 (m, 4H), 1.38 (m, 1H),
1.19 (s, 3H), 1.09 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm)
197.2, 189.4, 175.2, 166.0, 165.7, 162.4, 146.9, 145.4, 130.3,
130.1, 121.4, 116.2, 115.8, 74.4, 73.4, 71.3, 59.4, 50.7, 47.5, 45.9,
40.4, 36.3, 32.9, 32.2, 29.4, 23.5, 23.0, 19.1; MS (ESI) m/z: 509.2
[M + H]+, 526.2 [M + NH4]+; HR-MS (ESI) m/z: calcd for
C29H30FO7 [M + H]+ 509.1970, found 509.1966.
The synthesis routes for the preparation of target com-
pounds were outlined in Scheme 1, as reported earlier by our
group [10]. Starting from commercially available oridonin (1),
enmein-type diterpenoid analog 3 was obtained by treatment
with sodium periodate in water. Oxidation of compound 3 with
Jones reagent at 0 °C afforded corresponding ketone 5 in total
yields of 88%. This procedure was well-established and the
structure was confirmed by the key HMBC correlations of H-20
with C-6, C-1, C-9, C-10 and C-5 [11]. Enmein-type diterpenoids
3 and 5 provided good building blocks for the construction of
a small library of their 14-O-derivatives (4a–h, 6a–i) by con-
densation reaction with various acids.
Acetylated oridonin of 1-hydroxy (9), which is also a
naturally occurring compound named lasiokaurin [12] exhibited
a good MIC value of 1 μg/mL against M. phlei in our previous test
[9]. Therefore, its 14-O-derivative (10) was further synthesized
to investigate its antimycobacterical activity. As shown in
Scheme 1, treatment of 1 with 2,2-dimethoxypropane in the
presence of TsOH in acetone provided ketal 7 in 97% yield;
ent-1a-O-acetyl-6β,7β,-dihydroxy-(14β-O-p-florobenzoyl)-
15-oxo-7,20-epoxy-16-kaurene (Compound 10)
67% as white solid, mp. 124–126 °C; 1H NMR (CDCl3,
500 MHz): δ (ppm) 7.96 (m, 2H), 7.06 (d, J = 9.0 Hz, 2H),
6.20 (s, 1H), 6.17 (d, J = 11.0 Hz, 1H), 6.07 (s, 1H), 5.53 (s, 1H),
4.65 (m, 1H), 4.19, 4.33 (dd, JA = JB = 9.5 Hz, each 1H), 4.09
(s, 1H), 3.77 (m, 1H), 3.27 (d, J = 11.0 Hz, 1H), 2.58 (m, 1H),
2.14 (m, 3H), 2.02 (s, 3H), 1.78 (m, 2H), 1.62 (m, 1H), 1.49

304
S. Xu et al. / Fitoterapia 99 (2014) 300–306
Scheme 1. Synthesis of oridonin analogs and their derivatives. Reaction conditions:
(a). 4-Methoxycinnamic acid, EDCI, DMAP, r.t., overnight, 75%;
(b). NaIO4, H2O, rt, 24 h, 98%;
(c). Jones reagent, Acetone, 0 °C, 15 min, 90%;
(d). RCOOH, EDCI, DMAP, r.t., 8–24 h, 28–77%;
(e). RCOOH, EDCI, DMAP, r.t., 6–32 h, 31–81%;
(f). DMP, TsOH, Acetone, 65 °C, 10 min, 97%;
(g). Ac2O, DMAP, TEA, DCM, r.t., 3 h, 94%;
(h). 10% HCl, THF, 0.5 h, 97%; (i) 4-Fluorbenzoic acid, EDCI, DMAP, r.t., overnight, 67%.
compound 7 upon reaction with Ac2O/DMAP/TEA led to
acetylated compound 8 in the yield of 94%; deprotection of
8 with 10% HCl gave the corresponding alcohol 9 in almost
quantitative yield.
Compound 2 was proved to be potent against M. phlei,
M. smegmatis and M. marinum with MIC values of 0.5 μg/mL,
4 μg/mL and 4 μg/mL, respectively, in our previous research.
