New cholic acid analogs: Synthesis and 17β-hydroxydehydrogenase (17β-HSD) inhibition activity

Article abstract of DOI:10.1515/znb-2018-0192

The 17β-hydroxysteroid dehydrogenase (17β-HSD) enzyme family is involved in the biosynthesis of active steroids and its inhibition constitutes an interesting approach for treating estrogen-, androgen-dependent cancers and osteoporosis. In this study, a new series of cholic acid analogs was designed with the goal of improving the biological activity as 17β-HSD1 and 17β-HSD2 inhibitors. To this end, 23-cholyl amides 4-7, 3-O-p-toluenesulfonyl-23-cholyl amides 10-12, 23-cholyl-carbohydrazide 14, carbothioamide analog 15, and 23-cholyl-acylhydrazone derivatives 18-22 were synthesized from cholic acid (3) via coupling, sulfonation and substitution reactions. Basic treatment of keto group of 5 with p-bromoaniline afforded 8, meanwhile acidic treatment of 3 with thiosemicarbazide furnished the 23-cholyl-thiadiazole derivative 16. The synthesized compounds were evaluated for their inhibition activity against 17β-HSD1 and 17β-HSD2, and were found inactive at 1.0 μm concentration (inhibition <10%). However, the steroids 12, 21 and 22 showed inhibition of 21.1, 23.9 and 21.3%, respectively, against 17β-HSD2 at the same concentration. Therefore, these steroidal analogs can be further structurally modified to optimize their inhibition activity against 17β-HSD2 for the development of potential therapeutics.

Full text of DOI:10.1515/znb-2018-0192

Z. Naturforsch. 2018; 73(3–4)b: 211–223
Najim A. Al-Masoudia,*, Abbas Sami, Nabeel A. Abdul-Rida and Martin Fortscher
New cholic acid analogs: synthesis and
17β-hydroxydehydrogenase (17β-HSD)
inhibition activity
https://doi.org/10.1515/znb-2018-0192
Received November 16, 2017; accepted December 7, 2017
1 Introduction
Abstract: The 17β-hydroxysteroid dehydrogenase (17β- Cholic acid is a major primary bile acid produced in the
HSD) enzyme family is involved in the biosynthesis of liver and found in the bile of mammals and other verte-
active steroids and its inhibition constitutes an interesting brates and usually conjugated with glycine or taurine. It
approach for treating estrogen-, androgen-dependent can- facilitates fat absorption and cholesterol excretion [1]. A
cers and osteoporosis. In this study, a new series of cholic number of bile acid derivatives possess diverse pharmaco-
acid analogs was designed with the goal of improving the logical activities, notably antimicrobial [2–4], antifungal
biological activity as 17β-HSD1 and 17β-HSD2 inhibitors. [4–6], carbonic anhydrase inhibition [7, 8] and potential
To this end, 23-cholyl amides 4–7, 3-O-p-toluenesulfonyl- antioxidant [9]. One of the most promising applications
23-cholyl amides 10–12, 23-cholyl-carbohydrazide 14, of bile acid derivatives is as drug carriers, a direct con-
carbothioamide analog 15, and 23-cholyl-acylhydrazone sequence of their amphiphilic character [5]. They have
derivatives 18–22 were synthesized from cholic acid (3) already been reported to improve the permeability of cell
via coupling, sulfonation and substitution reactions. membranes, including the bacterial wall [2, 10]. Therefore,
Basic treatment of keto group of 5 with p-bromoaniline conjugation of active substances with bile acid derivatives
afforded 8, meanwhile acidic treatment of 3 with thiosem- through chemical modifications at the 3- and 24-positions
icarbazide furnished the 23-cholyl-thiadiazole derivative of the sterol nucleus led to various potential active analogs
16. The synthesized compounds were evaluated for their [11–21]. Brossard et al. [22] have synthesized several bile
inhibition activity against 17β-HSD1 and 17β-HSD2, and acid derivatives like N-(4-N-cinnamylpiperazin-1-yl)-
were found inactive at 1.0 μm concentration (inhibition 3α,7α-dihydroxy-5β-cholan-24-amide and its 7β-analog
<10%). However, the steroids 12, 21 and 22 showed inhibi- showed significant activity against three human cancer
tion of 21.1, 23.9 and 21.3%, respectively, against 17β-HSD2 cells [multiple myeloma (KMS-11), glioblastoma multi-
at the same concentration. Therefore, these steroidal ana- forme (GBM) and colonic carcinoma (HCT-116) human
logs can be further structurally modified to optimize their cell lines], within IC50ꢀ=ꢀ8.5–31.4 μm. Additionally, Zhang
inhibition activity against 17β-HSD2 for the development et al. [23] reported that cholic acid n-butyl ester had lower
of potential therapeutics.
IC50 against MCF-7 human breast cancer (BC) but it did
not have cytotoxicity to normal cell at low concentration.
Keywords: breast cancer; cholic acid; coupling reaction;
17β-hydroxydehydrogenase (17β-HSD); osteoporosis.
Diastereoisomeric-cholic-acid-derived
1,2,4,5-tetraox-
anes were also tested and found to have high anticancer
activity against human melanoma (Fem X), and cervix
cancer (HeLa) [24] cis stereoisomers were twofold more
active. Recently, Sakhuja et al. [25] have synthesized a
series of cholanamide derivatives linked via α-amino
acid, and some of these analogs exhibited fairly good
activity against the BC cell line (IC50ꢀ=ꢀ1.35–4.52 μm) (e.g.:
1, Fig. 1). Furthermore, cholic acid derivatives displayed
significant activity against some viral infections. Salunke
et al. [26] have reported the first examples of C-11 azido/
amino functionalized cholic acid derivatives induce HIV
replication and syncytia formation in T cells. Moreover,
bile acids have been suggested as useful moieties to liver
organotropic drugs, since cisplatin-bile acid derivatives
aPresent address: Am Tannenhof 8, 78457 Konstanz, Germany
*Corresponding author: Najim A. Al-Masoudi, Department of
Chemistry, College of Science, University of Basrah, Basrah, Iraq,
e-mail: najim.al-masoudi@gmx.de
Abbas Sami: Department of Chemistry, College of Education,
University of Qadisiyah, Qadisiyah, Iraq
Nabeel A. Abdul-Rida: Department of Chemistry, College of Science,
University of Qadisiyah, Qadisiyah, Iraq
Martin Fortscher: Rega Institute for Medical Research, Katholieke
Universiteit Leuven, Leuven B-3000, Belgium
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ꢀN.A. Al-Masoudi et al.: New cholic acid analogs
Br
O
O
H
N
N
N
N
OH
H
OH
H
H
H
O
H
H
H
H
HO
OH
HO
OH
H
H
1. Cytotoxicity (GI50 = 1.35
human breast adenocarcinoma cell lines
µ
M
) against
2. Cytotoxicity (GI50 = 1.96
µM) against
Gram positive bacteria E. faecalis
Fig. 1:ꢀSome potentially active steroid analogs.
(Bamets) have been used in vitro to determine the produc- acid analogs as inhibitors of 17β-HSD1, 17β-HSD2 enzymes
tion of virions by HBV-transfected hepatoblastoma cells or antimicrobial candidates (Fig. 1).
(HepG2) [27] and effects of DNA-reactive bile acid deriva-
tives (Bamets) on hepatitis B virus life cycle [28]. However,
number of cholic acid analogs revealed significant anti-
2 Result and discussion
microbial activity [4, 29–33]. Recently, Al-Qawasmeh et al.
[34] have reported the synthesis and antimicrobial activity
of cholic acid hydrazone analogs, as some of the deriva- 2.1 Chemistry
tives showed stronger antimicrobial activity against Gram-
positive bacteria than Cefaclor and Cefixime, for example Treatment of cholic acid 3 with the appropriate amines
analog 2 (IC50ꢀ=ꢀ1.96 μm) (Fig. 1) against Gram positive bac- (e.g.: 4-bromoaniline-, N′-aminoacetophenoe, 4-ami-
teria Enterococcus faecalis.
noantipyrine and safranine) in the presence of DCC as
In respect with the biological significance of bile acid coupling reagent and DMAP as catalyst in CH2Cl2-DMF
derivatives and in continuation of our work on the 17β- gave N-(substituted-aryl)-3α,7α,12α-trihydroxy-5β-cholan-
hydroxydehydrogenase inhibition activity [35], we report 24-amide derivatives 4–7 in 68%–88% yield. The amide
here the synthesis and the biological activity of new cholic derivative 4 has been selected as a key intermediate for
O
O
24
21
Me
NH R
Me
OH
HO
12
HO
H
Me
22
Me
17
19
Me
1
Me
H
8
RNH2
15
10
H
3
H
H
i
H
HO
OH
HO
OH
6
H
H
3
R
4
5
6
7
4-Br-C6H4
4-COMe-C6H4
O
5'
Me
ii
6''
Me
Me
antipyrine
NH
HO
1'
Br
N
safranin-7-yl
3'
2''
Me
H
H
8
H
H
9
10a
2
HO
OH
9a
5a
N
N
CH3
NH
H3C
Me
4
NH
X
1
N
Me
H2N
4a
7
3
N
N
Cl
=
RNH
H
1''
3
O
5''
3''
Safraninyl
X: Br, COMe
Aminoantipyrinyl
Scheme 1:ꢀReagents and conditions: (i) DCC, DMAP, CH2Cl2-DMF, r.t., 16–20 h; (ii) 4-Br-C6H4-NH2, AcOH, EtOH, reflux, 12 h.
