Synthesis of erythrodiol C-ring derivatives and their activity against Chlamydia trachomatis

Article abstract of DOI:10.1016/j.steroids.2021.108912

To develop new potential agents against Chlamydia trachomatis among oleanane type triterpenoids the synthesis, spectral and X-ray analysis as well as antimicrobial screening of C-12 oxygen and nitrogen derivatives of erythrodiol is presented. The reduction of methyl 3β-acetoxy-12-oxo-oleanoate with LiAlH₄ led to isomeric erythrodiol 12β- and 12α-hydroxy-derivatives, their stereochemistry with respect to the position of hydroxyl-group at C-12 was determined based on the multiplets splitting patterns, the magnitude of the spin–spin interaction, and NOESY interactions. Methyl 3β-acetoxy-12-oxo-oleanoate was transformed to 12E-hydroxyimino- and 12E-methoxyimino-derivatives by the interaction with NH₂OH?HCl or CH₃ONH₂?HCl, respectively. By Beckmann rearrangement with SOCl₂ in dioxane 12E-oxime was converted to C-lactame and its following reduction with LiAlH₄ in THF or dioxane led to erythrodiol C-azepanone or C-azepane derivatives. The structure 3-O,12-N-bis-acetyl-derivative of C-azepane-erythrodiol was confirmed by the single crystal X-ray analysis. Erythrodiol 12β-hydroxy- and C-azepane derivatives were found to be lead compounds with significant activity against C. trachomatis with MIC 1.56 and 3.125 μg/mL. Molecular docking was employed to suggest potential binding interaction, the tested compounds are likely to act as Cdu1 protein inhibitors while 12β-hydroxy-erythrodiol exhibited the highest affinity towards this respective target protein. These results indicated that C-ring oxygen and nitrogen erythrodiol derivatives might be considered for further research in the design of antibacterial agents against Chlamydia trachomatis.

Full text of DOI:10.1016/j.steroids.2021.108912

Steroids 175 (2021) 108912
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of erythrodiol C-ring derivatives and their activity against
Chlamydia trachomatis
,
Oxana Kazakovaa *, Liudmila Rubanikb, Alexander Lobova, Nikolai Poleshchukb,
Irina Baikovaa, Yuliya Kapustinab, Anastasiya Petrovaa, Tatyana Korzunb, Tatyana Lopatinaa,
Alexandra Fedorovaa, Tatyana Rybalovac, Dmitri Polovianenkoc, Marius Miocd,
Codruța Șoicad
a Ufa Institute of Chemistry of the Ufa Federal Research Centre of the Russian Academy of Sciences, Ufa 450054, Russian Federation
b Republican Research and Practical Center for Epidemiology and Microbiology, Minsk 220114, Belarus
c N.N. Vorozhtzov Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk 630090, Russian Federation
d Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy, 2nd Eftimie Murgu Square, Timisoara 300041, Romania
A R T I C L E I N F O
A B S T R A C T
Keywords:
To develop new potential agents against Chlamydia trachomatis among oleanane type triterpenoids the synthesis,
spectral and X-ray analysis as well as antimicrobial screening of C-12 oxygen and nitrogen derivatives of
erythrodiol is presented. The reduction of methyl 3β-acetoxy-12-oxo-oleanoate with LiAlH4 led to isomeric
Triterpenoids
Oleanolic acid
Erythrodiol
Oxime
erythrodiol 12β- and 12α-hydroxy-derivatives, their stereochemistry with respect to the position of hydroxyl-
group at C-12 was determined based on the multiplets splitting patterns, the magnitude of the spin–spin inter-
action, and NOESY interactions. Methyl 3β-acetoxy-12-oxo-oleanoate was transformed to 12E-hydroxyimino-
and 12E-methoxyimino-derivatives by the interaction with NH2OH∙HCl or CH3ONH2∙HCl, respectively. By
Beckmann rearrangement with SOCl2 in dioxane 12E-oxime was converted to C-lactame and its following
reduction with LiAlH4 in THF or dioxane led to erythrodiol C-azepanone or C-azepane derivatives. The structure
3-O,12-N-bis-acetyl-derivative of C-azepane-erythrodiol was confirmed by the single crystal X-ray analysis.
Erythrodiol 12β-hydroxy- and C-azepane derivatives were found to be lead compounds with significant activity
Lactam
Azepane
Chlamydia trachomatis
against C. trachomatis with MIC 1.56 and 3.125 μg/mL. Molecular docking was employed to suggest potential
binding interaction, the tested compounds are likely to act as Cdu1 protein inhibitors while 12β-hydroxy-
erythrodiol exhibited the highest affinity towards this respective target protein. These results indicated that C-
ring oxygen and nitrogen erythrodiol derivatives might be considered for further research in the design of
antibacterial agents against Chlamydia trachomatis.
1. Introduction
presented by oxidized at C11 and C12 compounds, 12-hydroxy-, 12-
halogenated and saturated/unsaturated lactones, dienes, or five-
Oleanolic acid (OA) and erythrodiol (ED) (Fig. 1) belong to oleanane
type triterpenoids that widely distributed throughout the plant kingdom
[1,2]. OA possesses different pharmacological activities such as anti-
inflammatory, antimicrobial, antioxidant, antitumour, anti-diabetic,
etc. [3–5]. Several studies have shown favorable properties of eryth-
rodiol including diverse endogenous antioxidative and cancer activities
[6,7]. OA and ED have been employed as substrates for numerous
chemical transformations usually by modification of the key C3 and C28
positions [8–10]. The C-ring derivatives of OA are predominantly
membered cycle analogs [11–13]. It was shown that the introduction
of an oxygen substituent into C-ring increased the biological activity, for
example, the presence of
α,β-unsaturated system in the structure of
CDDO (2-cyano-3,12-diooxoolean-1,9-dien-28-oic acid) has a sufficient
influence on anti-inflammatory, anti-diabetic nephropathy, and cyto-
toxic activities [14,15].
Oleanolic acid 12E-oxime and its Beckmann rearrangement products
such as C-lactam (12-oxo-12a-aza-C-homoolean) and C-seco- (12-nitrile-
12,13-seco-15(14 → 13)-abeoolean-14(27)-en) derivatives have been
* Corresponding author.
E-mail address: obf@anrb.ru (O. Kazakova).
https://doi.org/10.1016/j.steroids.2021.108912
Received 24 May 2021; Received in revised form 4 August 2021; Accepted 25 August 2021
Available online 1 September 2021
0039-128X/© 2021 Elsevier Inc. All rights reserved.

O. Kazakova et al.
Steroids 175 (2021) 108912
2.1.2. Methyl 3β-acetoxy-12-oxo-olean-28-oate (3)
A solution of compound 2 (0.51 g, 1.0 mmol) in CH2Cl2 (50 mL) at ꢀ
40 ◦C was purged with ozone. The solvent was evaporated in vacuo, the
solid was purified by column chromatography with elution hexane-
20
EtOAc (10:1). Yield 87%, m.p. lit. [16] 198–199 ◦C, [
α
]
lit. [19]
6.54◦ (c 0.10, CHCl3). Spectral data corresponded to [27]. DFound, %: C
74.85; H 9.84. C33H52O5. Calculated, %: C 74.96; H 9.91.
2.1.3. Synthesis of compounds 4 and 5
To a solution of compound 3 (0.53 g, 1.0 mmol) in dry THF (50 mL)
LiAlH4 (0.15 g, 4 mmol) was added and a mixture was refluxed for 3 h,
after cooling H2O (5 mL) was added drop wise, then quenched with 5%
HCl (50 mL), the aqueous layer was extracted with CH2Cl2 (2 × 15 mL),
the combined extracts were washed with 5% HCl (2 × 40 mL), H2O (2 ×
15 mL), dried over CaCl2, and the solvent was evaporated in vacuo. The
product was purified by column chromatography on SiO2 by elution
with CHCl3-EtOH (100:1, 50:1, 25:1, 10:1, 5:1).
Fig. 1. Structures of oleanolic acid (OA) and erythrodiol (ED).
reported by Bednarczyk-Cwynar and coworkers [16–19]. The intro-
duction of seven-membered lactam to the C-ring of OA gives a possibility
to enhance their ability to interact with biological membranes [20]. At
the same time, erythrodiol C-ring derivatives are practically not
described and their activity is not reported.
Recently we have shown that OA derivatives such as 3-nicotinoy-
loxy-oleanolic acid, A-azepano-erythrodiol as well as conjugates with
homopyperazine and diethylenetriamine demonstrated high inhibitory
activity against C. trachomatis with chemotherapeutic index > 8, while
3-amino-3,4-seco-4(23)-en-erythrodiol was found to be a lead antibac-
Olean-3β,12β,28-triol (4).