Thus, in this paper, compound 2 was prepared to further test its
antitubercular activity against M. tuberculosis H37Rv.
screened against Mycobacterium phlei (M. Phlei, ATCC 355),
Mycobacterium smegmatis (M. smegmatis, ATCC19420) and
Mycobacterium marinum (M. marinum, ATCC 927) using Micro-
plate Alamar Blue Assay (MABA). As evident form Table 1,
among all the screened compounds, except compounds 3 and
5, all were active against M. phlei with MIC values between
0.5 and 8 μg/mL. Especially, compounds 2, 6g and 6i exhibited
potent antimycobacterial activity against M. phlei at a con-
centration of 0.5 μg/mL, which was comparable to that of
positive drug streptomycin (MIC = 0.5 μg/mL). However, all
the compounds were less active against M. smegmatis and
M. marinum, suggesting that these compounds possess differ-
ent selectivity characteristics.
3.1.1. Antibacterial activity
The preliminary antimycobacterial activity (minimum in-
hibitory concentration; MIC) of the title compounds were first

S. Xu et al. / Fitoterapia 99 (2014) 300–306
305
Table 1
Structures and antimycobacterial activities of oridonin derivatives.
Compd
R
MPa
MSb
MMc
ClogPd
PSAd(Å2)
1
2
3
4a
4b
4c
4d
4e
4f
4g
4h
5
6a
6b
6c
6d
6e
6f
6g
6h
6i
9
10
–
16
0.5
32
8
1
4
4
1
2
1
2
16
1
1
1
4
2
1
0.5
1
N64
4
N64
64
4
16
N64
8
16
8
64
64N
4
8
8
16
N64
32
8
32
4
16
4
0.25
N64
4
N64
64
4
8
N64
8
32
16
N64
64N
4
8
16
8
N64
32
8
64
4
32
8
0.5
−0.62
2.37
0.53
2.79
2.91
3.05
0.12
2.89
2.54
2.31
1.87
0.20
2.54
2.79
2.96
2.93
0.01
1.44
3.19
1.75
3.38
0.25
3.02
–
107.22
122.52
93.06
99.13
99.13
99.13
123.85
81.7
111.49
111.49
111.49
89.9
95.97
95.97
105.21
95.97
120.69
108.33
108.00
108.33
95.97
113.29
119.36
–
4-Methoxylphenylvinyl
–
Cyclopentyl
Phenyl
4-Fluorophenyl
2-Pyrazinyl
2-Indolyl
2-Quinolyl
2-Chloro-4-pyridyl
2-Chloro-3-pyridyl
–
Cyclopentyl
Phenyl
4-Methoxylphenyl
4-Fluorophenyl
2-Pyrazinyl
3-Pyridyl
2-Indolyl
2-Chloro-3-pyridyl
4-Fluorophenylvinyl
–
0.5
1
4
4-Fluorophenyl
Streptomycin
0.5
a
Mycobacterium phlei (ATCC 355).
b
c
Mycobacterium smegmatis (ATCC19420).
Mycobacterium marinum (ATCC 927).
Calculated using chemdraw ultra 12.0.
d
Due to the fact that the cell wall of mycobacteria contains
4. Conclusion
lipophilic substances, more lipophilic substances are likely to
penetrate more easily into the cell. It has been reported that the
modulation of ClogP values often contributes better antituber-
cular activity for some compounds [13]. As shown in Table 1,
the ClogP values of building blocks 1, 3 and 5 revealed
moderate potency (16, 32, and 16 μg/mL against M. phlei,
respectively) as well as general lipophilicity. As an attempt
to increase the lipophilicity of these building blocks, a small
library of 14-O-derivatives of enmine-type diterpenoid was
constructed by introduction of a series of different side chains
into the 14-O-position. Therefore, a remarkable improvement
in lipophilicity of the synthesized enemine-type derivatives
was evidenced by ClogP values, and to our delight, the same
increase in potency was witnessed as well.
