Synthesis, in-vitro antiprotozoal activity and molecular docking study of isothiocyanate derivatives

Article abstract of DOI:10.1016/j.bmc.2019.115185

Novel isothiocyanate derivatives were synthesized starting from noscapine, bile acids, amino acids, and some aromatic compounds. Antiparasitic activities of the synthesized derivatives were tested against four unicellular protozoa, i.e., Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium falciparum. Interestingly, seven isothiocyanate analogues displayed promising antiparasitic activity against Leishmania donovani with IC₅₀ values between 0.4 and 1.0 μM and selectivity index (SI) ranged from 7.8 to 18.4, comparable to the standard drug miltefosine (IC₅₀ = 0.7 μM). Compound 7h demonstrated the best antileishmanial activity with an IC₅₀ value of 0.4 μM. Seven products exhibited inhibition activity against T. brucei rhodesiense with IC₅₀s below 2.0 μM and SI between 2.7 and 29.3. Four primary amine derivatives of noscapine and five isothiocyanate derivatives exhibited antiplasmodial activity with IC₅₀s in the range of 1.1–2.7 μM and SI values between 1.1 and 14.5. The isothiocyanate derivative 7c showed against T. cruzi with an IC₅₀ value of 1.9 μM and SI 4. Molecular docking and ADMET studies were performed to investigate the interaction between active ligands and T. brucei trypanothione reductase active site. The docking studies showed significant binding affinity of noscapine derivatives to enzyme active site and good compatibility with experimental data.

Full text of DOI:10.1016/j.bmc.2019.115185

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Synthesis, in-vitro antiprotozoal activity and molecular docking study of iso-
thiocyanate derivatives
Kosar Babanezhad Harikandei, Peyman Salehi, Samad Nejad Ebrahimi,
Morteza Bararjanian, Marcel Kaiser, Ahmed Al-Harrasi
PII:
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Bioorganic & Medicinal Chemistry
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Please cite this article as: K. Babanezhad Harikandei, P. Salehi, S. Nejad Ebrahimi, M. Bararjanian, M. Kaiser, A.
Al-Harrasi, Synthesis, in-vitro antiprotozoal activity and molecular docking study of isothiocyanate derivatives,
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Synthesis, in-vitro antiprotozoal activity and molecular docking
study of isothiocyanate derivatives
Kosar Babanezhad Harikandei,a Peyman Salehi,a,* Samad Nejad Ebrahimi,a Morteza
Bararjanian,a Marcel Kaiser,b,c Ahmed Al-Harrasid
a Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid
Beheshti University, G. C., Evin, 1983963113 Tehran, Iran
b Swiss Tropical and Public Health Institute, Basel, Switzerland
c University of Basel, Basel, Switzerland
d National and Medical Sciences Research Center, University of Nizwa, P.O. Box 33, Birkat Al-
Mauz, Nizwa 611, Oman
*Corresponding author
Lead contact: Prof. Peyman Salehi, Tel.: +98 21 29904049, fax: +98 21 22431783, e-mail: p-
salehi@sbu.ac.ir. Department of Phytochemistry, Medicinal Plants and Drugs Research Institute,
Shahid Beheshti University, G. C., Evin, 1983963113 Tehran, Iran.
1