Yield 42% (0.19 g), mp: 159 ◦C, [
α
]
+ 106◦ (c 0.01, CHCl3). 1H
20
D
NMR (CDCl3 + Py-D5), 500 MHz): 3.99 (1H, ddd, 3J12-13 = 11.0, 3J12ax-
11ax = 10.3, 3J12ax-13 = 11.0, 3J12ax-9eq = 5.6, Hax-12), 3.79 (1H, d, 2J =
10.8, HA-28), 3.82 (1H, d, 2J = 10.8, HB-28), 3.39 (1H, dd, 3J3-2ax = 10.4,
3J3-2eq = 5.8, H-3), 2.58 (1H, ddd, 3J18-19ax = 13.4, 3J13-18 = 5.2, 3J18-
terial agent with MIC 3.125 μg/mL [21,22]. Taking into account the
= 4.0, H-18), 2.16 (1H, dd, 3J13-12ax = 11.0, 3J13-18 = 5.2, H-13),
above results, in this paper we present the synthesis, spectral and X-ray
analysis, antimicrobial screening of C-12 oxygen, nitrogen as well as
azepanone and azepane derivatives of erythrodiol obtained from ole-
anolic acid.
19eq
2.12 (1H, ddd, 2J = 13.3, 3J22ax-21ax = 13.0, 3J22ax-21eq = 4.1, Hax-22),
2.09 (1H, ddd, 2J = 12.1, 3J11eq-12ax = 5.6, 3J11eq-9 = 2.7, Heq-11), 1.91
(1H, ddd, 2J = 13.7, 3J16ax-15eq = 13.3, 3J16ax-15eq = 4.4, Hax-16), 1.83
(1H, ddd, 2J = 13.2, 3J2ax-1ax = 13.3, 3J2ax-1ax = 4.1, Hax-2), 1.80 (1H, m,
Heq-2), 1.79 (1H, ddd, 2J = 13.7, J15ax-16eq = 13.3, 3J15ax-16ax = 3.0,
3
2. Experimental part
Hax-15), 1.73 (1H, dt, 2J = 13.1, 3J1eq-2ax = 3.7, Heq-1), 1.69 (1H, dd, 2J
= 12.9, 3J19eq-18 = 4.0, Heq-19), 1.58 (1H, m, Heq-6), 1.56 (1H, ddd, 2J =
12.1, 3J11ax-9 = 13.3, 3J11ax-12ax = 10.3, Hax-11), 1.55 (1H, dd, 2J = 12.9,
3J19ax-18 = 13.5, Hax-19), 1.45 (1H, m, Heq-22), 1.47 (1H, m, Hax-7), 1.42
(1H, m, Hax-21), 1.40 (1H, m, Hax-6), 1.38 (1H, dd, 3J9-11ax = 13.3, 3J9-
2.1. Chemistry
2.1.1. General
1H, 13C, and 15N NMR spectra and two-dimensional correlation
spectra of compounds 3–11 were recorded on a Bruker Avance-III 500
MHz spectrometer (500 MHz for 1H, 125 MHz for 13C, and 50 MHz for
15N) in CDCl3 and MeOD using TMS as internal standard at the Center
for the Collective Use “Chemistry” of the UIC UFRC RAS and RCCU
“Agidel” of the UFRC RAS. The X-ray diffraction experiment for com-
pound 11 was carried out at the Multi-Access Chemical Research Center
SB RAS at ambient temperature on a Bruker KAPPA APEX II diffrac-
11eq = 2.7, H-9), 1.32 (1H, ddd, 2J = 13.7, 3J16eq-15eq = 4.4, 3J16eq-15ax
=
2
3
3.0, Heq-16), 1.27 (1H, dd, J = 13.6, J21eq-22ax = 4.1, Heq-21), 1.24
(1H, m, Heq-7), 1.19 (3H, s, H-23), 1.10 (3H, s, H-27), 1.01 (3H, s, H-30),
1.00 (1H, m, Hax-1), 0.99 (3H, s, H-26), 0.98 (3H, s, H-29), 0.98 (3H, s,
H-24), 0.94 (1H, ddd, 2J = 13.7, 3J15eq-16ax = 4.4, 3J15eq-16eq = 3.0, Heq-
15), 0.87 (3H, s, H-25), 0.78 (1H, dd, 3J5-6ax = 11.6, 3J5-6eq = 2.3, H-5).
13C NMR (CDCl3 + Py-D5, 500 MHz): 77.9 (C-3), 67. 5 (C-28), 67.3 (C-
12), 55.7 (C-5), 49.5 (C-9), 42.0 (C-8), 41.2 (C-14), 40.9 (C-13), 39.4 (C-
4), 39.1 (C-1), 37.5 (C-17), 37.2 (C-10), 36.7 (C-19), 35.0 (C-21), 33.9
(C-29), 32.9 (C-7), 32.7 (C-11), 32.0 (C-22), 31.1 (C-18), 30.9 (C-20),
28.6 (C-23), 28.0 (C-2), 27.0 (C-15), 23.9 (C-30), 23.8 (C-16), 18.7 (C-
6), 18.1 (C-27), 16.5 (C-26), 16.2 (C-25), 16.2 (C-24). Found, %: C
78.17; H 11.42. C30H52O3. Calculated, %: C 78.21; H 11.38.
tometer (graphite-monochromated Mo K
α radiation). Reflection in-
tensities were corrected for absorption by the SADABS program [23].
The structure was solved by direct methods using the SHELXS-97 pro-
gram [24] and refined by anisotropic (isotropic for all H atoms) full-
matrix least-squares method against F2 of all reflections by the
SHELXL2014/7 programs set [25]. The positions of the hydrogen were
calculated geometrically and refined in the riding model. Free solvent-
accessible volume equaled to 328 Å3 (10% of cell volume) is occupied
by unidentified solvent molecules. We employed PLATON/SQUEEZE
procedure [26] to calculate the contribution to the diffraction from the
solvent region and thereby produced a set of solvent-free diffraction
intensities. Melting points were measured on a microtable Boetius.
Optical rotations were measured on a Perkin-Elmer 241MC polarimeter
(Germany) in a 1 dm tube. Elemental analysis was performed on a Euro
EA-3000 CHNS analyzer (Eurovector, Milan, Italy); the main standard is
acetanilide. Mass spectra were obtained on a liquid chromatog-
raph–mass spectrometer LCMS-2010 EV (Shimadzu, Kyoto, Japan).
Thin-layer chromatography analyses were performed on Sorbfil plates
(Sorbpolimer, Krasnodar, Russian Federation), using the solvent system
chloroform–ethyl acetate, 40:1. Substances were detected by a 10%
solution of a sulfuric acid solution with subsequent heating at
Olean-3β,12
α,28-triol (5).
20
Yield 42% (0.19 g), mp: 126 ◦C, [
α]
D
+ 189◦ (c 0.01, CHCl3). 1H
NMR (CDCl3, 500 MHz): 3.97 (1H, q, 3J = 2.7, Heq-12), 3.56 (1H, d, 2J =
10.8, HB-28), 3.51 (1H, d, 2J = 10.8, HA-28), 3.22 (1H, dd, 3J3-2ax = 10.4,
3J3-2eq = 5.8, H-3), 1.85 (1H, dd, 3J13-12eq = 2.7, 3J13-18 = 7.0, H-13),
1.82 (1H, ddd, 2J = 13.8, 3J16ax-15eq = 13.6, 3J16ax-15eq = 4.3, Hax-16),
1.84 (1H, m, Heq-19), 1.72 (1H, m, Hax-19), 1.72 (1H, m, H-18), 1.71
(1H, m, Hax-15), 1.70 (1H, dt, 2J = 13.1, 3J1eq-2ax = 3.7, 3J1eq-2eq = 3.7,
Heq-1), 1.69 (1H, dd, 3J9-11ax = 13.3, 3J9-11eq = 2.70, H-9), 1.67 (1H, m,
Hax-22), 1.63 (1H, m, Heq-2), 1.59 (1H, ddd, J11ax-12eq = 2.7, 2J =
3
12.80, 3J11ax-9 = 13.3, Hax-11), 1.58 (1H, m, Hax-2), 1.55 (1H, m, Heq-6),
1.52 (1H, m, Hax-7), 1.49 (1H, td, 2J = 12.8, 3J11eq-9eq = 2.7, Heq-11),
1.37 (1H, m, Hax-6), 1.34 (3H, s, H-27), 1.33 (1H, m, Heq-7), 1.30 (1H,
m, Hax-21), 1.18 (1H, m, Heq-22), 1.17 (1H, m, Heq-21), 1.05 (1H, ddd,
2J = 13.8, 3J16eq-15ax = 3.7, 3J16eq-15eq = 3.1, Heq-16), 1.02 (3H, s, H-26),
◦
0.99 (3H, s, H-23), 0.97 (1H, ddd, 2J = 13.1, 3J1ax-2ax = 13.2, 3J1-2eq
=
=
100–120 C for 2–3 min. Oleanolic acid was purchased from Sigma –
4.1, Hax-1), 0.93 (3H, s, H-29), 0.91 (1H, ddd, 2J = 13.8, 3J15eq-16ax
Aldrich. Compound 2 was synthesized according to the method
3
described in [16].