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ꢁꢀꢀꢀ
N.A. Al-Masoudi et al.: New cholic acid analogsꢂ
ꢂ213
the synthesis of new imine analog 8 in 74% yield, by treat- the synthesis of new carbohydrazide derivatives aiming
ment with 4-bromoaniline (Scheme 1).
to examine their anti-HSD and antimicrobial activities in
A common feature of bile acid-derived antimicrobials comparison to those of the analogs 4–7. Thus, treatment of
is their potential to exhibit a facially amphiphilic nature 13 with 2,4-dinitrophenylhydrazine or thiosemicarbazide
due to both a hydrophobic and a hydrophilic face [36]. afforded the carbohydrazide analogs 14 and 15 in 73 and
Therefore, our work was modified by selective tosylation 81% yield, respectively. Further, the broad and potent
of 9 at C-3 [37] followed by amidation of carboxylic group activity of thiadiazole and their derivatives has estab-
leading to a facial amphilphile analog with two oriented lished them as pharmacologically significant scaffolds
hydroxy groups at C-7 and C-12 that may be exploited in [39]. In this study, the carboxylic acid moiety has been
podant-type receptors. Thus, the tosylate 9 was subjected converted into a 1,3,4-thiazdiazole group by treatment of
to an amide forming reaction using DCC and amines cholic acid (3) with thiosemicarbazide in alcoholic H2SO4
(e.g.: i-propyl-, n-butylamines and thiourea), following furnishing 16 in 83% yield (Scheme 3).
the procedure described previously in preparation of
PAC-1 (2-(4-benzylpiperazin-1-yl)-N′-[(Z)-(6-oxo-5-prop-
4–7, furnished the 3α-p-toluenesulfonyl cholan-24-amide 2-enylcyclohexa-2,4-dien-1-ylidene) methyl]acetohydrazide)
derivatives 10–12 in 75, 73 and 71% yield, respectively is the first procaspase activating compound that selectively
(Scheme 2).
induces apoptosis in cancerous cells having the potential
Our work was modified by selecting methyl cholate 13, N-acylhydrazone pharmacophore [40, 41]. In an attempt
prepared previously by Shen et al. [38], as a precursor for to discover new antitumor agents via the inhibition of
O
O
O
Me
Me
Me
Me
Me
Me
OH
NH R
OH
HO
HO
H
HO
H
Me
Me
Me
H
i
ii
H
H
H
H
H
H
O
O
S
HO
OH
OH
OH
H
H
H
O
S
O
O
O
3
R
9
10 NHiPr2
11 NHBu2
12 -CS-NH2
Scheme 2:ꢀReagents and conditions: (i) TsCl, pyridine, 0°C, (30 min), 55°C, 5 h; (ii) RNH2, DCC, DMAP, CH2Cl2-DMF, r.t., 16–20 h.
O
O
Me
Me
Me
Me
OR
NH NHR
HO
H
HO
H
ii
Me
Me
+
RNHNH2
H
H
H
H
HO
HO
OH
OH
H
H
3. R = H
13. R = Me
R
Entry
i
iii
2,4-NO2-C6H4
-CS-NH2
14
15
NH2
S
N
N
Me
Me
HO
Me
H
H
H
16
HO
OH
H
Scheme 3:ꢀReagents and conditions: (i) MeOH, HCl, reflux, 5 h; (ii) EtOH, DMF, reflux, 12 h; (iii) NH2NHCSNH2, EtOH, conc.
H2SO4, reflux, 5 h.
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ꢀꢀꢀꢁ
214ꢀ
ꢀN.A. Al-Masoudi et al.: New cholic acid analogs
O
O
N
R'
Me
R
Me
N
H
Me
Me
HO
HO
H
COR'
R
ii
Me
+
Me
H
H
R
H
H
H
H
HO
i
OH
HO
OH
H
Entry
18
R
R'
13: R = Me
17: R = NHNH2
H
H
H
Me
Me
OH
N(Me)2
Cl
OH
NO2
NH2
H
O
N
N
19
20
21
22
N
N-acylhydrazone
N
PAC-1
Scheme 4:ꢀReagents and conditions: (i) NH2NH2. H2O, EtOH, reflux, 12 h; (ii) EtOH, AcOH, 10–12 h.
17β-HSD enzymes or for the treatment of osteoporosis, we group (CONH), whereas the aromatic carbon atoms
combined the cholic acid moiety and N-acylhydrazone appeared at the regions δꢀ=ꢀ160.2–106.0 ppm. The signals
based in a hybrid pharmacophore design [42]. The car- at the lower fields δꢀ=ꢀ180.0 and 183.8 ppm were assigned
bohydrazide derivative 17 was prepared according to the to Cꢀ=ꢀS carbon atoms of 12 and 15, whereas the reso-
procedure of Al-Qawasmeh et al. [34] from methyl cholate nances at δꢀ=ꢀ173.3 and 170.2 ppm were attributed to C-2′
13 and hydrazine hydrate. Condensation of 17 with the and C-5′ of the thiadiazole moiety of 16. The resonances at
carbonyl compounds (e.g.: 4-N,N-dimethyl-, 4-choloro-, the region δꢀ=ꢀ150.2–142.8 and 169.2 ppm were assigned for
and 4-hydroxybenzaldehydes as with as well 4-nitro- and Cꢀ=ꢀN carbon atoms of 18–22 and 8, respectively. C-22 and
4-aminoacetophenones) in refluxing acidic EtOH gave the C-23 and C-24 appeared at the regions δꢀ=ꢀ33.3–31.0 and
desired benzylidene and ethylidene analogs 18–22 in 71%– 32.7–26.7 ppm, respectively. The other aliphatic carbon
90% yield (Scheme 4).
atoms and the substituents have been fully identified (c.f.
The structures of 4–16 and 18–22 were assigned on Experimental section).
the basis of their IR and NMR (1H, 13C and 2D) spectra,
Compound 6 was selected for further NMR studies.
which showed rather similar patterns of the aliphatic From a gradient heteronuclear multiple bond correlation
proton and carbon atoms. The H NMR spectra of 4–8, (HMBC) [43] NMR spectrum, 3JC,H couplings between 23a-H,
1
14–16 (ok?) and 18–22 were characterized by the presence 23b-H protons at δꢀ=ꢀ2.09, 2.02 ppm and carbonyl carbon
of aromatic protons and carbon atoms, indicative for ami- atoms of the amide group at δꢀ=ꢀ171.5 ppm, was observed.
dation of the cholic acid backbone. The aromatic protons Further, 22a-H and 22b-H at δꢀ=ꢀ1.67, 1.23 ppm showed 2JC,H
3′-H and 5′-H of all analogs, except 6 and 14, appeared couplings with the same carbonyl carbon atom of the
as doublets or at the regions δꢀ=ꢀ8.49–6.58 ppm (Jꢀ=ꢀ8.8– amide group. Additionally, C-4′ and C-5′ of the pyrazolone
7.5 Hz), while 2′-H and 6′-H were resonated at the regions ring at δꢀ=ꢀ107.8 and 135.0 ppm showed two 2JC,H couplings
δꢀ=ꢀ8.17–6.55 ppm. The multiplet at δꢀ=ꢀ7.35–7.49 ppm with methylene protons at δꢀ=ꢀ2.73 and 3.02 ppm, respec-
assigned for the five aromatic protons of 6, whereas the tively (Fig. 2). All the compounds have been identified by
singlet at δ 8.60 ppm and two doublets at δꢀ=ꢀ8.37 and their 1H,13C HSQC NMR spectra [44].
7.65 ppm (Jꢀ=ꢀ7.9 Hz) attributed to aromatic protons 3′-H,
5′-H and 6′-H of 14, respectively. The multiplets at δꢀ=ꢀ1.36–
1.29 ppm were assigned to 20-H, while the doublets at the
3JC5',Me
Me
3JC4',Me
regions at δꢀ=ꢀ0.94–0.91 ppm (JMe21,H20ꢀ=ꢀ6.6–6.1 Hz) were
attributed to the methylene protons (21-Me). The Nꢀ=ꢀCH
protons of 18–20 resonated as singlets at δꢀ=ꢀ8.08, 8.42
and 8.52 ppm, respectively, while methylene protons of
Nꢀ=ꢀCMe group of 21 and 22 appeared as singlets at δꢀ=ꢀ2.32
and 2.23 ppm, respectively. The other aliphatic protons
3JCO,H22
Me
21
1'
N
N
H
Me
O
H
OH
12
22
N
4'
H
17
1
HH
O
2JCO,H23
10
13
were fully analyzed (c.f. Experimental section). In the C
HO
OH
3
7
NMR, the resonances at the regions δꢀ=ꢀ173.7–167.8 ppm
were assigned for the carbonyl carbon atoms of the amide Fig. 2:ꢀJC,H correlations in the NMR HMBC spectrum of 6.
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ꢂ215
derivatives showed less than 10% inhibition against 17β-
HSD1 at 1.0 μm and were considered to be inactive. On the
other hand, compounds 12, 20 and 21 exhibited inhibition
2.2 In vitro inhibition activity of 17β-HSD1
and 17β-HSD2
17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) cata- activities against 17β-HSD2 of 21.1%, 23.9% and 21.3%,
lyzes the conversion of the weakly active estrone (E1) to the respectively, at 1.0 μm concentration, and thus turned out
highly active estradiol (E2), meanwhile 17β-hydroxysteroid to be promising analogs for treatment of osteoporosis. In
dehydrogenase type 2 (17β-HSD2) catalyzes the conversion general, the introduction of arylated acylhydrazone group
of E2 and testosterone (T) into the E1 and δ4-androstene- via derivatization of the carboxylic group of cholic acid,
3,17-dione (δ4-AD), respectively, [45]. The enzyme 17β- had a positive impact on inhibitory activity, in comparison
HSD1 came into the focus of interest as a novel therapeutic for those of other cholic acid derivatives.
target for the treatment of estrogen dependent diseases
like BC and endometriosis [46–48]. 17β-HSD2 is expressed
in human osteoblastic (OB) cells [49], therefore its inhi-
bition can lead to the desired increase of E2, formed by
3 Conclusion
reduction of E1, and T levels in the bone tissue and may
A new series of amides, tosylates, carbohydrazide, car-
thus be a novel approach for the treatment of osteopo-
bothioamide analog and acylhydrazone analogs derived
rosis [50]. As 17β-HSD2 catalyzes the inactivation of E2
cholic acid were synthesized. The synthesized compounds
into E1, inhibitory activity toward this enzyme must be
were screened for their inhibitory activity against 17β-
avoided. However, 17β-HSD1 inhibitors should not inhibit
HSD1 and 17β-HSD2 at 1.0 μm concentration. Only com-
17β-HSD2 and, of course, should not be estrogenic. Our
pounds 12, 20 and 21 showed inhibition against 17β-HSD2
new synthesized compounds were tested for their ability
(21.1, 23.9 and 21.3%, respectively). These analogs can be
to inhibit 17β-HSD1 and 17β-HSD2. The inhibition values
considered as promising agents for treatment of osteopo-
of the tested compounds are shown in Table 1. All these
rosis waiting for further structural modification.
Table 1:ꢀ17β-HSD1 and 17β-HSD2 inhibitory activity of some cholic
acid analogsa.
4 Experimental
Comp
17β-HSD1a (% inhib.)
(1.0 μm)
17β-HSD2b (% inhib.)