Abstract
Novel isothiocyanate derivatives were synthesized starting from noscapine, bile acids, amino
acids, and some aromatic compounds. Antiparasitic activities of the synthesized derivatives were
tested against four unicellular protozoa, i.e., Trypanosoma brucei rhodesiense, T. cruzi,
Leishmania donovani, and Plasmodium falciparum. Interestingly, seven isothiocyanate
analogues displayed promising antiparasitic activity against Leishmania donovani with IC50
values between 0.4 -1.0 µM and selectivity index (SI) ranged from 7.8 to 18.4, comparable to the
standard drug miltefosine (IC50 = 0.7 μM ). Compound 7h demonstrated the best antileishmanial
activity with an IC50 value of 0.4 µM. Seven products exhibited inhibition activity against T.
brucei rhodesiense with IC50s below 2.0 μM and SI between 2.7 - 29.3. Four primary amine
derivatives of noscapine and five isothiocyanate derivatives exhibited antiplasmodial activity
with IC50s in the range of 1.1 - 2.7 µM and SI values between 1.1 to 14.5. The isothiocyanate
derivative 7c showed against T. cruzi with an IC50 value of 1.9 µM and SI 4. Molecular docking
and ADMET studies were performed to investigate the interaction between active ligands and T.
brucei trypanothione reductase active site. The docking studies showed significant binding
affinity of noscapine derivatives to enzyme active site and good compatibility with experimental
data.
Keywords: Isothiocyanate, Noscapine, Bile acid, Antiprotozoal activity, Trypanosoma brucei,
Plasmodium falciparum, Molecular docking.
2

1.
Introduction
Malaria and several neglected diseases such as human African trypanosomiasis (HAT, sleeping
sickness), Chagas disease, and leishmaniasis which caused by protozoan parasites, threaten
millions of people health in the world especially in developing regions of Asia, Africa, and Latin
America.1 Malaria is the first responsible for the death caused by parasitic diseases of which over
219 million infections and 435,000 death were reported in 2017.2 Four species of Plasmodium
parasites are responsible for human malaria and transmitted by anopheline mosquitos. Plasmodium
falciparum is the most widespread and fatal among them that results in severe infections and death
3
in most cases. Malaria is endemic in the southeastern provinces of Iran, and according to the
World health organization (WHO) report, 828,672 people are at risk of malaria in these areas.2
Currently, artemisinin-based combination therapies (ACTs) are recommended treatment for
malaria. However, the efficacy of ACTs is being threatened by the emergence of parasite resistance
to them.2,4
Human African trypanosomiasis is caused by the two subspecies of parasitic protozoa of
Trypanosoma brucei (T. brucei gambiense and T. brucei rhodesiense) that spread by tsetse fly.5 T.
b. rhodesiense infection is endemic in East Africa and responsible for an acute infection whereas
T. b. gambiense occurs in western and central Africa and causes a chronic infection. HAT is a fatal
disease if it is not treated.6,7
Leishmaniasis, caused by Leishmania protozoan parasites, is the second widespread parasitic
disease.8 The disease occurs in three main forms, visceral leishmaniasis (also known as kala-azar
and the most serious form of the disease), cutaneous leishmaniasis (the most common), and
mucocutaneous leishmaniasis. There are 12 million cases infected with Leishmania species
worldwide according to the WHO report.9,10
T. cruzi strains are responsible for Chagas diseases or American trypanosomiasis in tropical and
subtropical countries in Latin America.11 This disease includes two phases: acute phase and
chronic phase that are usually asymptomatic or present non-specific symptoms. If Chagas disease
is not diagnosed and left untreated can lead to death.12
The available treatments have significant drawbacks and high costs and also, emerging
resistance of parasites result in decreasing the efficacy of these treatment strategies. All of these
reasons make the needs to develop new antiprotozoal agents urgent.13 Natural products and their
semi-synthetic derivatives are known as a primary source in drug development.14,15 In our effort
3