3.7, J15eq-16eq = 3.1, Heq-15), 0.86 (3H, s, H-30), 0.87 (3H, s, H-25),
0.77 (3H, s, H-24), 0.77 (1H, dd, 3J5-6ax = 11.9, 3J5-6eq = 1.9, H-5). 13
C
2

O. Kazakova et al.
Steroids 175 (2021) 108912
NMR (CDCl3, 500 MHz): 78.9 (C-3), 74.1 (C-12), 68.5 (C-28), 55.5 (C-5),
45.5 (C-9), 42.4 (C-8), 41.5 (C-14), 39.7 (C-19), 38.9 (C-4), 38.8 (C-1),
38.5 (C-13), 38.4 (C-18), 37.8 (C-17), 36.8 (C-10), 33.9 (C-21), 33.5
(C29), 33.4 (C7), 32.1 (C11), 31.3 (C22), 30.8 (C20), 28.2 (C15), 28.0
(C23), 27.3 (C-2), 23.8 (C-30), 23.5 (C-16), 21.1 (C-27), 18.3 (C-6), 16.6
(C-25), 16.1 (C-26), 15.5 (C-24). Found, %: C 78.30; H 11.46. C30H52O3.
Calculated, %: C 78.21; H 11.38.
(C-30), 22.9 (C-16), 21.3 (C-2′’), 20.8 (C-11), 19.7 (C-27), 18.1 (C-6),
16.5 (C-24), 15.8 (C-26), 15.2 (C-25). 15N NMR (CDCl3, 500 MHz):
372.29 (NOCH3). Found, %: C 73.30; H 9.85; N 2.47. C34H55NO5.
Calculated, %: C 73.21; H 9.94; N 2.51.
2.1.5. Methyl 3β-acetoxy-12-oxo-12-aza-C-homo-olean-28-oate (8)
To a solution of compound 6 (0.54 g, 1.0 mmol) in anhydrous 1,4-
dioxane (10 mL) SOCl2 (0.5 mL) was added dropwise, the mixture was
stirred for 30 min, poured into H2O (100 mL), the precipitate was
filtered, washed with water, dried and purified by column chromatog-
raphy on SiO2 by elution with CHCl3. Yield 85% (0.46 g), mp: lit. [16]
2.1.4. Synthesis of compounds (6) and (7)
A mixture of compound 3 (0.53 g, 1.0 mmol) and NH2OH⋅HCl (0.07
g, 1.0 mmol; for compound 6) or CH3ONH2⋅HCl (0.16 g, 2.0 mmol; for
compound 7) in pyridine (20 mL) was refluxed for 4 h, then poured into
H2O (100 mL). The precipitate was filtered, washed with 5% HCl, water
and dried.
289–290 ◦C, [
α]
20 lit. [13] ꢀ 14.0◦ (c 0.58, CHCl3). 1H NMR (CDCl3,
D
500 MHz): 6.18 (1H, br.s, NH), 4.49 (1H, dd, 3 J3-2ax = 11.7, 3 J3-2 eq
= 4.8, H-3), 4.05 (t, 3J13-18 ¼ 5.7, 3J13-NH = 5.7, H-13), 3.73 (3H, s, H-
1′), 2.54 (1H, dt, 3J18-19ax = 12.5, 3J18-13 = 5.3, 3J18-19eq = 5.3, H-18),
2.48 (1H, dd, 2J = 13.9, 3J11B-9 = 10.5, HB-11), 2.12 (1H, d, 2J = 13.9,
HA-11), 2.04 (3H, s, H-2′’), 1.93 (1H, dt, 2J = 13.4, 3J1eq-2ax = 3.5, 3J1eq-
2eq = 3.5, Heq-1), 1.82 (1H, ddd, 2J = 13.8, 3J16ax-15ax = 14.8, 3J16ax-15ax
Methyl 3β-acetoxy-12E-hydroxyimino-olean-28-oate (6).
The product was crystallized from CHCl3-EtOH (100:1). Yield 85%
20
(0.46 g), mp: 195 ◦C, [
α]
ꢀ 109◦ (c 0.01, CHCl3), mp: lit [18]
D
196–198.5 ◦C [18]. 1H NMR (CDCl3, 500 MHz): 4.46 (1H, dd, 3J3-2ax
=
3
2
11.3, J3-2eq = 4.8, H-3), 3.66 (3H, s, H-1′), 3.06 (1H, dd, J = 17.8,
3J11eq-9 = 5.4, Heq-11), 2.85 (1H, dt 3J18-19ax = 13.6, 3J18-13 = 3.8, 3J18-
19eq = 3.8, H-18), 2.54 (1H, d, 3J13-18 = 3.8, H-13), 2.03 (3H, m, H-2′’),
1.93 (1H, dd, 2J = 13.6, 3J19eq-18 = 3.8, Heq-19), 1.81 (1H, m, Hax-22),
1.79 (1H, dd, 2J = 17.8, 3J11ax-9 = 12.8, Hax-11), 1.69 (1H, dt, 2J = 13.2,
3J1eq-2ax = 3.6, 3J1eq-2eq = 3.6, Heq-1), 1.64 (1H, m, Heq-16), 1.62 (1H, m,
Heq-2), 1.61 (1H, m, Hax-15), 1.59 (1H, m, Hax-2), 1.56 (1H, m, Heq-6),
1.42 (1H, m, Heq-22), 1.41 (1H, m, Hax-6), 1.36 (1H, dd, 3J9-11ax = 12.8,
3J9-11eq = 5.4, H-9), 1.35 (1H, m, Hax-7), 1.33 (1H, td, 2J = 13.7, 3J22ax-
= 4.4, Hax-16), 1.71 (1H, ddd, 2J = 13.5, 3J22ax-21ax = 13.9, 3J22ax-21eq
=
2
3
3
4.5, Hax-22), 1.69 (1H, dddd, J = 13.4, J2eq-3 = 4.8, J2eq-1ax = 4.3
3J2eq-1eq = 3.5, Heq-2), 1.60 (1H, tdd, 2J = 13.4, 3J2ax-1ax = 13.4, 3J2ax-3
= 11.7, 3J2ax-1eq = 3.5, 13.50, Hax-2), 1.58 (1H, m, Hax-15), 1.57 (1H, m,
Heq-22), 1.57 (1H, m, Heq-16), 1.53 (1H, m, Heq-6), 1.49 (1H, dt, 2J =
13.3, 3J7eq-6ax = 3.2, 3J7eq-6eq = 3.2, Heq-7), 1.43 (1H, d, 3J9-11B = 10.5,
H-9), 1.42 (1H, ddd, 2J = 13.3, 3J7ax-6ax = 13.8, 3J7ax-6eq = 3.7, Hax-7),
1.41 (1H, m, Hax-19), 1.38 (1H, m, Heq-19), 1.36 (1H, ddd, 2J = 14.2,
3J22ax-21ax = 14.2, 3J21ax-22eq = 4.6, Hax-21), 1.33 (1H, m, Hax-6), 1.32
21ax = 13.7, 3J21ax-22eq = 4.1, Hax-21), 1.25 (1H, t, 2J = 13.6, 3J19ax-18
=
(1H, ddd, J = 13.8, J15eq-16ax = 4.6, J15eq-16eq = 2.2, Heq-15), 1.24
(1H, m, Heq-21), 1.11 (1H, td, 2J = 13.4, 3J1ax-2ax = 13.4, 3J1ax-2eq = 4.3,
Hax-1), 1.01 (3H, s, H-26), 0.99 (3H, s, H-27), 0.97 (3H, s, H-29), 0.88
(3H, s, H-30), 0.86 (3H, s, H-23), 0.84 (3H, s, H-25), 0.82 (3H, s, H-24),
0.78 (1H, dd, 3J5-6ax = 11.7, 3J5-6eq = 2.0, H-5). 13C NMR (CDCl3, 500
MHz): 177.7 (C-12), 177.6 (C-28), 170.8 (C-1′’), 80.2 (C-3), 54.4 (C-5),
52.0 (C-1′), 51.0 (C-13), 48.6 (C-9), 47.3 (C-17), 45.2 (C-8), 42.6 (C-14),
38.6 (C-10), 38.5 (C-1), 37.7 (C-4), 36.1 (C-18), 35.8 (C-19), 34.8 (C-7),
33.6 (C-21), 33.3 (C-29), 32.8 (C-11), 32.6 (C-22), 30.8 (C-20), 28.4 (C-
15), 27.9 (C-23), 23.4 (C-30), 23.4 (C-2), 21.7 (C-16), 21.3 (C-2′’), 17.8
(C-6), 17.6 (C-27), 16.9 (C-26), 16.6 (C-24), 15.9 (C-25). 15N NMR
(CDCl3, 500 MHz): 125.38 (NH). Found, %: C 72.79; H 9.90; N 2.65.