(1.0 μm)
4.1 General
4
3.0
2.4
14.4
3.6
0.9
8.5
10.2
4.9
2.0
21.1
15.2
11.5
4.9
8.1
ni
12.5
4.2
23.9
21.3
5.7
Melting points are uncorrected and were measured on a
Büchi melting point apparatus B-545 (Büchi Labortech-
nik AG, Switzerland). Microanalytical data were obtained
with a Vario Elemental Analyzer (Shimadzu, Japan). NMR
spectra were obtained at 400 and 600 MHz (1H) as well as
100 MHz and 150:91 MHz (13C) spectrometers (Avance III,
Bruker, Germany) with TMS as internal standard and on
δ scale in ppm. Signal assignments for protons were per-
formed by selective proton decoupling or by COSY spectra.
Heteronuclear assignments were verified by HSQC and
HMBC experiments. TLC plates 60 F254 were purchased
from Merck.
5
7.5
6
ni
7
−8.6
−0.3
7.1
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
−3.3
0.2
2.2
6.2
8.5
−0.8
3.1
ni
−4.6
−8.5
−2.2
−7.3
ni
4.2 General procedure for the synthesis of
amides analogs of cholic acid (4–7)
ani: no inhibition. Human placenta, cytosolic fraction, substrate
[3H]E1ꢁ+ꢁE1 [500 nm], cofactor NADH [500 μm]; bhuman placenta,
microsomal fraction, substrate [3H]E2ꢁ+ꢁE2 [500 nm], cofactor NADꢁ+
[1500 μm]; cmean values of two determinations, standard deviation
less than 10%.
To a cold solution of cholic acid 3 (409 mg, 1.00 mmol)
in CH2Cl2-DMF (20 mL, 1:1 v/v) were added 4-(dimethyl-
amino)pyridine (DMAP) (16 mg, 0.13 mmol) and amines
(1.00mmol)withstirringfor10min, followedbyadditionof
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216ꢀ ꢀN.A. Al-Masoudi et al.: New cholic acid analogs
N,N′-dicyclohexylcarbodiimid (DCC) (206 mg, 1.00 mmol). 1H, Jꢀ=ꢀ7.9 Hz, OH), 5.58 (d, 1H, Jꢀ=ꢀ7.6 Hz, OH), 4.30 (bs, 1H,
The reaction mixture was stirred at room temperature for OH), 4.06 (m, 1H, 12-H), 3.78 (m, 1H, 7-H), 3.18 (m, 1H, 3-H),
16–20 h and completion of reaction was monitored by 2.58 (s, 3H, COMe), 2.24 (m, 1H, 4a-H), 2.15 (m, 2H, 9-H),
TLC. A white precipitate was filtered and the solvent was 2.12 (m, 1H, 23a-H), 2.06 (m, 1H, 23b-H), 1.94 (m, H, 14-H),
evaporated to dryness and the residue was dissolved in 1.80 (m, 1H, 6a-H), 1.78 (m, 1H, 17-H), 1.75 (m, 1H, 16a-H),
ethyl acetate (50 mL). The organic layer was washed with 1.67 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.64 (m, 2H, 2a-Hꢀ+ꢀ15a-H), 1.48
aqueous 4% HCl and saturated NaHCO3 (3ꢀ×ꢀ30 mL) then (m, 1H, 4b-H), 1.44 (m, 1H, 11a-H), 1.39 (m, 1H, 6b-H), 1.35
dried (Na2SO4) and evaporated to dryness. The residue (m, 1H, 11b-H), 1.33 (m, 2H, 8-Hꢀ+ꢀ20-H), 1.28 (m, 1H, 2b-H),
was purified on a SiO2 column chromatography using, in 1.25 (m, 1H, 5-H), 1.23 (m, 1H, 22b-H), 1.21 (m, 1H, 5-H), 1.17
gradient, MeOH (0%–10%) and CHCl3 as eluent to give the (m, 1H, 16b-H), 0.93 (d, 3H, JMe21,H20ꢀ=ꢀ6.3 Hz, 21-Me), 0.91
desired product.
(m, 1H, 15b-H), 0.83 (m, 1H, 1b-H), 0.80 (s, 3H, 19-Me), 0.57
(s, 3H, 18-Me). – 13C NMR ([D6]DMSO): δꢀ=ꢀ191.2 (C=O), 171.5
(CONH), 139.7 (C-1′), 138.7 (C-4′), 131.3 (C-3′ꢀ+ꢀC-5′), 120.8
(C-2′ꢀ+ꢀC-6′), 73.1 (C-12), 70.9 (C-3), 66.2 (C-7), 47.1 (C-17), 45.6
(C-13), 41.5 (C-5), 41.3 (C-14), 40.1 (C-4ꢀ+ꢀC-8), 37.7 (C-20),
36.7 (C-1), 35.4 (C-6), 35.2 (C-10), 31.7 (C-2ꢀ+ꢀC-22), 30.9 (C-23),
4.2.1 N-(4-bromophenyl)-3α,7α,12α-trihydroxy-5β-
cholan-24-amide (4)
From 4-bromoaniline (172 mg). Yield: 438 mg (78%) 28.4 (C-11), 27.2 (C-16), 26.1 (C-9), 24.5 (COMe), 18.4 (Me-21),
as colorless solid, m.p.: 85–87°C; Rfꢀ=ꢀ0.32. – IR (KBr): 12.2 (Me-18). – (C32H47NO5) (525.73): calcd. C 73.11, H 9.01, N
νꢀ=ꢀ3373(OH), 2931, 2854 (CH2), 1697 (C=O)amid, 1635 2.66; found C 72.90, H 8.91, N 2.43.
1
(C=C)arom. cm−1. – H NMR ([D6]DMSO): δꢀ=ꢀ9.95 (s, 1H,
NHamide), 7.58 (d, 2H, Jꢀ=ꢀ8.7 Hz, 3′-Harom.ꢀ+ꢀ5′-Harom.), 6.55 (d,
2H, Jꢀ=ꢀ8.7 Hz, 2′-Harom.ꢀ+ꢀ6′-Harom.), 6.00 (bs, 1H, OH), 5.58 (d, 4.2.3 N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
1H, Jꢀ=ꢀ8.0 Hz, OH), 4.34 (bs, 1H, OH), 3.98 (m, 1H, 12-H),
3.78 (m, 1H, 7-H), 3.19 (m, 1H, 3-H), 2.27 (m, 2H, CH2-4),
2.22 (m, 1H, 9-H), 2.15 (m, 1H, 23a-H), 2.13 (m, 1H, 23b-H),
pyrazol-4-yl)-3α,7α,12α-trihydroxy-5β-cholan-24-
amide (6)
1.94 (m, 1H, 14-H), 1.82 (m, 1H, 6-H), 1.79 (m, 1H, 7-H), From 4-aminoantipyrine (203 mg). Yield: 523 mg (88%)
1.77 (m, 1H, 16a-H), 1.67 (m, 2H, 1a-Hꢀ+ꢀ22-H), 1.64 (m, 2H, as orange solid, m.p.: 142–145°C, Rfꢀ=ꢀ0.64. – IR (KBr):
2a-Hꢀ+ꢀ15a-H), 1.47 (m, 1H, 4a-H), 1.43 (m, 1H, 11a-H), 1.39 νꢀ=ꢀ3404 (OH), 2931, 2854 (CH2), 1705 (C=O)antipy., 1681
1
(m, 1H, 6b-H), 1.35 (m, 1H, 11b-H), 1.33 (m, 2H, 8-Hꢀ+ꢀ20-H), (C=O)amid, 1666 (C=C)arom. cm−1. – H NMR ([D6]DMSO):
1.29 (m, 1H, 2b-H), 1.27 (m, 1H, 5-H), 1.22 (m, 1H, 22-H), 1.18 δꢀ=ꢀ8.98 (s, 1H, NHamide), 7.35–7.49 (m, 5H, Harom), 5.58 (d, 1H,
(m, 1H, 16b-H), 0.93 (d, 3H, JMe21,H20ꢀ=ꢀ6.3 Hz, 21-Me), 0.91 Jꢀ=ꢀ7.8 Hz, OH), 4.32 (m, 1H, OH), 4.07 (m, 1H, 17-H), 3.79
(m, 1H, 15b-H), 0.83 (m, 1H, 1b-H), 0.80 (s, 3H, 19-Me), 0.57 (m, 1H, 7-H), 3.19 (m, 1H, 3-H), 3.02 (s, 3H, C1pyrazolon-NMe),
(s, 3H, 18-Me). – 13C NMR ([D6]DMSO): δꢀ=ꢀ171.5 (C=O), 135.2 2.73 (s, 3H, C5pyrazolon-Me), 2.24 (m, 1H, 4a-H), 2.14 (m, 1H,
(Carom.-1′), 130.4 (Carom.-3′ꢀ+ꢀCarom.-5′), 124.8 (C-4′), 120.8 (Carom
-
9-H), 2.09 (m, 1H, 23a-H), 2.02 (m, 1H, 23b-H), 1.97 (m, 1H,
2′ꢀ+ꢀCarom.-6′), 73.9 (C-12), 70.9 (C-3), 66.2 (C-7), 47.4 (C-17), 14-H), 1.81 (m, 1H, 17-H), 1.76 (m, 1H, 16a-H), 1.75 (m, 1H,
46.1 (C-13), 45.2 (C-5), 41.4 (C-14), 40.1 (C-8), 39.8 (C-4), 37.7 16b-H), 1.67 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.64 (m, 2H, 2a-Hꢀ+ꢀ15a-
(C-20), 36.6 (C-1ꢀ+ꢀC-6), 35.2 (C-10), 31.8 (C-2ꢀ+ꢀC-22), 31.7 H), 1.48 (m, 1H, 4a-H), 1.42 (m, 1H, 11a-H), 1.39 (m, 1H,
(C-23), 28.5 (C-11), 27.2 (C-16), 26.6 (C-9), 22.5 (Me-19ꢀ+ꢀC-15), 6a-H), 1.36 (m, 1H, 11b-H), 1.34 (m, 2H, 8-Hꢀ+ꢀ20-H), 1.29
18.4 (Me-21), 12.2 (Me-18). – C30H44BrNO4 (562.59): calcd. C (m, 1H, 2b-H), 1.26 (m, 1H, 5-H), 1.23 (m, 1H, 22b-H), 1.18
64.05, H 7.88, N 2.49; found C 63.81, H 7.75, N 2.22.