to find new antiprotozoal natural products, we isolated an isothiocyanate glycoside compound (4-
(α-L-rhamnosyloxy) benzyl isothiocyanate (1)) from Moringa peregrina that showed high activity
against Trypanosoma brucei rhodesiense with an IC50 of 0.1μM and selectivity index of 73. The
mechanism of action of compound 1 (Fig. 1) revealed that isothiocyanate 1 acts as an irreversible
inhibitor of trypanothione reductase (TR), a key enzyme of the unique trypanothione-based redox
metabolism of trypanosomatids. 1 was bound to TR by a covalent interaction between the
isothiocyanate moiety and a cysteine residue of TR active site.16 The chemical reactive
isothiocyanate moiety can be considered as an interesting pharmacophore in the irreversible
inhibition of TR enzyme. So, we decided to semi-synthesize some natural compound derivatives
containing isothiocyanate functional group.
O
O
N
O
H
H
H
C
R1
H
N
O
S
OH
O
OMe
O
HO
HO
OH
H 7
H
R2
O
MeO
HO
OMe
H
1
2
3a R1=H, R2=H, 7R (Lithocholic acid)
3b R1=OH, R2=OH, 7R (Cholic acid)
3c R1=OH, R2=H, 7R (Deoxycholic acid)
3d R1=H, R2=OH, 7R (Chenodeoxycholic acid)
3e R1=H, R2=OH, 7S (Ursodeoxycholic acid)
Fig. 1. Chemical structures of isothiocyanate 1, noscapine 2 and bile acids 3a-e.
Noscapine (2) is a natural phthalideisoquinoline alkaloid that has been used as an antitussive
drug since 1960. In the recent decade, this natural compound and it′s derivatives were considered
as potent anticancer agents. 17–20 Many reports revealed several alkaloids have antiparasitic activity
with IC50 values in the micromolar concentrations (Fig. 2).5,21 These reports have encouraged us
to choose noscapine as a starting material and synthesize some noscapine derivatives with an
isothiocyanate group and investigate this group effect on their antiprotozoal activity.
H
N
N
O
O
H
H
H
N
H
H
N
N
OMe
HN
OH
OH
N
NH
NH
MeO
OMe
P. falciparum
T. b. brucei
T. b. brucei
IC50 = 8.0µM
IC50 = 2.8µM
IC50 = 6.2µM
Fig. 2. Structures of some alkaloids with antiprotozoal activity.
4

Bile acids (3a-3e) are natural steroids that are produced in mammals by cholesterol
catabolism. Digestion and absorption of fat and regulation of cholesterol metabolism are the
most familiar role of these compounds in the body.22 Bile acids are of interest in drug
development because of their amphiphilic property and play a major role in drug transportation
and adsorption. Moderate to good antiparasitic activities were reported for bile acids and their
derivatives in several studies.23–26 So, we chose them as another parent class for the synthesis of
isothiocyanate compounds.
In the present work, isothiocyanate derivatives were designed and synthesized from noscapine
derivatives, bile acids, dehydroabietylamine, two amino acids, and some simple primary amines.
The in vitro antiprotozoal activities of the synthesized compounds were evaluated against T. b.
rhodesiense, T. cruzi, L. donovani, and P. falciparum. Also, the cytotoxicities of the products
against L6 cells (rat skeletal myoblasts) were determined, and their selectivity indices were
calculated. The probable interaction of synthetic derivatives with Trypanosoma brucei
trypanothione reductase (TbTR) was studied by molecular docking (Glide module, Schrodinger
suite), and their ADMET properties were calculated by Qikprop 4.4.
2.
Results and discussion
2.1.
Chemistry
In pursuing our aim for synthesizing isothiocyanate compounds, we designed and synthesized
a series of nineteen isothiocyanate derivatives including five noscapine derivatives, five bile acid
derivatives and nine derivatives from other amines. The critical functional group needed in all
starting materials was a primary amine.
Synthesis of noscapine isothiocyanate derivatives was performed in six steps (Scheme 1). At
the first step, α-amino nitrile derivatives of noscapine (4a-4e) were synthesized by a three-
component Strecker reaction. This reaction was performed in the acidic condition and room
temperature. Then, the nitrile groups of these compound (4a-4e) were converted to amide (5a-
5e) in the presence of hydrogen peroxide and potassium carbonate in methanol and dimethyl
sulfoxide. In order to reduce amide moiety to a primary amine, the amido noscapine analogues
(5a-5e) were treated with NaBH4 and BF3.Et2O in dry THF and the corresponding primary
amines (6a-6e) were provided in good yields.27 Finally, treatment of these compounds (6a-6e)
with CS2, followed by using di-tert-butyl dicarbonate (Boc2O) and 3% mol of DMAP as
catalysts yielded the corresponding isothiocyanate derivatives (7a-7e).28
5