2
3
3
13.6, Hax-19), 1.24 (1H, m, Heq-7), 1.17 (1H, m, Heq-21), 1.06 (1H, m,
Heq-15), 1.01 (1H, ddd, 2J = 13.2, 3J1ax-2ax = 11.8, 3J1ax-2eq = 4.2, Hax-
1), 0.91 (3H, s, H-30), 0.90 (3H, s, H-27), 0.87 (3H, s, H-29), 0.86 (3H, s,
H-25), 0.84 (3H, s, H-23), 0.83 (3H, s, H-24), 0.81 (3H, s, H-26), 1.93
(1H, m, Hax-16), 8, 3J5-6eq = 2.1, H-5), 0.79 (1H, dd, 3J5-6ax = 11. 8, 3J5-
6eq = 2. 1, H-5). 13C NMR (CDCl3, 500 MHz): 178.7 (C-28), 171.0 (C-1′’),
160.4 (C-12), 80.6 (C-3), 55.2 (C-5), 51.8 (C-1′), 48.1 (C-17), 47.4 (C-9),
43.4 (C-13), 41.1 (C-8), 40.3 (C-14), 37.8 (C-4), 37.7 (C-1), 36.9 (C-10),
35.9 (C-19), 34.6 (C-21), 33.5 (C-29), 33.2 (C-22), 32.6 (C-18), 31.7 (C-
7), 30.8 (C-20), 27.9 (C-23), 27.3 (C-15), 23.5 (C-2), 22.9 (C-30), 22.7
(C-16), 21.3 (C-2′’), 20.6 (C-11), 19.8 (C-27), 18.1 (C-6), 16.5 (C-24),
15.8 (C-26), 15.2 (C-25). 15N NMR (CDCl3, 500 MHz): 338.77 (NOH).
Found, %: C 72.78; H 9.95; N 2.61. C33H53NO5. Calculated, %: C 72.89;
H 9.82; N 2.58.
C33H53NO5. Calcd, %: C 72.89; H 9.82; N 2.58.
2.1.6. Synthesis of compounds (9) and (10)
Methyl 3β-acetoxy-12E-methoxyimino-olean-28-oate (7).
To a solution of compound 8 (0.54 g, 1 mmol) in anhydrous THF (20
mL, for compound 9) or anhydrous dioxane (20 mL, for compound 10)
LiAlH4 (0.15 g, 4 mmol) was added and a mixture was refluxed 3 h (for
compound 9) or 14 h (for compound 10), after cooling H2O (5 mL) was
added dropwise, then quenched with 5% HCl (50 mL). The aqueous
layer was extracted with CH2Cl2 (2 × 15 mL), the combined extracts
were washed with 5% HCl (15 mL), H2O (2 × 20 mL), dried over CaCl2,
and the solvent was evaporated in vacuo.
The product was crystallized from hexane. Yield 84% (0.47 g), mp:
20
68 ◦C, [
α
]
D
+ 25◦ (c 0.01, CHCl3). 1H NMR (CDCl3, 500 MHz): 4.47
(1H, dd, 3J3-2ax = 11.4, 3J3-2eq = 4.9, H-3), 3.83 (3H, s, NOCH3), 3.68
(3H, s, H-1′), 2.93 (1H, dd, 2J = 17.5, 3J11eq-9 = 5.5, Heq-11), 2.90 (1H,
3
3
3
dt, J19ax-18 = 13.4, J18-13 = 3.8, J18-19eq = 3.8, H-18), 2.47 (1H, d,
3J13-18 = 3.8, H-13), 2.10 (1H, dd, 2J = 13.4, 3J19eq-18 = 3.8, Heq-19),
2.04 (3H, s, H-2′’), 1.91 (1H, td, 2J = 13.1, 3J16ax-15ax = 13.1, 3J16ax-15eq
= 4.5, Hax-16), 1.83 (1H, m, Hax-22), 1.72 (1H, dd, 2J = 17.5, 3J11ax-9
=
12-Oxo-12-aza-C-homo-olean-3β,28-diol (9).
12.8, Hax-11), 1.70 (1H, dt, 2J = 13.5, 3J1eq-2ax = 3.6, 3J1eq-2eq = 3.6, Heq-
1), 1.66 (1H, m, Heq-16), 1.63 (1H, m, Heq-2), 1.06 (1H, m, Heq-15), 1.60
(1H, m, Hax-2), 1.56 (1H, m, Heq-6), 1.43 (1H, m, Heq-22), 1.40 (1H, m,
Hax-6), 1.36 (1H, m, Hax-7), 1.32 (1H, td, 2J = 13.7, 3J22ax-21ax = 13.7,
3J21ax-22eq = 4.1, Hax-21), 1.25 (1H, m, Heq-7), 1.20 (1H, m, Heq-21),
1.19 (1H, t, 2J = 13.4, 3J19ax-18 = 13.4, Hax-19), 1.06 (1H, m, Heq-15),
1.02 (1H, ddd, 2J = 13.5, 3J1ax-2ax = 13.1, 3J1ax-2eq = 4.5, Hax-1), 0.90
(3H, s, H-29), 0.86 (3H, s, H-25), 0.85 (3H, s, H-23), 0.84 (3H, s, H-24),
0.81 (3H, s, H-26), 0.79 (1H, dd, 3J5-6ax = 11.6, 3J5-6eq = 2.3, H-5), 0.90
The product was crystallized from CHCl3-EtOH (20:1). Yield 76%
(0.36 g), mp: 53 ◦C, [
α
]
D
20 ꢀ 150◦ (c 0.01, CHCl3). 1H NMR (CDCl3, 500
MHz): 5.59 (1H, br.s, NH), 3.95 (1H, t, 3J13-18 = 5.6, 3J13-NH = 5.6, H-
13), 3.58 (1H, d, 2J = 10.9, HB-28), 3.52 (1H, d, 2J = 10.9, HA-28), 3.21
(1H, dd, 3J3-2ax = 11.7, 3J3-2eq = 4.7, H-3), 2.47 (1H, dd, 2J = 13.9, 3J11B-
9 = 10.5, HB-11), 2.11 (1H, d, 2J = 13.9, HA-11), 1.94 (1H, dt, 2J = 13.4,
3J1eq-2ax = 3.5, 3J1eq-2eq = 3.5, Heq-1), 1.94 (1H, ddd, 3J18-19ax = 13.3,
3J18-13 = 5.6, 3J18-19eq = 3.4, H-18), 1.79 (1H, td, 2J = 13.8, 3J16ax-15ax
=
13.8, 3J16ax-15ax = 4.6, Hax-16), 1.71 (1H, m, Hax-22), 1.68 (1H, m, Hax-
(3H, s, H-27), 0.94 (3H, s, H-30), 1.35 (1H, dd, 3J9-11ax = 12.7, 3J9-11eq
=
15), 1.67 (1H, m, Heq-2), 1.57 (1H, m, Hax-2), 1.55 (1H, m, Heq-6), 1.54
5.4, H-9). 13C NMR (CDCl3, 500 MHz): 178.9 (C-28), 170.9 (C-1′’), 157.8
(C-12), 80.6 (C-3), 61.3 (NOCH3), 55.2 (C-5), 51.7 (C-1′), 48.1 (C-17),
47.5 (C-9), 42.9 (C-13), 40.9 (C-8), 40.2 (C-14), 37.8 (C-1), 37.8 (C-4),
36.9 (C-10), 36.0 (C-19), 34.6 (C-21), 33.5 (C-29), 33.2 (C-22), 32.4 (C-
18), 31.7 (C-7), 30.9 (C-20), 27.9 (C-23), 27.4 (C-15), 23.5 (C-2), 23.1
(1H, m, Heq-7), 1.47 (1H, m, Hax-7), 1.46 (1H, t, 2J = 13.3, 3J19ax-18
=
13.3, Hax-19), 1.41 (1H, d, 3J9-11B = 10.5, H-9), 1.34 (1H, m, Hax-6), 1.33
(1H, m, Hax-21), 1.27 (1H, m, Heq-22), 1.23 (3H, s, H-26), 1.22 (1H, m,
Heq-15), 1.21 (1H, m, Heq-21), 1.18 (1H, dd, 2J = 13.3, 3J19eq-18 = 3.40,
Heq-19), 1.03 (1H, td, 2J = 13.4, 3J1ax-2ax = 13.4, 3J1ax-2eq = 4.3, Hax-1),
3

O. Kazakova et al.
Steroids 175 (2021) 108912
1.01 (1H, m, Heq-16), 1.00 (3H, s, H-27), 0.99 (3H, s, H-23), 0.95 (3H, s,
H-29), 0.84 (3H, s, H-25), 0.83 (3H, s, H-30), 0.76 (3H, s, H-24), 0.67
(1H, dd, 3J5-6ax = 11.7, 3J5-6eq = 1.7, H-5). 13C NMR (CDCl3, 500 MHz):
177.2 (C-12), 78.5 (C-3), 67.4 (C-28), 54.3 (C-5), 49.8 (C-13), 48.6 (C-
9), 45.7 (C-8), 42.9 (C-14), 38.9 (C-1), 38.8 (C-4), 38.7 (C-10), 37.6 (C-
17), 36.2 (C-19), 35.9 (C-18), 34.8 (C-7), 33.6 (C-21), 33.5 (C-29), 33.3
(C-11), 31.1 (C-20), 30.9 (C-22), 28.0 (C-23), 27.1 (C-2), 26.8 (C-15),
23.4 (C-30), 21.5 (C-16), 18.1 (C-6), 17.8 (C-27), 16.9 (C-26), 15.9 (C-
25), 15.5 (C-24). 15N NMR (CDCl3, 500 MHz): 123.15 (NH). Found, %: C
76.15; H 10.87; N 2.87. C30H51NO3. Calcd, %: C 76.06; H 10.85; N 2.96.