(m, 1H, 16b-H), 0.93 (d, 3H, JMe21,H20ꢀ=ꢀ6.3 Hz, 21-Me), 0.91
(m, 1H, 15b-H), 0.83 (m, 1H, 1b-H), 0.80 (s, 3H, 19-Me),
0.57 (s, 3H, 18-Me). – 13C NMR ([D6]DMSO): δꢀ=ꢀ171.5
(CONH), 162.2 (C=Opyrazolon), 135.0 (Cpyrazolon-5′ꢀ+ꢀCarom.-1′),
128.9, 123.2 (Carom.), 107.8 (Cpyrazolon-4′), 73.1 (C-12), 70.9
(C-3), 66.2 (C-7), 49.5 (C-17), 47.4 (C-13), 45.7 (C-5), 43.4
4.2.2 N-(4-acetylphenyl)-3α,7α,12α-trihydroxy-5β-
cholan-24-amide (5)
From 4-aminoacetophenone (135 mg). Yield: 357 mg (C-14), 41.3 (C-4ꢀ+ꢀC-8), 36.2 (C-20), 35.7 (C-1), 35.2 (C-6),
(68%) as dark yellow solid, m.p.: 128–130°C, Rfꢀ=ꢀ0.83. – 34.3 (C-10), 33.2 (NMe), 32.4 (C-22), 31.7 (C-2), 30.9 (C-23),
IR (KBr): νꢀ=ꢀ3394 (OH), 2931, 2854 (CH2), 1712 (C=O)acetyl
,
28.4 (C-11), 27.2 (C-16), 26.1 (C-9), 24.5 (C-15), 22.5 (Me-19),
1697 (C=O)amid, 1650 (C=C)arom. cm−1. – 1H NMR ([D6]DMSO): 18.4 (Me-21), 12.2 (Me-18), 11.1 (C5pyrazolon-Me). – r C35H51N3O5
δꢀ=ꢀ9.95 (s, 1H, NHamide), 7.57 (d, 2H, Jꢀ=ꢀ7.8 Hz, 3′-Harom.ꢀ+ꢀ5′- (593.81): calcd. C 70.79, H 8.66, N 7.08; found C 70.54, H
H
arom.), 7.44 (d, 2H, Jꢀ=ꢀ7.8 Hz, 32′-Harom.ꢀ+ꢀ56′-Harom.), 6.61 (d, 8.59, N 6.88.
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ꢁꢀꢀꢀ
N.A. Al-Masoudi et al.: New cholic acid analogsꢂ
ꢂ217
4.2.4 3-Amino-2,8-dimethyl-5-phenyl-7-yl-(N-
(3α,7α,12α-trihydroxy-5β-cholan-24-amido))-
phenazin-5-ium chloride (7)
3.78 (m, 1H, 7-H), 3.19 (m, 1H, 3-H), 2.22 (m, 1H, 4a-H),
2.14 (m, 1H, 9-H), 2.11 (s, 3H, N=CMe), 2.09 (m, 1H, 23a-H),
2.02 (m, 1H, 23b-H), 1.95 (m, 1H, 14-H), 1.80 (m, 1H, 6a-H),
1.77 (m, 1H, 17-H), 1.67 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.64 (m, 2H,
From safranine (351 mg). Yield: 615 mg (83%) as brown 2a-Hꢀ+ꢀ15a-H), 1.46 (m, 1H, 4b-H), 1.44 (m, 1H, 11a-H),
crystals, m.p.: 161–163°C, Rfꢀ=ꢀ0.42. – IR (KBr): νꢀ=ꢀ3420 1.39 (m, 1H, 6a-H), 1.35 (m, 1H, 11b-H), 1.33 (m, 1H, 8-H),
(OH), 2931, 2854 (CH2), 1690 (C=O)amid, 1650 (C=C)arom. cm−1. 1.30 (m, 1H, 20-H), 1.27 (m, 1H, 2b-H), 1.25 (m, 1H, 5-H),
– 1H NMR ([D6]DMSO): δꢀ=ꢀ8.49 (d, 1H, Jꢀ=ꢀ7.8 Hz, 9′-Harom.), 1.23 (m, 1H, 12-22b-H), 1.19 (m, 1H, 16b-H), 0.93 (d, 3H,
8.09 (d, 2H, Jꢀ=ꢀ7.9 Hz, 2″-Harom.ꢀ+ꢀ6″-Harom.), 7.95 (s, 1H, H-4′),
J
Me21,H20ꢀ=ꢀ6.5 Hz, 21-Me), 0.91 (m, 1H, 15b-H), 0.84 (m, 1H,
13
7.46 (bs, 2H, 1′-Harom.ꢀ+ꢀ6′-Harom.), 7.32 (m, 3H, 3″-Harom.ꢀ+ꢀ4″- 1b-H), 0.80 (s, 3H, 19-Me), 0.57 (s, 3H, 18-Me). – C NMR
H
arom.ꢀ+ꢀ5″-H), 5.69 (d, 1H, Jꢀ=ꢀ8.0 Hz, OH), 4.41 (d, 1H, ([D6]DMSO): δꢀ=ꢀ172.1 (CONH) 169.2 (N=CMe), 148.5 (Carom.-
Jꢀ=ꢀ7.8 Hz, OH), 4.00 (m, 1H, 12-H), 3.77 (m, 1H, 7-H), 3.20 1″), 139.2 (Carom.-1′), 135.1 (Carom.-4′), 131.8, 125.4, 122.4, 121.4,
(m, 1H, 3-H), 2.26 (m, 1H, 4a-H), 2.14 (m, 7H, H-9ꢀ+ꢀ(2ꢀ×ꢀMe- 119.8 (Carom.), 71.6 (C-12), 70.2 (C-3), 66.8 (C-7), 47.7 (C-17),
safranin)), 2.11 (m, 1H, 23a-H), 2.08 (m, 1H, 23b-H), 1.96 46.2 (C-13), 45.4 (C-5), 41.3 (C-14), 40.2 (C-4ꢀ+ꢀC-8), 36.1
(m, 1H, 14-H), 1.80 (m, 1H, 6a-H), 1.77 (m, 1H, 17-H), 1.73 (C-20), 35.8 (C-1), 35.6 (C-6), 34.9 (C-10), 32.3 (C-2ꢀ+ꢀC-22),
(m, 1H, 16a-H), 1.65 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.63 (m, 2H, 30.9 (C-23), 29.0 (C-11), 27.8 (C-16), 25.8 (C-9), 23.1 (C-15),
2a-Hꢀ+ꢀ15a-H), 1.48 (m, 1H, 4b-H), 1.42 (m, 2H, 11a-H), 1.37 22.7 (Me-19), 19.0 (Me-21), 19.0 (Me-21), 17.6 (N=CMe). –
(m, 1H, 6b-H), 1.35 (m, 1H, 11b-H), 1.30 (m, 1H, 8-Hꢀ+ꢀ20-H), C38H51BrN2O4 (679.74): calcd. C 67.15, H 7.56, N 4.12; found C
1.26 (m, 2H, 2b-Hꢀ+ꢀ5-H), 1.24 (m, 1H, 22b-H), 1.17 (m, 1H, 66.91, H 7.42, N 4.22.
16b-H), 0.92 (d, 3H, JMe21,H20ꢀ=ꢀ6.2 Hz, 21-Me), 0.91 (m, 1H,
15b-H), 0.83 (m, 1H, 1b-H), 0.80 (s, 3H, 19-Me), 0.58 (s, 3H,
18-Me). – 13C NMR ([D6]DMSO): δꢀ=ꢀ173.3 (C=O), 150.1 (C-4a′), 4.4 3 α-p-Toluenesulfonyl-7α,12α-dihydroxy-
141.4 (C7′-NH2), 138.8 (Carom.-1″), 136.9, 134.5, 131.2, 131.1,
129.4, 128.0, 127.7, 126.6, 125.6 (Carom.), 121.6 (C-10a′ꢀ+ꢀC-9a′),
5β-cholan-24-oic acid (9)
106.6 (C-4′), 71.8 (C-12), 70.3 (C-3), 66.1 (C-7), 47.4 (C-17), This compound was prepared according to the procedure
46.2 (C-13), 45.6 (C-5), 41.4 (C-14), 40.9 (C-8), 40.4 (C-4), 36.2 reported by Barnett and Reichstein [37]. To a solution of
(C-20), 35.6 (C-1), 35.4 (C-6), 35.1 (C-10), 31.8 (C-2ꢀ+ꢀC-22), 31.0 cholic acid 3 (613 mg, 1.50 mmol) in dry pyridine (15 mL)
(C-23), 28.0 (C-11), 27.1 (C-16), 26.1 (C-9), 22.6 (C-15), 22.5 (Me- was added a solution of p-toluenesulfonyl chloride
19), 19.3 (C2′-Me), 18.4 (Me-21), 16.9 (C8′-Me), 12.2 (Me-18). (300 mg, 1.58 mmol) in dry pyridine (5 mL) with stirring
– C44H57ClN4O4 (741.41): calcd. C 71.28, H 7.75, N 7.56; found at 0°C for 30 min. The solution was allowed to warm up,
C 71.01, H 7.65, N 7.26.
stirred at room temperature for 5 h, and the progress of
the reaction was monitored by TLC. The reaction mixture
was slowly added to 1.5 m HCl (100 mL). The white pre-
cipitate was collected by filtration and dried in vacuo.
The crude product was purified on a SiO2 column using
in gradient, MeOH (0%–20%) and CHCl3 as eluent to give
pure 13 (642 mg, 76%), m.p.: 130–133°C (Lit. [37], m.p.:
4.3 N-(4-(1-((4-bromophenyl)imino)ethyl)
phenyl)-3α,7α,12α-trihydroxy-5β-cholan-
24-amide (8)
To a solution of 5 (310 mg, 0.60 mmol) in EtOH (20 mL) 131°C).
and glacial acetic acid (1 mL) was added 4-bromoaniline
(100 mg, 0.60 mmol) and the mixture was heated under
reflux for 12 h. The reaction progress was monitored by 4.5 General procedure for synthesis of 3α-
TLC. After cooling, the mixture was poured into cold
tosyloxy-7α,12α-dihydroxy-5β-cholan-
water, and the solid product was collected by filtration
24-amide derivatives (10–12)
followed by recrystallization from EtOH to give 8 (230 mg,
74%) as red solid, m.p.: 180–182°C, Rfꢀ=ꢀ0.7. – IR (KBr): The amide analogs were prepared according to the proce-
νꢀ=ꢀ3409 (OH), 2931, 2854 (CH2), 1649 (C=N), 1595 (C=C)arom. dure for preparation of the analogs 4–7 from the tosylate
cm−1.ꢀꢀ−ꢀꢀ1H NMR ([D6]DMSO): δꢀ=ꢀ10.09 (s, 1H, NH), 7.88 (d, derivative 9 (281 mg, 0.50 mmol) and amines (0.50 mmol)
2H, Jꢀ=ꢀ7.9 Hz, 3′-Harom.ꢀ+ꢀ5′-Harom.), 7.65 (d, 2H, Jꢀ=ꢀ7.9 Hz, 3″- in CH2Cl2-DMF (10 mL), DMAP (0.07 mmol) and DCC
H
arom.ꢀ+ꢀ5″-Harom.), 7.57 (d, 2H, Jꢀ=ꢀ7.9 Hz, 2′-Harom.ꢀ+ꢀ6′-Harom.), (0.50 mmol). The products were purified from cyclohexy-
7.11 (d, 2H, Jꢀ=ꢀ7.9 Hz, 2″-Harom.ꢀ+ꢀ6″-Harom.), 5.61 (bs, 1H, OH), lurea by SiO2 column using, in gradient, MeOH (0%–10%)
5.21 (bs, 1H, OH), 4.44 (bs, 1H, OH), 4.04 (m, 1H, 12-H), and CHCl3 as eluent.