Based on the promising results of the preliminary anti-
mycobacterial screening, compounds with good activities were
further tested for their in vitro antimycobacterial activity
against M. tuberculosis H37Rv using isoniazid (INH) and
rifampicin (RIF) as standards. The results are depicted in
Table 2, the parent compound oridonin was found to be less
active even above 32 μg/mL against M. tuberculosis H37Rv, while
all of the derivatives are potent with MIC values ranging from
17.1 to 31.7 μg/mL. Compound 10, the 14-O-derivtive of natural
product lasiokaurin was found to be the most
potent compound in terms of its activity against
M. tuberculosis H37Rv (17.1 μg/mL), and compound 4b also
showed intriguing antitubercular activity (24.0 μg/mL). Besides,
the ADME/T of the these promising compounds were also
predicated by Discovery Studio v2.5, the results indicated the
potential of these compounds as antitubercular agents (please
see the supporting information).
In summary, enmein-type tetracyclic diterpenoid deriva-
tives have been synthesized by the simple and efficient syn-
thetic protocols and their antimycobacterial activity was also
evaluated. It is found that the introduction of lipophilic frac-
tion to the framework of enmein-type diterpenoid as lipo-
philicity control was achieved successfully. The results revealed
that most of the compounds showed moderate to potent
antimycobacterial activity against M. phlei. The further antitu-
bercular evaluation against M. tuberculosis H37Rv demonstrated
that compounds 10 and 4b showed intriguing antitubercular
activity, which deserved further investigation. Therefore, we
have identified a series of natural oridonin derivatives, which
could be further modified to exert stronger potency. These
findings could provide new insights into the development of
novel antitubercular agents.
Table 2
Antitubercular activity of oridonin derivatives.
Compd
H37Rv MIC (μg/mL)
CLogPa
PSAa(Å2)
1
2
N32
31.7
24.0
28.8
17.1
0.04
0.05
−0.62
2.37
2.91
3.38
3.02
–
107.22
122.52
99.13
95.97
119.36
–
4b
6i
10
INHb
RFPc
–
–
a
Calculated using chemdraw ultra 12.0.
Isoniazid.
Rifampicin.
b
c

306
S. Xu et al. / Fitoterapia 99 (2014) 300–306
[4] Bueno J, Coy ED, Stashenko E. Antimycobacterial natural products-an
Acknowledgment
opportunity for the Colombian biodiversity. Rev Esp Quimioter 2011;24:
175–83.
This study was supported by grants from the National
Natural Science Foundation (Nos. 30973610; 81373280),
Young Teachers' Scientific Research Fund Project of Shenyang
Pharmaceutical University (QNJJ 2013501), and the Project
Program of State Key Laboratory of Natural Medicines, China
Pharmaceutical University (No. SKLNMZZCX201404).
[5] Koehn FE, Carter GT. The evolving role of natural products in drug
discovery. Nat Rev Drug Discov 2005;4:206–20.
[6] Stavrakov G, Philipova I, Valcheva V, Momekov G. Synthesis and
antimycobacterial activity of novel camphene-based agents. Bioorg Med
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[7] Sun HD, Huang SX, Han QB. Diterpenoids from Isodon species and their
biological activities. Nat Prod Rep 2006;23:673–98.
[8] Fujita E, Nagao Y, Kaneko K, Nakazawa S, Kuroda H. The antitumor and
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Appendix A. Supplementary data
[9] Xu ST, Li DH, Pei LL, Yao H, Wang CQ, Cai H, et al. Design, synthesis and
antimycobacterial activity evaluation of natural oridonin derivatives.
Bioorg Med Chem Lett 2014;24:2811–4.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.fitote.2014.10.005.
[10] Wang L, Li DH, Xu ST, Cai H, Yao HQ, Zhang YH, et al. The conversion
of oridonin to spirolactone-type or enmein-type diterpenoid: synthesis
and biological evaluation of ent-6,7-seco-oridonin derivatives as novel
potential anticancer agents. Eur J Med Chem 2012;52:242–50.
[11] Li DH, Xu ST, Cai H, Pei LL, Zhang HY, Wang L, et al. Enmein-type
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