12-Deoxy-12-aza-C-homo-olean-3β,28-diol (10).
44.4 (C-8), 43.7 (C-14), 39.5 (C-1), 38.3 (C-10), 37.7 (C-17), 37.7 (C-4),
37.2 (C-19), 35.2 (C-7), 35.0 (C-18), 34.9 (C-21), 33.7 (C-29), 31.1 (C-
15), 30.8 (C-20), 30.8 (C-22), 27.9 (C-23), 27.2 (C-11), 24.9 (C-16), 23.8
(C-2), 22.8 (C-30), 21.8 (C-2′’), 21.3 (C-2′), 20.2 (C-27), 18.4 (C-6), 16.6
(C-24), 15.7 (C-25), 14.8 (C-27). 15N NMR (CDCl3, 500 MHz): 139.44
(N). Found, %: C 76.64; H 11.11; N 2.65. C32H55NO3. Calcd, %: C 76.60;
H 11.05; N 2.79.
2.1.8. Crystallographic data for compound 11
C
34H57NO4 + [CHCl3], M 543.80, orthorombic, P212121, a 7.6796
(7), b 16.230(2), c 27.138(3) Å, V 3382.5(6) Å3, Z 4, Dcalcd 1.068 g⋅cmꢀ 3
,
The product was crystallized from CHCl3-THF (5:1). Yield 63% (0.29
μ
(Mo-K
α
) 0.068 mmꢀ 1, F(000) 1200, T 296(2) K, colorless, (0.30 ×
g), mp: 148 ◦C, [
α]
20 ꢀ 14◦ (c 0.01, CHCl3). 1H NMR (MeOD, δ ppm, J
0.20 × 0.02) mm3, transmission 0.8070–0.9280, 37,988 measured re-
flections (θ 2.5 – 25.1◦, completeness 99.8%) in index range ꢀ 9≤h≤9,
ꢀ 19≤k≤19, –32≤l≤30, 5992 independent (Rint 0.0888), 362 parame-
D
Hz): 0.67 (dd, 1H, 3J5-6ax = 12.0, 3J5-6eq = 3.3, H-5); 0.77 (s, 3H, H-24);
0.85 (s, 3H, H-30); 0.90 (s, 3H, H-25); 0.92 (s, 3H, H-29); 0.97 (m, 1H,
Hax-1); 0.97 (m, 1H, Heq-16); 0.98 (s, 3H, H-23); 1.02 (s, 3H, H-27); 1.09
(s, 3H, H-26); 1.17 (m, 1H, Heq-22); 1.18 (m, 1H, Heq-21); 1.21 (m, 1H,
Heq-15); 1.29 (m, 1H, Hax-21); 1.30 (m, 1H, Heq-19); 1.31 (dd, 1H, 3J9-
11B = 8.1, 3J9-11A = 5.2, H-9); 1.38 (dd, 1H, 2J = 12.0, 3J19ax-18 = 14.1,
Hax-19); 1.39 (m, 1H, Hax-6); 1.41 (m, 1H, Hax-7); 1.43 (m, 1H, Heq-7);
1.53 (m, 1H, Heq-6); 1.58 (m, 2H, Hax-2, Heq-2); 1.59 (m, 1H, HA-11);
1.67 (dddd, 1H, 2J = 14.4, 3J11B-9 = 8.1, 3J11B-12A = 7.0, 3J11B-12B = 5.8,
HB-11); 1.59 (m, 1H, Hax-15); 1.73 (m, 1H, Hax-22); 1.75 (m, 1H, Hax-
16); 1.78 (dt, 1H, 2J = 13.0, 3J1eq-2ax = 3.5, 3J1eq-2eq = 3.5, Heq-1); 1.86
(dddd, 1H, 3J18-19ax = 14.1, 3J18-13 = 5.4, 3J18-19eq = 3.2, 4J18-16eq = 1.3,
H-18); 2.92 (ddd, 1H, 2J = 13.5, 3J12A-11B = 7.0, 3J12A-11A = 6.2, HA-12);
2.99 (ddd, 1H, 2J = 13.5, 3J12B-11A = 7.8, 3J12B-11B = 5.8, HB-12); 3.08
(d, 3J13-18 = 5.4, H-13); 3.18 (dd, 1H, 3J3-2ax = 10.1, 3J3-2eq = 6.1, H-3);
3.41 (d, 1H, 2J = 11.2, HA-28); 3.57 (d, 1H, 2J = 11.2, HB-28). 13C NMR
(MeOD, δ ppm): 15.7 (C-24); 15.9 (C-25); 17.8 (C-27); 18.5 (C-26); 18.7
(C-6); 22.6 (C-16); 23.2 (C-30); 26.9 (C-11); 26.9 (C-2); 27.8 (C-15);
28.2 (C-23); 30.7 (C-22); 30.9 (C-20); 33.7 (C-29); 34.2 (C-21); 35.9 (C-
7); 36.2 (C-19); 37.8 (C-17); 38.8 (C-18); 38.9 (C-4); 39.1 (C-1); 39.4 (C-
10); 44.9 (C-14); 45.2 (C-8); 47.2 (C-12); 51.4 (C-9); 54.8 (C-5); 55.5 (C-
13); 67.5 (C-28); 78.6 (C-3). 15N NMR (MeOD, δ ppm): 43.49 (NH).
Found, %: 78.42; H 11.55; N 3.12. C30H53NO2. Calcd, %: 78.37; H 11.62;
N 3.05.
ters, 0 restraintes, R1 0.0598 (for 3323 observed I > 2σ(I)), wR2 0.186
(all data), GOOF 1.005, largest diff. peak and hole 0.252 and ꢀ 0.185 e.
A-3. Supplementary publication no. CCDC 2070951. Copy of the data can
be obtained free of charge, on application to CCDC, 12 Union Road,
Cambridge CB21EZ, UK (fax: +44 122 3,336,033 or e-mail: depos-
it@ccdc.cam.ac.uk; internet: www.ccdc.cam.ac.uk).
2.2. Antichlamydial activity
Strain C. trachomatis F-3271/Belarus/2015 specialized collection of
viruses and bacteria pathogenic for humans https://www.belriem.by/
o-tsentre/spetsializirovannaya-kollektsiya-virusov-i-bakteriy-patogenn
ykh-dlya-cheloveka/ (Research Center for Epidemiology and Microbi-
ology, Minsk, Belarus) were used. Sequencing of the ompA gene using
primers P1/OMP2, NL-F/NL-R, CT6F/OMP2, P1/CT6R [28] showed
that this strain corresponds to the serovar F and has 100% homology
with the sequence of the strain F/IC-CAL3 https://www.ncbi.nlm.nih.
gov/nuccore/DQ064287.1. The nucleotide sequence of the ompA gene
of strain C. trachomatis F-3271/Belarus/2015 is deposited in the Gen-
Bank database https://www.ncbi.nlm.nih.gov/nuccore/MG733343.
Cell Culture: C. trachomatis were propagated in McCoy cells grown in
DMEM with 10% heat-inactivated fetal bovine serum (HyClone, USA)
supplemented with 2 mM L-glutamine, 10 µg/mL gentamicin, 5 mg/ml
amphotericin B and 1 µg/mL cycloheximide (AppliChem, Germany).