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ꢀꢀꢀꢁ
218ꢀ ꢀN.A. Al-Masoudi et al.: New cholic acid analogs
4.5.1 (N,N-diisopropyl)- 7α,12α-dihydroxy-3α-tosyloxy-
5β-cholan-24-amide (10)
1.18 (m, 1H, 16b-H), 0.92 (d, 3H, JMe21,H20ꢀ=ꢀ6.5 Hz, Me-21),
0.91 (m, 1H, 15b-H), 0.89 (m, 6H, 2ꢀꢀ×ꢀꢀN(CH2)3Me), 0.83 (m,
1H, 1b-H), 0.80 (s, 3H, 19-Me), 0.57 (s, 3H, 18-Me). – 13C
From N,N-diisopropylamine (51 mg). Yield: 242 mg (75%) NMR ([D6]DMSO): δꢀ=ꢀ171.4 (C=O), 145.6 (C-1′), 137.5 (C-4′),
as yellow solid, m.p.: 155–156°C, Rfꢀ=ꢀ0.28. – IR (KBr): 130.1 (C-2′ꢀ+ꢀC-6′), 127.9 (C-3′ꢀ+ꢀC-5′), 71.8 (C-12), 70.9 (C-3),
νꢀ=ꢀ3410 (OH), 2931, 2854 (CH2), 1695 (C=O)amid, 1650 66.1 (C-7), 50.3 (2ꢀ×ꢀNCH2), 47.1 (C-17), 46.1 (C-13), 45.6 (C-5),
1
(C=C)arom, 1314, 1338 (S=O) cm−1. – H NMR ([D6]DMSO): 41.4 (C-14), 40.5 (C-8), 40.0 (C-4), 36.1 (C-20), 35.6 (C-1),
δꢀ=ꢀ7.60 (d, 2H, Jꢀ=ꢀ7.8 Hz, 2′-Harom.ꢀ+ꢀ6′-Harom.), 7.38 (d, 2H, 35.5 (C-6), 35.2 (C-10), 31.7 (C-2ꢀ+ꢀC-22), 30.9 (C-23), 30.3
Jꢀ=ꢀ7.8 Hz, 3′-Harom.ꢀ+ꢀ5′-Harom.), 5.66 (m, 1H, OH), 3.99, 4.00 (NCH2CH2CH2CH3), 28.4 (C-11), 27.2 (C-16), 26.0 (C-9), 22.7
(d, 1H, Jꢀ=ꢀ3.2 Hz, OH), 3.78 (m, 1H, 12-H), 3.61 (m, 1H, 7-H), (C-15), 22.5 (Me-19), 22.0 (Me-Ar), 20.7 (NCH2CH2CH2CH3),
3.49 (m, 2H, 2ꢀ×ꢀCHMe2), 3.18 (m, 1H, 3-H), 2.39 (s, 3H, 17.0 (Me-21), 13.6 (N(CH2)3Me), 12.2 (Me-18). – C39H63NO6S
Me-Ar), 2.23 (m, 1H, 4a-H), 2.14 (m, 1H, 9-H), 2.12 (m, 1H, (673.99): calcd. C 69.50, H 9.42, N 2.08, found C 69.28, H
23a-H), 2.04 (m, 1H, 23b-H), 1.96 (m, 1H, 14-H), 1.78 (m, 9.30, N 1.82.
1H, 6a-H), 1.73 (m, 1H, 17-H), 1.70 (m, 1H, 16a-H), 1.67 (m,
2H, 1a-Hꢀ+ꢀ22a-H), 1.63 (m, 2H, 2a-Hꢀ+ꢀ15a-H), 1.46 (m, 1H,
4b-H), 1.43 (m, 1H, 11a-H), 1.41 (m, 2H, 6b-Hꢀ+ꢀ11b-H), 1.38
4.5.3 N-carbamothioyl-7α,12α-dihydroxy-3α-tosyloxy-
(m, 2H, 8-Hꢀ+ꢀ11b-H), 1.36 (m, 1H, 20-H), 1.33 (m, 1H, 2b-H),
5β-cholanamide (12)
1.28 (m, 1H, 5-H), 1.24 (m, 12H, 2ꢀꢀ×ꢀꢀCHMe2), 1.22 (m, 1H,
22b-H), 1.20 (m, 1H, 16b-H), 0.93 (d, 3H, JMe21,H20ꢀ=ꢀ6.1 Hz,
From thiourea (38 mg). Yield: 220 mg (71%) as yellow
21-Me), 0.91 (m, 1H, H-15b), 0.83 (m, 1H, 1b-Hb), 0.80 (s,
solid, m.p.: 137–139°C; Rfꢀ=ꢀ0.23. – IR (KBr): νꢀ=ꢀ3420 (OH),
3H, 19-Me), 0.57 (s, 3H, 18-Me). – 13C NMR ([D6]DMSO):
2931, 2854 (CH2), 1728 (C=O)amid, 1629 (C=C)arom., 1321, 1342
δꢀ=ꢀ171.5 (C=O), 148.0 (C-1′), 138.7 (C-4′), 130.2 (C-2′ꢀ+ꢀC-6′),
(S=O) cm−1. 1H NMR ([D6]DMSO): δꢀ=ꢀ9.66 (bs, 2H, NH2), 8.48
128.3 (C-3′ꢀ+ꢀC-5′), 71.8 (C-12), 70.3 (C-3), 66.1 (C-7), 53.8
(s, 1H, NH), 7.64 (d, 2H, Jꢀ=ꢀ8.4 Hz, 2′-Harom.ꢀ+ꢀ6′-Harom.), 7.47
(CHMe2), 47.5 (C-17), 45.6 (C-13), 45.6 (C-5), 41.4 (C-14), 40.5
(d, 2H, Jꢀ=ꢀ8.4 Hz, 3′-Harom.ꢀ+ꢀ5′-Harom.), 5.70 (bs, 1H, OH), 4.03
(C-8), 40.2 (C-4), 36.0 (C-20), 35.7 (C-1), 35.5 (C-6), 35.2
(bs, 1H, OH), 3.78 (m, 1H, 12-H), 3.61 (m, 1H, 7-H), 3.18 (m,
(C-10), 31.7 (C-2ꢀ+ꢀC-22), 30.9 (C-23), 28.4 (C-11), 27.2 (C-16),
1H, 3-H), 2.39 (s, 3H, Me-Ar), 2.22 (m, 1H, 4a-H), 2.14 (m,
26.1 (C-9), 24.5 (2ꢀꢀ×ꢀꢀCHMe2), 22.7 (C-15), 22.5 (Me-19), 21.9
1H, 9-H), 2.12 (m, 1H, 23a-H), 2.04 (m, 1H, 23b-H), 1.98
(Me-Ar), 17.0 (Me-21), 12.2 (Me-18). – C37H59NO6S (645.94):
(m, 1H, 14-H), 1.82 (m, 1H, 6a-H), 1.77 (m, 1H, 17-H), 1.71
calcd. C 68.80, H 9.21, N 2.17; found C 68.58, H 9.10, N 1.92.
(m, 1H, 16a-H), 1.65 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.63 (m, 2H,
2a-Hꢀ+ꢀ15a-H), 1.47 (m, 1H, 4b-H), 1.43 (m, 1H, 11a-H), 1.37
(m, 1H, 6b-H), 1.35 (m, 1H, 11b-H), 1.33 (m, 1H, 8-H) 1.30
4.5.2 (N,N-dibutyl)-7α,12α-dihydroxy-3α-tosyloxy-5β-
(m, 1H, 20-H), 1.27 (m, 1H, 2b-H), 1.24 (m, 1H, 5-H), 1.21 (m,
cholan-24-amide (11)
1H, 22b-H), 1.17 (m, 1H, 16b-H), 0.93 (d, 3H, JMe21,H20ꢀ=ꢀ6.6 Hz,
21-Me), 0.92 (m, 1H, 15b-H), 0.83 (m, 1H, 1b-H), 0.80 (s,
From N,N-dibutylamine (65 mg). Yield: 246 mg (73%) as
3H, 19-Me), 0.58 (s, 3H, 18-Me). – 13C NMR ([D6]DMSO):
light brown solid, m.p.: 133–132°C, Rfꢀ=ꢀ0.41. – IR (KBr):
δꢀ=ꢀ180.0 (C=S), 172.4 (C=O), 146.9 (C-1′), 138.6 (C-4′), 129.4
νꢀ=ꢀ3407 (OH), 2931, 2854 (CH2),, 1699 (C=O)amid, 1649
(C-2′ꢀ+ꢀC-6′), 128.1 (C-3′ꢀ+ꢀC-5′), 72.0 (C-12), 70.4 (C-3), 66.2
1
(C=C)arom., 1314, 1338 (S=O) cm−1. – H NMR ([D6]DMSO):
(C-7), 46.0 (C-17), 45.7 (C-13), 41.7 (C-5), 41.4 (C-14), 40.5
δꢀ=ꢀ7.59 (d, 2H, Jꢀ=ꢀ7.9 Hz, 2′-Harom.ꢀ+ꢀ6′-Harom.), 7.48 (d, 2H,
(C-8), 40.0 (C-4), 36.2 (C-20), 35.8 (C-1), 35.5 (C-6), 35.2
Jꢀ=ꢀ7.9 Hz, 3′-Harom.ꢀ+ꢀ5′-Harom.), 5.54 (m, 1H, OH), 4.07 (m,
(C-10), 31.4 (C-22), 31.0 (C-2), 30.3 (C-23), 28.5 (C-11), 27.2
1H, OH), 3.78 (m, 1H, 12-H), 3.61 (m, 1H, 7-H), 3.34 (m,
(C-16), 26.1 (C-9), 23.7 (C-15), 22.5 (Me-19), 21.9 (Me-Ar), 18.5
4H, 2ꢀꢀ×ꢀꢀNCH2CH2CH2Me), 3.20 (m, 1H, 3-H), 2.39 (s, 3H,
(Me-21), 12.3 (Me-18). – C32H48N2O6S (620.86): calcd. C 61.91,
Me-Ar), 2.22 (m, 1H, 4a-H), 2.15 (m, 1H, 9-H), 2.13 (m, 1H,
H 7.79, N 4.51; found: C 61.70, H 7.67, N 4.29.