McCoy cells were cultured in glass flasks for 72 h. The cells were sus-
pended after the addition of 0.02% ethylenediamine tetraacetate
(Lonza, USA). Cell suspension with a volume of 1 mL, containing 105
cells, was transferred to flat–bottomed plastic tubes containing a
coverslip (13 mm in diameter). The cells were incubated at 37 ◦C for 24
h to obtain a confluent cell layer. Then 80–90% confluent McCoy cell
monolayer was infected stock suspension of C. trachomatis at a multi-
plicity rate of 1:1. The plate was centrifuged for 1 h at 1500x g to syn-
chronize the infection. After centrifugation, the tubes were incubated for
2 h at 37 ◦C, and thereafter a wide range of concentrations of compounds
3, 4, 6, 9–11, additions to the culture medium was performed. Tested
compounds were preliminarily dissolved in 10% ethanol. Each test was
performed in triplicate. All experiments were supplemented with
negative (intact McCoy cells) and positive controls (McCoy cells infected
by strain C. trachomatis). As quantitative criteria of the observable
antichlamydial action, a decrease in the titer C. trachomatis was calcu-
lated in comparison with the control. Median Tissue Culture Infectious
Dose (TCID50) of the compounds, Chemotherapeutic index (CTI) and
Minimum Inhibitory Concentration (MIC) were determined. Maximum
tolerated concentration (MTC), Minimum Inhibitory Concentration
(MIC) and Chemotherapeutic index (CTI) were determined. The CTI was
defined as the ratio between the MTC and the MIC. CTI equal to 0 (zero)
indicated that the substance had no antichlamydial activity, from > 0 to
< 2 – inactive, from 2 to < 4 – weakly active, from 4 to < 8 – medium
active, ≥8 – highly active compound. The MTC is the maximum of the
studied concentration of a substance that does not cause morphological
changes in cells and impairments in the absorption of vital dye by them
2.1.7. 3β-Acetoxy-12-deoxy-12-acetyl-aza-C-homo-olean-28-ol (11)
A mixture of compound 10 (0.46 g, 1 mmol) in pyridine (15 mL) and
AcCl (0.24 mL, 3 mmol) was heated for 3 h, then poured into 5%HCl
(100 mL), the precipitate was filtered, washed with water until neutral,
dried. The compound was crystallized from a mixture CHCl3-EtOH for X-
20
RAY experiments. Yield 56% (0.28 g), mp: 48 ◦C, [
α]
–22◦ (c 0.01,
D
CHCl3). 1H NMR (CDCl3, 500 MHz): 4.48 (1H, dd, 3J3-2ax = 11.4, 3J3-2eq
= 4.9, H-3), 4.23 (1H, d, 3J13-18 = 4.2, H-13), 4.15 (1H, d, 2J = 12.0, HB-
28), 3.68 (1H, ddd, 2J = 15.8, 3J12B-11A = 4.5, 3J12B-11A = 1.8, HB-12),
3.44 (1H, m, HA-12), 2.92 (1H, d, 2J = 12.0, HA-28), 2.24 (1H, dtd, 3J18-
3
3
4
= 13.8, J18-13 = 4.2, J18-19eq = 4.2, J18-16eq = 1.3, H-18), 2.15
19ax
(3H, s, H-2′’), 2.05 (3H, s, H-2′), 1.98 (1H, ddd, 2J = 14.2, 3J22ax-21ax
=
=
14.2, 3J22ax-21eq = 4.6, Hax-22), 1.90 (1H, ddd, 2J = 13.5, 3J16ax-15ax
14.5, 3J16ax-15ax = 4.2, Hax-16), 1.81 (1H, m, HB-11), 1.77 (1H, dt, 2J =
3
3
2
13.0, J1eq-2ax = 3.5, J1eq-2eq = 3.5, Heq-1), 1.68 (1H, dd, J = 12.7,
3J19ax-18 = 13.8, Hax-19), 1.66 (1H, m, Heq-2), 1.61 (1H, m, Hax-2), 1.60
(1H, m, Hax-15), 1.54 (1H, m, Heq-6), 1.48 (1H, m, H-9), 1.47 (1H, m,
HA-11), 1.41 (1H, m, Hax-6), 1.41 (1H, m, Heq-7), 1.32 (3H, s, H-27),
1.29 (1H, m, Hax-7), 1.28 (1H, ddd, 2J = 14.2, 3J22ax-21ax = 14.2, 3J21ax-
22eq = 4.2, Hax-21), 1.17 (1H, ddt, 2J = 14.2, 3J22ax-21eq = 4.6, 3J21eq-22eq
= 2.3, 4J21eq-19eq = 2.3, Heq-21), 1.06 (1H, ddd, 2J = 13.2, 3J15eq-16ax
=
4.2, 3J15eq-16eq = 2.6, Heq-15), 1.04 (1H, m, Hax-1), 1.03 (1H, ddd, 2J =
12.7, 3J19eq-18 = 4.2, 4J19eq-21eq = 2.3, Heq-19), 0.90 (1H, ddd, 2J = 14.2,
3J22ax-21eq = 4.2, 3J22eq-21eq = 2.3, Heq-22), 0.89 (3H, s, H-25), 0.89 (3H,
s, H-29), 0.87 (3H, s, H-23), 0.85 (3H, s, H-24), 0.83 (3H, s, H-26), 0.82
(1H, m, Heq-16), 0.80 (1H, dd, 3J5-6ax = 12.0, 3J5-6eq = 2.5, H-5), 0.79
(3H, s, H-30). 13C NMR (CDCl3, 500 MHz): 171.0 (C-1′), 169.4 (C-1′’),
80.4 (C-3), 67.9 (C-28), 54.9 (C-5), 54.2 (C-13), 51.6 (C-9), 44.4 (C-12),
4

O. Kazakova et al.
Steroids 175 (2021) 108912
Table 1
Molecular docking parameters and protein targets.
Protein
PDB ID
3DPM
Grid box center coordinates
Grid box size
Conformers generated per ligand
8
Chlamydial protease/proteasome-like activity factor (CPAF)
center_x = ꢀ 9.08393780322
center_y = 7.40593576658
center_z = 19.3976320361
center_x = 101.936143183
center_y = ꢀ 7.62802522143
center_z = ꢀ 9.07770324231
center_x = 10.3045908649
center_y = 106.13570343
center_z = 28.3098829277
center_x = ꢀ 44.5807131385
center_y = ꢀ 2.78174416703
center_z = 159.985219975
center_x = 36.6418108862
center_y = ꢀ 28.7066849522
center_z = 20.9777390168
center_x = ꢀ 0.883706331416
center_y = ꢀ 0.477122415468
center_z = 47.0660652213
size_x = 8.11019083445
size_y = 15.390752709
size_z = 9.41527016223
size_x = 17.2483876337
size_y = 14.6599818341
size_z = 11.7878620968
size_x = 16.621609331
size_y = 15.6222118727
size_z = 12.3409942145
size_x = 15.10633792
size_y = 13.7376610667
size_z = 15.4027590679
size_x = 13.1220599942
size_y = 13.7376610667
size_z = 12.104885424
size_x = 11.3182981068
size_y = 13.9624190931
size_z = 18.465444806
Chlamydia trachomatis enoyl-acp reductase (Fabi)
Chlamydia trachomatis CT263-D161N hydrolase
Chlamydial deubiquitinase ChlaDUB1 (Cdu1)
Chlamydia trachomatis penicillin-binding protein 3 (PBP3)
Chlamydia trachomatis lysyl-tRNA synthetase
4Q9N
4QAT
6FDU
6I1F
8
8
8
8
8
6NS0
in comparison with the control intact cells. The MIC is the minimum
dose of a substance that reduces the titer of Chlamydia by at least 1.25 lg
TCID50 compared with positive controls (McCoy cells infected by strain
C. trachomatis F-3271/Belarus/2015 without adding test substance).
Concentrations of the tested compounds providing suppression of the
Chlamydia reproduction by 50% (the average effective concentration,
EC50) were also calculated, as well as the ratios of maximum tolerated
unnecessary protein chains and the co-crystalized ligands were removed
from the protein structure file after which Gesteiger charges were added.
The target was saved as the required .pdbqt file format, when the soft-
ware automatically adds polar hydrogen atoms to the structure or
merges nonpolar hydrogens, where applicable. Subjected ligand mole-
cules corresponding to compounds 3, 4, 6, 9-11 were sketched as mol
files using Biovia Draw (Dassault Systems Biovia, San Diego, CA, USA)
and were after converted into 3D structures using PyRx’s embedded
Open Babel function, using the ghemical force field for compound ge-
ometry optimization. Molecular docking was performed with PyRx v0.8
(The Scripps Research Institute, La Jolla, CA, USA), using Autodock
Vina’s embedded scoring function [30]. Docking method validation was
achieved by re-docking the co-crystalized ligands into their original
protein binding sites. The predicted docking pose was compared with
the experimental co-crystallized binding pose. Docking studies were
performed for protein targets were the root mean square deviation
(RMSD) values between the co-crystalized ligand’s experimental and
docked pose didn’t exceed 2 Å. The grid box used to delimitate the
search space, was defined in terms of coordinates and size (Table 1) to
best fit the active binding site. Obtained data for docked molecules was
recorded as free binding energy values (ΔG, kcal/mol). Ligand-protein
binding features were analyzed using Accelerys Discovery Studio 4.1
(Dassault Systems Biovia, San Diego, CA, USA).
concentration (MTC) to EC50
.