23a-H), 2.05 (m, 1H, 23b-H), 1.94 (m, 1H, 14-H), 1.80 (m,
1H, 6a-H), 1.78 (m, 1H, 17-H), 1.75 (m, 1H, 16a-H), 1.67 (m,
2H, 1a-Hꢀ+ꢀ22a-H), 1.64 (m, 2H, 2a-Hꢀ+ꢀ15a-H), 1.53 (m,
4H, 2ꢀꢀ×ꢀꢀNCH2CH2CH2Me), 1.46 (m, 1H, 4a-H), 1.44 (m, 1H, 4.6 Methyl cholate (13)
11a-H), 1.38 (m, 1H, 6b-H), 1.36 (m, 1H, 11b-H), 1.33 (m, 1H,
8-H) 1.30 (m, 4H, 2ꢀꢀ×ꢀꢀN(CH2)2CH2Me), 1.29 (m, 1H, 20-H), This analog was prepared by typical procedure described
1.28 (m, 1H, 2b-H), 1.24 (m, 1H, 5-H), 1.22 (m, 1H, 22b-H), Shen et al. [38] (m.p.: 155–156°C).
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ꢂ219
2a-Hꢀ+ꢀ15a-H), 1.47 (m, 1H, 4a-H), 1.43 (m, 1H, 11a-H), 1.38
(m, 2H, 6b-H), 1.35 (m, 1H, 11b-H), 1.33 (m, 1H, 8-H), 1.31
(m, 1H, 20-H), 1.28 (m, 1H, 2b-H), 1.25 (m, 1H, 5-H), 1.23 (m,
1H, 22b-H), 1.18 (m, 1H, 16b-H), 0.92 (d, 3H, JMe21,H20ꢀ=ꢀ6.4 Hz,
4.7 General procedure for synthesis of
carbohydrazide analogs of cholic acid
(14 and 15)
A mixture of methyl cholate (9) (211 mg, 0.50 mmol) and 21-Me), 0.91 (m, 1H, 15b-H), 0.84 (m, 1H, 1b-H), 0.80 (s,
13
hydrazide (0.50 mmol) in abs. EtOH (15 mL) was heated 3H, 19-Me). – C NMR ([D6]DMSO): δꢀ=ꢀ183.8 (C=S), 173.7
under reflux for 12 h and completion of reaction was moni- (CONH), 71.8 (C-12), 70.3 (C-3), 66.2 (C-7), 47.5 (C-17), 45.9
tored by TLC. The precipitate formed was filtered off, dried (C-13), 45.2 (C-5), 41.4 (C-14), 40.1 (C-4ꢀ+ꢀC-8), 36.0 (C-20),
and recrystallized from EtOH to give the desired product.
35.8 (C-1), 35.2 (C-6), 34.9 (C-10), 32.7 (C-2ꢀ+ꢀC-22), 30.6
(C-23), 28.4 (C-11), 27.2 (C-16), 26.1 (C-9), 22.7 (C-15ꢀ+ꢀMe-19),
17.0 (Me-21), 12.2 (Me-18). – C25H43N3O4S (481.70): calcd. C
62.34, H 9.00, N 8.72; found C 62.05, H 8.78, N 8.50.
4.7.1 N′-(2,4-dinitrophenyl)-3α,7α,12α-trihydroxy-5β-
cholan-24-carbohydrazide (14)
From 2,4-dinitrophenylhydrazide (100 mg). Yield: 215 mg 4.8 5-Amino-2-(3α,7α,12α-trihydroxy-5β-
(73%) as orange solid, m.p.: 165–167°C, Rfꢀ=ꢀ0.33. – IR
cholyl-23-yl)-1,3,4-thiadiazole (16)
(KBr): νꢀ=ꢀ3412 (OH), 2931, 2854 (CH2), 1726 (C=O)amid, 1641
1
(C=C)arom., 1301 (NO2) cm−1. – H NMR ([D6]DMSO): δꢀ=ꢀ8.60 To a stirred mixture of cholic acid 3 (409 mg, 1.0 mmol)
(s, 1H, 3′-Harom), 8.37 (d, 1H, Jꢀ=ꢀ7.9 Hz, 5′-Harom.), 7.64 (d, 1H, and thiosemicarbazide (91 mg, 1.0 mmol) in EtOH
Jꢀ=ꢀ7.9 Hz, 6′-Harom.), 4.02 (m, 1H, 12-H), 3.77 (m, 1H, 7-H), 3.17 (25 mL) was added conc. H2SO4 (1.0 mL) dropwise and
(m, 1H, 3-H), 2.25 (m, 1H, 4a-H), 2.15 (m, 1H, 9-H), 2.10 (m, heated under reflux for 5 h and the reaction progress
1H, 23a-H), 2.02 (m, 1H, 23b-H), 1.97 (m, 1H, 14-H), 1.79 (m, was monitored by TLC. The mixture was poured into a
1H, 6a-H), 1.76 (m, 1H, 17-H), 1.70 (m, 1H, 16a-H), 1.65 (m, 2H, crushed ice and the solid was filtered, washed with cold
1a-Hꢀ+ꢀ22a-H), 1.62 (m, 2H, 2a-Hꢀ+ꢀ15a-H), 1.48 (m, 1H, 4b-H), water and recrystallized from EtOH to give 12 as brown
1.42 (m, 1H, H-11a), 1.39 (m, 1H, 6b-H), 1.36 (m, 1H, 11b-H), solid (385 mg, 83%), m.p.: 210–212°C, Rfꢀ=ꢀ0.36. – IR
1.34 (m, 1H, 8-H) 1.31 (m, 1H, 20-H), 1.29 (m, 1H, 2b-H), 1.25 (KBr): νꢀ=ꢀ3413 (OH), 2931, 2866 (CH2), 1594 (C=N), 690
1
(m, 1H, 5-H), 1.21 (m, 1H, 22b-H), 1.16 (m, 1H, 16b-H), 0.92 (C-S-C) cm−1. – H NMR ([D6]DMSO): δꢀ=ꢀ9.01 (s, 1H, NH2),
(d, 3H, JMe21,H20ꢀ=ꢀ6.4 Hz, 21-Me), 0.90 (m, 1H, 15b-H), 0.82 (m, 5.55 (bs, 1H, OH), 4.00 (bs, 1H, OH), 3.75 (m, 1H, 12-H),
1H, 1b-H), 0.79 (s, 3H, 19-Me), 0.57 (s, 3H, 18-Me). – 13C NMR 3.62 (m, 1H, 7-H), 3.45 (bs, 1H, OH), 3.16 (m, 1H, 3-H), 3.00
([D6]DMSO): δꢀ=ꢀ173.7 (CONH), 153.4 (Carom.-1′), 140.0 (C4′- (m, 2H, 23a,b-H), 2.23 (m, 1H, 4a-H), 2.16 (m, 1H, 9-H),
NO2), 135.4 (C2′-NO2), 128.7 (Carom.-5′), 121.9 (Carom.-3′), 116.6 1.90 (m, 1H, 14-H), 1.80 (m, 1H, 6a-H), 1.73 (m, 1H, 16a-H),
(Carom.-6′), 71.5 (C-12), 70.4 (C-3), 66.2 (C-7), 47.5 (C-17), 45.7 1.71 (m, 1H, 17-H), 1.68 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.63 (m, 2H,
(C-13), 41.5 (C-5), 41.3 (C-14), 40.4 (C-4ꢀ+ꢀC-8), 38.1 (C-20), 2a-Hꢀ+ꢀ15a-H), 1.46 (m, 1H, 4b-H), 1.44 (m, 1H, 11a-H), 1.35
36.1 (C-1), 34.3 (C-6), 33.3 (C-22), 32.7 (C-23) 30.4 (C-2), 28.4 (m, 3H, H-6bꢀ+ꢀH-8ꢀ+ꢀH-11b), 1.31 (m, 1H, 20-H), 1.28 (m,
(C-11ꢀ+ꢀC-16), 27.2 (C-9), 23.7 (C-15), 22.5 (Me-19), 18.5 (Me-21), 1H, 2b-H), 1.25 (m, 1H, 5-H), 1.23 (m, 1H, 22b-H), 1.17 (m,
12.2 (Me-18). – C30H44N4O8 (588.70): calcd. C 61.21, H 7.53, N 1H, 16b-H), 1.11 (m, 1H, 17-H), 0.96 (d, 3H, JMe21,H20ꢀ=ꢀ6.2 Hz,
9.52; found C 60.90, H 7.41, N 9.32.
21-Me), 0.94 (m, 1H, 15b-H), 0.84 (m, 1H, 1b-H), 0.80 (s,
3H, 19-Me), 0.56 (s, 3H, 18-Me). – 13C NMR ([D6]DMSO):
δꢀ=ꢀ173.3 (Cthiadiazole-2′), 170.2 (Cthiadiazole-5′), 71.7 (C-12), 70.4
(C-7), 66.2 (C-3), 47.4 (C-17), 45.3 (C-13), 41.9 (C-5), 40.6
(C-8ꢀ+ꢀC-4), 38.7 (C-20), 35.7 (C-1), 35.4 (C-6), 35.1 (C-10),
31.7 (C-2ꢀ+ꢀC-22), 29.0 (C-11), 27.8 (C-16), 26.7 (C-23), 26.0
(C-9), 22.9 (C-15), 22.7 (Me-19), 19.0 (Me-21); 12.8 (Me-18). –
C25H41N3O3S (463.68): calcd. C 64.76, H 8.91, N 9.06; found
C 64.53, H 8.80, N 8.78.
4.7.2 2-(3α,7α,12α-Trihydroxy-5β-cholanoyl)-hydrazine-
1-carbothioamide (15)
From thiosemicarbazide (50 mg). Yield: 195 mg (81%)
as brown crystals, m.p.: 135–137°C, Rfꢀ=ꢀ0.46. – IR (KBr):
νꢀ=ꢀ3406 (OH), 2931, 2854 (CH2), 1726 (Cꢀ=ꢀO)amid, 1641
1
(C=C)arom. cm−1. – H NMR ([D6]DMSO): δꢀ=ꢀ5.01 (br s., 1H,
OH), 4.30 (bs, 1H, Jꢀ=ꢀ7.8 Hz, OH), 4.09 (m, 1H, 12-H), 3.77
(m, 1H, 7-H), 3.17 (m, 1H, 3-H), 2.19 (m, 1H, 4-H), 2.15 (m, 4.9 Cholylhydrazide (17)
1H, 9-H), 2.10 (m, 2H, 23a-H), 2.03 (m, 1H, 23b-H), 1.97
(m, 1H, 14-H), 1.80 (m, 1H, 6a-H), 1.76 (m, 1H, 17-H), 1.72 This compound was prepared by typical procedure
(m, 1H, 16a-H), 1.67 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.65 (m, 2H, described by Al-Qawasmeh et al. [34] from methyl cholate
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13 and hydrazine hydrate, and characterized by melting
point comparison with literature value (m.p.: 160–162°C)
4.10.2 N′-[(1E)-4-Cholorobenzylidene]-3α,7α,12α-
trihydroxy-5β-cholan-24-ohydrazide (19)
From 4-chlorobenzaldehyde (77 mg). Yield: 234 mg (86%)
as white solid, m.p.: 259–261°C (Lit. [36] 260–261°C),
Rfꢀ=ꢀ0.50. – IR (KBr): νꢀ=ꢀ3040 (OH), 2931, 2862 (CH2), 1694
4.10 General procedure for the synthesis
of N′-[(1E)-4-substituted-benzylidene
or ethylidene]-3α,7α,12α-trihydroxy-
5β-cholan-24-ohydrazide derivatives
(18–22)
1
(C=O)amid, 1650 (C=N), 1604 (C=C)arom. cm−1. – H NMR ([D6]
DMSO): δꢀ=ꢀ9.61 (s, 1H, NH), 8.42 (s, 1H, N=CH), 7.94 (d,
2H, Jꢀ=ꢀ7.5 Hz, 2′-Harom.ꢀ+ꢀ6′-Harom.), 7.56 (d, 2H, Jꢀ=ꢀ7.5 Hz,
3′-Harom.ꢀ+ꢀ5′-Harom.), 6.21 (bs, 1H, OH), 4.09 (bs, 1H, OH),
3.85 (m, 1H, 12-H), 3.61 (m, 1H, 7-H), 3.18 (m, 1H, 3-H), 2.22
(m, 1H, 4a-H), 2.14 (m, 1H, 9-H), 2.12 (m, 1H, 23a-H), 2.03 (m,
1H, 23b-H), 1.98 (m, 1H, 14-H), 1.83 (m, 1H, 6a-H), 1.79 (m,
1H, 17-H), 1.72 (m, 1H, 16a-H), 1.65 (m, 2H, 1a-Hꢀ+ꢀ22a-H),
1.62 (m, 2H, 2-Hꢀ+ꢀ15a-H), 1.46 (m, 1H, 4b-H), 1.44 (m, 1H,
11a-H), 1.37 (m, 1H, 6b-H), 1.35 (m, 1H, 11b-H), 1.33 (m,
1H, 8-H), 1.30 (m, 1H, 20-H), 1.29 (m, 1H, 2b-H), 1.24 (m, 1H,
5-H), 1.22 (m, 1H, 22b-H), 1.16 (m, 1H, 16b-H), 0.93 (d, 3H,
To a solution of 17 (211 mg, 0.50 mmol) in abs. EtOH
(15 mL) and glacial acetic acid (1 mL) was added the cor-
responding aromatic aldehyde or ketone (0.55 mmol) and
the mixture was heated under reflux for 10–12 h and the
reaction progress was monitored by TLC. After cooling,
the mixture poured into cold water and left standing for
10 h in order to complete precipitation. The solid product
was collected by filtration, washed by petroleum ether
and recrystallized from EtOH.
J
Me21,H20ꢀ=ꢀ6.4 Hz, 21-Me), 0.92 (m, 1H, 15b-H), 0.83 (m, 1H,
13
1b-H), 0.80 (s, 3H, 19-Me), 0.58 (s, 3H, 18-Me). – C NMR
([D6]DMSO): δꢀ=ꢀ167.8 (CONH), 144.5 (N=CH), 137.7 (C4′-Cl),
131.8 (C-1′), 130.8 (Carom.-2′ꢀ+ꢀCarom.-6′), 128.7 (Carom.-3′ꢀ+ꢀCarom.
-
4.10.1 N′-[(1E)-4-Dimethylaminobenzylidene]-3α,7α,12α- 5′), 71.9 (C-12), 70.3 (C-3), 66.1 (C-7), 46.3 (C-17), 45.6 (C-13),
trihydroxy-5β-cholan-24-ohydrazide (18)
41.4 (C-5ꢀ+ꢀC-14), 40.4 (C-4ꢀ+ꢀC-8), 36.3 (C-20), 35.8 (C-1),
35.3 (C-6), 35.2 (C-10), 31.3 (C-22), 31.1 (C-2), 30.3 (C-23),
From 4-(dimethylamino)benzaldehyde (82 mg). Yield: 28.4 (C-11), 27.2 (C-16), 26.2 (C-9), 22.7 (C-15), 22.5 (Me-19),
249 mg (90%) as yellow solid, m.p.: 190–192°C; Rfꢀ=ꢀ0.53. 17.1 (Me-21), 12.2 (Me-18). – C31H45ClN2O4 (545.16): calcd. C
– IR (KBr): νꢀ=ꢀ3049 (OH), 2931, 2862 (CH2), 1698 (C=O)amid
,
68.30, H 8.32, N 5.14; found C 60.12, H 8.23, N 4.89.
1
1650 (C=N), 1604 (C=C)arom. cm−1. – H NMR ([D6]DMSO):
δꢀ=ꢀ8.08 (s, 1H, N=CH), 7.30 (d, 2H, Jꢀ=ꢀ7.9 Hz, 2′-Harom.ꢀ+ꢀ6′-
H
arom.’), 6.58 (d, 2H, Jꢀ=ꢀ7.9 Hz, 3′-Harom.ꢀ+ꢀ5 ′-Harom.), 5.64 (bs, 4.10.3 N′-[(1E)-4-hydroxybenzylidene]-3α,7α,12α-
1H, OH), 3.96 (bs, 1H, OH), 3.78 (m, 1H, 12-H), 3.57 (m, 1H,
H-7), 3.19 (m, 1H, 3-H), 2.94 (s, 6H, NMe2), 2.29 (m, 1H,
trihydroxy-5β-cholan-24-ohydrazide (20)
4a-H), 2.15 (m, 1H, 9-H), 2.12 (m, 1H, 23a-H), 2.06 (m, 1H, From 4-hydroxyaldehyde (67 mg). Yield: 200 mg (76%) as
23b-H), 1.97 (m, 1H, 14-H), 1.83 (m, 1H, 6a-H), 1.76 (m, light brown solid, m.p.: 206–208°C, Rfꢀ=ꢀ0.61. – IR (KBr):
1H, 17-H), 1.72 (m, 1H, 16a-H), 1.63 (m, 2H, 1a-Hꢀ+ꢀ22a-H), νꢀ=ꢀ3046 (OH), 2935, 2868 (CH2), 1696 (C=O)amid, 1658
1
1.65 (m, 2H, 2a-Hꢀ+ꢀ15a-H), 1.54 (m, 1H, 4b-H), 1.52 (m, (C=N), 1558 (C=C)arom. cm−1. – H NMR ([D6]DMSO): δꢀ=ꢀ9.63
1H, 11a-H), 1.39 (m, 1H, 6b-H), 1.37 (m, 2H, 6a-Hꢀ+ꢀ11b- (bs, 1H, NH), 8.53 (s, 1H, N=CH), 7.67 (d, 2H, Jꢀ=ꢀ8.4 Hz, 2′-
H), 1.35 (m, 1H, 8-H), 1.30 (m, 1H, 20-H), 1.29 (m, 1H,
Harom.ꢀ+ꢀ6′-Harom.), 6.87 (d, 2H, Jꢀ=ꢀ8.4 Hz, 3′-Harom.ꢀ+ꢀ5′-Harom.),
2b-H), 1.22 (m, 1H, 5-H), 1.18 (m, 1H, 22b-H), 0.92 (d, 3H, 4.00 (bs, 1H, OH), 3.78 (m, 1H, 12-H), 3.61 (m, 1H, 7-H),
J
Me21,H20ꢀ=ꢀ6.5 Hz, 21-Me), 0.91 (m, 1H, 15b-H), 0.83 (m, 1H, 3.43 (s, 1H, Ar-OH), 3.19 (m, 1H, 3-H), 2.21 (m, 1H, 4a-H),
1b-H), 0.80 (s, 3H, 19-Me), 0.58 (s, 3H, 18-Me). – 13C NMR 2.15 (m, 1H, 9-H), 2.13 (m, 1H, 23a-H), 2.01 (m, 1H, 23b-H),
([D6]DMSO): δꢀ=ꢀ171.2 (CONH), 156.5 (C-4′), 149.2 (N=CH), 1.91 (m, 1H, 14-H), 1.83 (m, 1H, 6a-H), 1.78 (m, 1H, 17-H),
127.9 (Carom.-2′ꢀ+ꢀCarom.-6′), 122.2 (Carom.-1′), 106.6 (Carom.
-
1.71 (m, 1H, 16a-H), 1.65 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.63 (m, 2H,
3′ꢀ+ꢀCarom.-5′), 71.4 (C-12), 70.8 (C-3), 66.7 (C-7), 46.4 (C-17), 2a-Hꢀ+ꢀ15a-H) 1.44 (m, 1H, 4b-H), 1.42 (m, 1H, 11a-H), 1.38
45.4 (C-13), 42.0 (NMe2), 41.3 (C-5ꢀ+ꢀC-14), 40.3 (C-4ꢀ+ꢀC-8), (m, 1H, 6b-H), 1.35 (m, 1H, 11b-H), 1.33 (m, 1H, 8-H), 1.31
36.1 (C-20), 35.6 (C-1), 35.2 (C-6), 35.0 (C-10), 31.3 (C-22), (m, 1H, 20-H), 1.28 (m, 1H, 2b-H), 1.24 (m, 1H, 5-H), 1.22 (m,
31.1 (C-2), 30.3 (C-23), 28.4 (C-11), 27.2 (C-16), 26.2 (C-9), 22.7 1H, 22b-H), 1.17 (m, 1H, 16b-H), 0.94 (d, 3H, JMe21,H20ꢀ=ꢀ6.4 Hz,
(C-15), 22.5 (Me-19), 17.1 (Me-21), 12.2 (Me-18). – C33H51N3O4 21-Me), 0.93 (m, 1H, 15b-H), 0.83 (m, 1H, 1b-H), 0.80 (s,
(553.79): calcd. C 71.57, H 9.28, N 7.59; found C 71.29, H 3H, 19-Me), 0.58 (s, 3H, 18-Me). – 13C NMR ([D6]DMSO):
9.11, N 7.38.