Assessment of Infective Progeny: The infection rate in the McCoy cells
and infective progeny formation were estimated 72 h after pathogen
inoculation. Titers were determined by infecting cell monolayers with
10-fold dilutions of the stock suspension. All monolayers were stained
using FITC-conjugated monoclonal antibody against chlamydial lipo-
polysaccharide (ChlamyScan, OOO Agrobiomed, Russian Federation).
Inclusion-containing cells were visualized using the Nikon Eclipse 50i
microscope at x200 and x400 magnifications.
2.3. Docking studies
Crystalographic 3D protein structures used as docking targets were
obtained from the RCSB Protein Data Bank [29] and are listed in Table 1
along with their PDB entry ID. These structures were prepared as suit-
able targets for the docking software, using Autodock Tools v1.5.6 (The
Scripps Research Institute, La Jolla, CA, USA). All water molecules,
iii
ii
i
iv
v
i
vi
iii
Scheme 1. Reagent and conditions: i. 1. AcCl, pyridine, reflux; 2. CH2N2, diethyl ester, 22 ◦C; ii. O3, CH2Cl2, ꢀ 40 ◦C; iii. LiAlH4, THF, reflux, 3 h; iv. NH2OH∙HCl (for
compound 6) or CH3ONH2∙HCl (for compound 7), pyridine, reflux 4 h; v. SOCl2, 1,4-dioxane, 22 ◦C, 30 min; vi. LiAlH4, 1,4-dioxane, reflux, 14 h.
5

O. Kazakova et al.
Steroids 175 (2021) 108912
Fig. 2. Fragment of 1H NMR spectrum and multiplet splitting of the signal at C-12 for 12(R)-isomer (compound 4 (a)) and 12(S)-isomer (compound 5 (b)), 500 MHz.
3. Results and discussion
3.1. Chemistry
Oleanolic acid 1, a commercially available material, was taken as the
starting compound for the synthesis of a series erythrodiol derivatives
(Scheme 1). Methyl 3β-acetoxy-12-oxo-oleanoate 3 was obtained by
◦
ozonolysis of compound 2 in DCM at ꢀ 40 C with yield of 87%. The
treatment of ketone 3 with LiAlH4 in anhydrous THF under reflux led to
a mixture of erythrodiol 12β- 4 and 12α-hydroxy- 5 derivatives with
yields of 42% after CC isolation. The structures of compounds 4 and 5
have been noticed in [31] as inhibitors of nitric oxide production in
mouse macrophages, but no experimental data were provided. The
synthesis of compound 4 using 1 M LiAlH4 solution in THF with yield of
17% was described in [32]. Our method allows the synthesis of isomeric
compounds with better yield in simplified ways.
The stereochemistry for isomeric compounds 4 and 5 with respect to
the position of hydroxyl-group at C-12 was determined based on the
multiplets splitting patterns, the magnitude of the spin–spin interaction,
and NOESY interactions (Fig. 2). In the 1H NMR spectrum for the 12(R)-
diastereomer, a triplet doublet signal (δH 3.99 ppm) of the methine
proton is observed at the oxygen-containing carbon C-12 with 3J = 10.7
and 5.6 Hz (Fig. 2a). Based on the transaxial COSY cross-peaks between
H-12/Hax-11 (δH 1.56 ppm) and H-12 / H-13 (δH 2.16 ppm) with
spin–spin coupling constants of 10.7 Hz, it was found that that the
proton at C-12 is in the axial position. The NOESY correlation between
Fig. 3. HMBC, COSY and NOESY correlations of compounds 6, 7.
position C-12 was determined based on cross-peaks in the {1H, 15N}
HMBC spectrum of nearby protons H-13, Hax-11, and Heq-11 with a ni-
trogen signal at δN 372.29 ppm (for compound 7) and 338.77 ppm (for
compound 6). The β-orientation of the methine protons H-13 and H-18
3
was established based on the transaxial binding constants J18-19ax
=
13.6 Hz and a number of NOESY interactions: Hax-11 and H-26 with H-
13, as well as H-30 and Hax-22 with H-18 (Fig. 3). The NOESY cross peak
between proton signals δH 3.83 (CH3ON) and 2.93 ppm (Heq-11) showed
that in the molecule of compound 7 methoxyimino group in position C-
12 of the C-ring displays E configuration [18].
H-12 and the α-oriented methyl group of H-27 (δH 1.10 ppm) confirmed
the β-orientation of the hydroxyl-group for the 12(R)-isomer 4. In the 1H
NMR spectrum (Fig. 2b) of the 12(S)-diastereomer 5, the signal of the
methine proton at C-12 (δH 3.97 ppm) is represented by a quartet with
the coupling constants (3J = 2.7 Hz), which indicates its equatorial
Oxime 6 was subjected to Beckmann rearrangement with SOCl2 in
dioxane to give lactame 8 in good yield. The treatment of 8 with LiAlH4
in anhydrous THF under reflux for 3 h accompanied only two ester
groups at C-3 and C-28 while azepanone fragment was not involved into
the reduction. By these conditions erythrodiol C-lactame derivative 9
was obtained. When lactame 8 was refluxed with LiAlH4 in anhydrous
dioxane for 14 h erythrodiol C-azepane derivative 10 was successfully
synthesized.
position and, therefore, α-orientation of the hydroxyl group.
On the other hand, methyl 3β-acetoxy-12-oxo-oleanoate 3 was
transformed to 12-hydroxyimino- 6 and 12-methoxyimino- 7 derivatives
by the interaction with hydroxylamine or methoxyhydroxylamine hy-
drochlorides in pyridine under reflux. The synthesis of compound 7 was
designed to further involve into Griesbaum co-ozonolysis with ketone in
order to obtain a derivative with 1,2,4-trioxolane ring at C-12 atom.
Recently we have demonstrated a possibility of diastereoselective syn-
thesis of triterpenic 1,2,4-trioxolanes on the basis of methyl 2-
methoxyimino-oleanoate [33]. But unexpectedly the co-ozonolysis of
12-methoxyoxime 7 in the presence of 1,1,1-trifluoroacetone gave
neither 1,2,4-trioxolane nor methoxy-lactame derivative (data not
shown). These results indicate that oleanane 12-methoxyoxime is an
unreactive compound for this type of transformaions.
For compounds 8, 9 the presence of a carbamide group in position C-
12 (δC 177.67 and 177.20 ppm, respectively) is confirmed by {1H, 13C}
HMBC cross peaks with the H-9 and H-13 nodal protons, and also
characteristic chemical shifts of the neighboring methylene group C-11
(δC 32.87 and 33.20 ppm). The intracyclic character of the carbamide
group is confirmed by the cross-peaks of the H-13 and HA-11 protons
with a δN signal of 125.38 ppm (for compound 8) and δN 123.15 ppm
For compounds 6 and 7 the presence of an oxime substituent at
6

O. Kazakova et al.
Steroids 175 (2021) 108912
vary from 111 to 118◦ with alongated intercyclic bonds C8-C9, C8-C14,
C14-C13 equaling to 1.572(7), 1.612(7), 1.562(7) Å accordingly. The
similar alongation of C8-C9 and C8-C14 bonds is observed in 3β-acetoxy-
12-oxo-12a-aza-C-homoolean-13(18)-en-28-oic acid [18]. The orienta-
tion of planar acetoxy group in C-3 position is determined by torsion
angle C2C3O1C31 equaling to 98.6(6)◦ with eclipsed O1-C3 and
C31=O2 bonds that accompanies the forming of week intramolecular
hydrogen bond C3-H3… O2 (Table 2). The carbonyl group of acetyl in
N1 is eclipsed with N1-C13 bond that is favourable for conjugation of
NOE
NOE
Гц
3.4
5.7
13.4
NOE
Гц
Гц
NOE
π
-system on N1, C33, O3. The group C28O4H is staggered with C17-C18
bond (torsion angle C18C17C28O4 is ꢀ 56.1(8)◦) and hydroxyl group
hydrogen is directed according to intramolecular h-bond O4-H… O3.
There exist also some other weak hydrogen bonds in the molecule of
compound 11 [26].
8: R1 = Ac, R2 = COOMe
9: R1 = H, R2 = CH2OH
Fig. 4. HMBC, COSY and NOESY correlations of compounds 8, 9.
(for compound 9) in the spectrum of {1H, 15N} HMBC, as well as NOESY
interactions of the amide proton with Heq-19 and H-27 (Fig. 4).
The formation of an azepane ring in compound 10 is confirmed by
the δN values characteristic of cyclic amines. In the {1H, 15N} HMBC
spectrum, cross peaks of protons at positions C-11 (δH 1.60 and 1.67
ppm) and C-12 (δH 2.92 and 2.99 ppm) with an amine signal at δN 43.49
ppm are observed (Fig. 5).