δꢀ=ꢀ171.5 (CONH), 160.2 (C4′-OH), 142.8 (N=CH), 129.9
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(Carom.-2′ꢀ+ꢀCarom.-6′), 128.2 (C-1′), 115.7 (Carom.-3′ꢀ+ꢀCarom.-5′), (m, 1H, 23a-H), 2.05 (m, 1H, 23b-H), 1.98 (m, 1H, 14-H), 1.83
71.8 (C-12), 70.4 (C-3), 66.2 (C-7), 46.5 (C-17), 45.7 (C-13), (m, 1H, 6a-H), 1.75 (m, 1H, 17-H), 1.72 (m, 1H, 16a-H), 1.66
41.5 (C-5ꢀ+ꢀC-14), 40.3 (C-4ꢀ+ꢀC-8), 36.2 (C-20), 35.9 (C-1), 35.3 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 1.63 (m, 2H, 2a-Hꢀ+ꢀ15a-H), 1.47 (m,
(C-6), 35.1 (C-10), 31.4 (C-22), 31.2 (C-2), 30.3 (C-23), 28.5 1H, 4b-H), 1.43 (m, 1H, 11a-H), 1.38 (m, 1H, 6b-H), 1.36 (m,
(C-11), 27.2 (C-16), 26.1 (C-9), 23.1 (C-15), 22.7 (Me-19), 18.5 1H, 11b-H), 1.33 (m, 1H, 8-H), 1.31 (m, 1H, 20-H), 1.28 (m,
(Me-21), 12.3 (Me-18). – C31H46N2O5 (526.72): calcd. C 70.69, 1H, 2b-H), 1.25 (m, 1H, 5-H), 1.23 (m, 1H, 22b-H), 1.18 (m,
H 8.80, N 5.32; found C 70.38, H 8.67, N 5.03.
1H, 16b-H), 0.94 (d, 3H, JMe21,H20ꢀ=ꢀ6.2 Hz, 21-Me), 0.92 (m,
1H, 15b-H), 0.84 (m, 1H, 1b-H), 0.81 (s, 3H, 19-Me), 0.60
(s, 3H, 18-Me). – 13C NMR ([D6]DMSO): δꢀ=ꢀ171.5 (CONH),
158.0 (C4′-NH2), 150.2 (N=CMe), 123.5 (Carom.-2′ꢀ+ꢀCarom.-6′),
127.5 (C-1′), 113.0 (Carom.-3′ꢀ+ꢀCarom.-5′), 71.5 (C-12), 70.3 (C-3),
66.2 (C-7), 47.5 (C-17), 45.7 (C-13), 41.7 (C-5), 41.6 (C-14), 40.8
4.10.4 N′-(1-(4-nitrophenyl)ethylidene)-3α,7α,12α-
trihydroxy-5β-cholanhydrazide (21)
From 4-nitroacetophenone (108 mg). Yield: 202 mg (71%) (C-4ꢀ+ꢀC-8), 36.2 (C-20), 35.9 (C-1), 35.2 (C-6), 34.8 (C-10),
as red solid, m.p.: 188–190°C, Rfꢀ=ꢀ0.71. – IR (KBr): νꢀ=ꢀ3051 31.9 (C-22), 31.4 (C-2), 30.9 (C-23), 28.5 (C-11), 27.3 (C-16), 26.2
(OH), 2933, 2866 (CH2), 1672 (C=O)amid, 1595 (C=N), 1558 (C-9), 23.0 (C-15), 22.7 (N=CMe), 22.5 (Me-19), 18.5 (Me-21),
1
(C=C)arom. cm−1. – H NMR ([D6]DMSO): δꢀ=ꢀ10.60 (bs, 1H, 13.9 (Me-18). – C32H49N3O4 (539.76): calcd. C 71.21, H 9.15, N
NH), 8.31 (d, 2H, Jꢀ=ꢀ8.8 Hz, 3′-Harom.ꢀ+ꢀ5′-Harom.), 8.17 (d, 2H, 7.79; found C 70.89, H 9.01, N 7.55.
Jꢀ=ꢀ8.8 Hz, 2′-Harom.ꢀ+ꢀ6′-Harom.), 6.61 (bs, 1H, OH), 5.62 (bs, 1H,
OH), 4.00 (bs, 1H, OH), 3.79 (m, 1H, 12-H), 3.60 (m, 1H, 7-H),
3.18 (m, 1H, 3-H), 2.32 (s, 3H, N=CMe), 2.23 (m, 1H, 4a-H), 4.11 Bioactivity assays
2.15 (m, 1H, 9-H), 2.13 (m, 1H, 23a-H), 2.07 (m, 1H, 14-H),
1.96 (m, 1H, 23b-H), 1.83 (m, 1H, 6a-H), 1.79 (m, 1H, 17-H), 4.11.1 Inhibition assay of human 17β-HSD1
1.69 (m, 1H, 16a-H), 1.65 (m, 2H, 1a-Hꢀ+ꢀ22a-H), 163 (m, 2H,
2a-Hꢀ+ꢀ15a-H), 1.46 (m, 1H, 4b-H), 1.44 (m, 1H, 11a-H), 1.38 Inhibitory activities were evaluated by an established
(m, 1H, 6b-H), 1.35 (m, 1H, 11b-H), 1.33 (m, 1H, 8-H), 1.30 method with minor modifications [51]. Briefly, the enzyme
(m, 1H, 20-H), 1.28 (m, 1H, 2b-H), 1.24 (m, 1H, 5-H), 1.21 (m, preparation was incubated with NADPH (500 μm) in the
1H, 22b-H), 1.18 (m, 1H, 16b-H), 0.94 (d, 3H, JMe21,H20ꢀ=ꢀ6.6 Hz, presence of potential inhibitors at 37°C in a phosphate
21-Me), 0.93 (m, 1H, 15b-H), 0.83 (m, 1H, 1b-H), 0.80 (s, 3H, buffer (50 mm) supplemented with 20% of glycerol and
19-Me), 0.59 (s, 3H, 18-Me). – 13C NMR ([D6]DMSO): δꢀ=ꢀ170.9 EDTA (1 mm). Synthesized compounds stock solutions
(CONH), 156.0 (C4′-NO2), 148.0 (N=CMe), 143.2 (C-1′), 127.8 were prepared in DMSO. The final concentration of DMSO
(Carom.-3′ꢀ+ꢀCarom.5′), 123.5 (Carom.-2′ꢀ+ꢀCarom.6′), 71.5 (C-12), 70.3 was adjusted to 1% in all samples. The enzymatic reaction
(C-3), 66.1 (C-7), 47.5 (C-17), 45.7 (C-13), 41.9 (C-5), 41.6 (C-14), was started by addition of a mixture of unlabeled- and
3
40.4 (C-8), 39.8 (C-4), 36.5 (C-20), 35.8 (C-1), 35.4 (C-6), 35.2 [2,4,6,7- H]-E1 (from Perkin Elmer, Boston and Quicksz-
(C-10), 31.7 (C-2ꢀ+ꢀC-22), 31.0 (C-23), 28.5 (C-11), 27.2 (C-16), int Flow 302 scintillator fluid was from Zinsser Analytic,
26.1 (C-9), 22.8 (C-15ꢀ+ꢀN=CMe), 22.5 (Me-19), 18.4 (Me-21), Frankfurt) (final concentration: 500 nm, 0.15 μCi). After
12.2 (Me-18). – C32H47N3O6 (569.74): calcd. C 67.46, H 8.32, N 10 min, the incubation was stopped with HgCl2 and the
7.38; found C 67.19, H 8.15, N 7.12.
mixture was extracted with diethylether. After evapora-
tion, the steroids were dissolved in acetonitrile. E1 and
E2 were separated using acetonitrile-water (45:55) as
mobile phase in a C18 reverse phase chromatography
column (Nucleodur C18 125/3 100-5, Macherey-Nagel)
connected to a HPLC-system (Agilent 1200 Series, Agilent
4.10.5 N′-(1-(4-aminophenyl)ethylidene)-3α,7α,12α-
trihydroxy-5β-cholanhydrazide (22)
From 4-aminoacetophenone (74 mg). Yield: 232 mg (86%) Technologies). Detection and quantification of the ster-
as dark red solid, m.p.: 198–200°C, Rfꢀ=ꢀ0.66. – IR (KBr): oids were performed using a radioflow detector (Ramona,
νꢀ=ꢀ3049 (OH), 2931, 2866 (CH2), 1896 (C=O)amid, 1631 (C=N), raytest). The conversion rate was calculated after analysis
1564 (C=C)arom. cm−1. – 1H NMR ([D6]DMSO): δꢀ=ꢀ10.04 (d, 2H, of the resulting chromatograms according to the following
Jꢀ=ꢀ8.6 Hz, NH2), 8.92 (s, 1H, NH), 7.62 (d, 2H, Jꢀ=ꢀ8.7 Hz, 2′- equation:
H
arom.ꢀ+ꢀ6′-Harom.), 6.58 (d, 2H, Jꢀ=ꢀ8.7 Hz, 3′-Harom.ꢀ+ꢀ5′-Harom.),
% conversion = %E2 /(%E2 + %E1)×100
5.37 (bs, 1H, OH), 4.00 (bs, 1H, OH), 3.80 (m, 1H, 12-H),
3.61 (m, 1H, 7-H), 3.44 (bs, 1H, OH), 3.19 (m, 1H, 3-H), 2.23
Each value was calculated from at least three inde-
(s, 3H, N=CMe), 2.21 (m, 1H, 4-H), 2.14 (m, 1H, 9-H), 2.11 pendent experiments in triplicate.
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222ꢀ ꢀN.A. Al-Masoudi et al.: New cholic acid analogs
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3
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