Acylation of C-azepane 10 with AcCl led to 3-O,12-N-bis-acetyl-de-
rivative 11. The presence of an N-acyl fragment in C-ring of compound
11 was established using cross peaks in the {1H, 15N} HMBC spectrum
for protons HB-11 and H-13 (δH 1.81 and 4.23 ppm), as well as acetyl
methyl (δH 2.15 ppm) with a signal of amide nitrogen at δN 139.44 ppm
(Fig. 5). In addition, interactions are observed in the {1H, 13C} HMBC
spectrum of the H-13 knot proton and protons (δH 3.44 and 3.68 ppm) of
the neighboring methylene group at position C-12 (δC 44.4 ppm) with a
carbamide signal at δC 169.4 ppm. The Z (or syn) orientation of the N-
acyl fragment is supported by the interaction of the methyl group of the
acetyl residue with the HB-12 proton observed in the NOESY spectrum.
For the nodal protons H-13 and H-18, the β-orientation is retained,
which is confirmed by the NOESY spectrum: H-13 / H-27, H-13 / Hax-15
and H-18 / H-30, H-18 / Hax-22.
Fig. 6. The molecular structure and atom numbering of compound 11.
Table 2
The parameters of intramolecular hydrogen bonds for compound 11.
Intramolecular h-bonds
H… A, (Å)
D… A, (Å)
D-H… A,(◦)
The structure of compound 11 was determined by the single crystal
X-ray analysis (Fig. 6). The analysis of the molecular geometries and
intramolecular hydrogen bonds was performed using PLATON program
[26]. The five-ring backbone of compound 11 includes one trans- and
one cis-fused decaline fragments with 7-membered heterocycle between.
The six-membered rings of both decalines reveal chair conformation.
Seven-membered heterocycle has slightly distorted chair conformation.
To decries steric hindrance the Csp3 atom bond angles are increased and
O4-H4O… O3
C3-H3… O2
2.57
2.25
2.26
2.53
2.48
2.48
2.55
2.58
3.23(1)
138
104
105
118
110
109
102
122
2.662(8)
2.683(7)
3.108(7)
2.96(1)
C13-H13… O3
C18-H18… O3
C18-H18… O4
C22-H22… O4
C24-H24… O1
C26-H26… N1
2.95(1)
2.899(7)
3.195(7)
Fig. 5. HMBC, COSY and NOESY correlations of compounds 10, 11.
7

O. Kazakova et al.
Steroids 175 (2021) 108912
better understanding of potential action mechanisms exhibited by in
vitro biological active compounds. Herein we employed molecular
docking to assess the presumable antichlamydial mechanism of action of
compounds 3, 4, 6, 9–11. For this sole purpose we chose to investigate
the ability of these structures to act as potential C. trachomatis specific
protein inhibitors. Proteins such as CPAF, FabI, CT263-D161N, Cdu1,
PBP3, lysyl-tRNA synthetase, exhibit sustainable biological functions
correlated with bacteria viability/infectivity and represent viable tar-
gets for antichlamydial drug development [34–37]. Therefore we
investigated the in silico inhibitory effect of compounds 3, 4, 6, 9–11
against the above mentioned C. trachomatis specific protein targets.
Docking scores are reported as free binding energy values (ΔG, kcal/
mol). The lowest numerical value indicates the highest affinity for a
certain target protein (Table 4). Given the fact that compounds 3, 4, 6,
9–11 share a high structural similarity, docking scores were depicted as
a three-colored scheme (red-yellow-green) heat map table, to easily
identify a clear tendency of a set of compounds to act as potential in-
hibitors for a certain protein. For each protein, the color range was set
from red as the lowest energy value (in most cases corresponding to the
docking score of the native ligand) to green, representing the highest
energy value (Table 4). Docking results for CPAF (PDB ID: 3DPM) were
not taken into consideration since the docking validation method gave a
RMSD value above the 2 Å threshold, between the experimental and
predicted pose of the native ligand.
Table 3
An activity of compounds 3, 4, 6, 9, 10 and 11 against Chlamydia trachomatis in
McCoy cells test.
Compound
CTI
MIC
μ
g/mL
EC50 (CI95
)
μ
g/mL
MTC*/EC50
3
>8
>8
>8
25
24.89 (26.14 ÷ 23.64)
1.23 (1.29 ÷ 1.17)
32.14
650.41
18.74
4
1.56
50
6
42.68 (44.81 ÷ 40.55)
9
not active substance
10
11
>8
>8
3.125
3.125
3.25 (3.41 ÷ 3.09)
3.05 (3.20 ÷ 2.90)
246.15
262.30
CI95 – 95% confidence interval.
*MTC – for all compounds was 800 μg/mL.
3.2. Biological activity
3.2.1. Antichlamydial activity
Compounds 3, 4, 6, 9–11 were screened in vitro toward
C. trachomatis strain F-3271/Belarus/2015. TCID50 assay method to
determine the viability of Chlamydia under compounds action was used.
The degree of the pathogen sensitivity to tested compounds was deter-
mined on the basis of the chemotherapeutic index (CTI) and Minimum
Inhibitory Concentration (MIC) (Table 3). It was revealed that studied
oleanane triterpenoids have different potential. Compound 9 was not
active, while compounds 3, 4, 6, 10 and 11 demonstrated a high
inhibitory activity with chemotherapeutic index > 8. The compounds 3
Docking results clearly show a strong tendency of all six analyzed
compounds to target the Cdu1 protein (PDB ID: 6FDU). In terms of
binding energy values, all compounds showed higher affinity for this
specific protein target as compared to the calculated score for the native
ligand. Obtained data also suggest that the highest inhibitory activity is
attributed to compound 4. These results evidently correlated with the in
vitro antichlamydial activity of tested compounds, according to which
compound 4 displayed the highest antibacterial potency.
and 6 were characterized by the MIC equal to 25 and 50
μg/mL. De-
rivatives 4, 10 and 11 were found to be lead compounds with significant
activity against C. trachomatis with MIC 1.56 μg/mL and 3.125 μg/mL.
As can be seen from presented data, the leaders in the rating of
antichlamydial activity are the compounds 4, 10 и 11 (EC50 1.23, 3.25
and 3.05
μg/mL respectively), which can be selected as promising
compounds for further research.
Chlamydial effector protein Cdu1 plays an important role in the
pathogenicity and infectivity mechanism correlated to C. trachomatis.
Since ubiquitylation and deubiquitylation represent key cellular process
3.2.2. Docking studies
Molecular docking represents a useful approach that can lead to a
Table 4
Heat map of the recorded docking scores (binding free energy, ΔG kcal/mol) for compounds 3, 4, 6, 9–111.
1Color scale varies, for each set (column), from red (lowest recorded ΔG) to green (highest recorded ΔG).
8

O. Kazakova et al.
Steroids 175 (2021) 108912
Fig. 7. Structure of C. trachomatis Cdu1 protein (6FDU) in complex with compound 4 (blue) and compound 11 (orange); hydrogen bond interactions are depicted as
green dotted lines; interacting aminoacids are shown as gray sticks. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
regulators of protein degradation, transcription regulation and cell cycle
progression, these mechanisms correlated with Cdu1 activity are ach-
ieved through the alteration of the ubiquitin–proteasome pathway [37].
Key stabilizing binding features suggest that a Ser168 H-acceptor
hydrogen bond (HB) is essential for a potent inhibition of Cdu1 [37].
Binding mode analysis of the highest active biological compounds 4, 10
and 11 revealed that this key HB is also present (Fig. 7). Compounds are
well stabilized in the binding pocket through the formation of two
additional HB. Results also suggest that the C-ring modification in
compounds 10 and 11 does not add to supplementary ligand–protein
interactions, which are also reflected by the obtained docking scores for
these respective structures (Fig. 7). Considering all of the above,
compound 4, 10 and 11 could exhert their antibacterial activity against
C. trachomatis by the inhibition of the Cdu1 protein.
4. Conclusions
In summary, a series of oleanolic acid derivatives have been syn-
thesized and evaluated for their antichlamydial activity. Compounds 3,
4, 6, 10 and 11 demonstrated a high inhibitory activity against
C. trachomatis with chemotherapeutic index > 8, while 4, 10 and 11
were found to be lead derivatives with significant activity (EC50 1.23,
3.25 and 3.05
μg/mL, respectively). Molecular docking was employed to
suggest potential binding interaction of the tested compounds. Retrieved
9

O. Kazakova et al.
Steroids 175 (2021) 108912
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[18] A. Froelich, B. Bednarczyk-Cwynar, L. Zaprutko, A. Gzella, Beckmann
rearrangement within the ring C of oleanolic acid lactone: synthesis, structural
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The reported study was funded by RFBR and BRFBR (project no. 20-
53-00014) and the Belarusian Republican Foundation for Fundamental
Research (project no.M20P-275). Registration and analysis of NMR
spectra was carried out in accordance with state assignment No. AAAA-
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