O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
N
O
NH
NH
O
N
O
R
1. H2O2, CH3CN
NaBH4, BF3.Et2O
THF
RCHO
KCN ,CH3COOH
O
O
O
O
CN
2. FeSO4·7H2O, MeOH
O
O
O
O
O
O
O
O
O
O
O
4a 4e
-
2a
2b
2
O
O
O
O
O
H
H
N
H
N
N
R
R
R
O
.
1.CS2, Et3N, EtOH
NaBH4, BF3 Et2O
THF
H2O2, K2CO3
MeOH,DMSO
O
O
O
O
O
2. Boc2O, DMAP, EtOH
NH2
NCS
NH2
O
H
O
H
H
O
O
O
O
O
O
5a 5e
-
7a-7e
6a 6e
-
R:
N
F
4c
4d
4e
4b
4a
Scheme 1. Synthesis of isothiocyanate derivatives (7a-7e) from noscapine.
To convert bile acids to primary amines, in first steps the corresponding bile amides (5f-5j)
were synthesized by addition of ammonium chloride, O-(benzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium tetrafluoroborate (TBTU), and triethylamine to bile acids in acetonitrile.29,30
The amidation of bile acids was performed at room temperature under a mild condition and was
completed in 1-3 hr with good to high yields. According to the method described above to reduce
the carbonyl group of noscapine derivatives, related amines of bile acids (6f-6j) were produced
from amides 5f-5j with more than 75% yield. The isothiocyanate analogues (7f-7j) of resulting
amines 6f-6j were synthesized similar to noscapine derivatives 7a-7e in 1 hr and good yields
(Scheme 2).
6

O
O
H
H
H
R1
H
R1
H
R1
H
NH2
NH2
OH
NaBH4, BF3.Et2O
THF
NH4Cl, TBTU
Et3N, CH3CN
H
H
R2
H 7
H
R2
H 7
H
R2
HO
HO
5f-5j
HO
3a-3e
H
H
H
6f-6j
1.CS2, Et3N, EtOH
2.Boc2O, DMAP, EtOH
H
3a, 5f, 6f, 7f: R1=H, R2=H, 7R
R1
NCS
3b, 5g, 6g,7g: R1=OH, R2=OH, 7R
3c, 5h, 6h, 7h: R1=OH, R2=H, 7R
3d, 5i, 6i, 7i: R1=H, R2=OH, 7R
3e, 5j, 6j, 7j: R1=H, R2=OH, 7S
H
H
H
R2
HO
H
7f-7j
Scheme 2. Synthesis of isothiocyanate derivatives (7f-7j) from bile acids.
Nine isothiocyanate derivatives (7k-7s) were synthesized using commercially available primary
amines including dehydroabietylamine, tryptamine, L-tryptophan methyl ester, phenylalanine
methyl ester, benzylamine, 2-ethyl aniline, 4-fluoro aniline, 4-methyl aniline, and 3,4-dimethoxy
aniline by treatment with CS2 in excellent yields (Fig 3.).
NCS
CO2CH3
NCS
CO2CH3
NCS
N
N
H
H
H
SCN
7k
7l
7m
7n
NCS
NCS
NCS
F
NCS
NCS
O
O
7o
7p
7q
7r
7s
Fig. 3. Structure of the synthesized isothiocyanate derivatives from commercially available primary amines.
All novel synthetic compounds were characterized using HRMS, 1H NMR, and 13C NMR
spectra and known compounds were identified by comparison of their physical properties with
those of the literature.
2.2.
Biological assay
The synthesized compounds (5f-5j, 6a-6e, and 7a-7s) were tested for growth inhibition
against four parasites namely, Trypanosoma brucei rhodesiense strain STIB900, T. cruzi strain
7

Tulahuen C4, Leishmania donovani axenic amastigotes strain MHOM-ET-67/L82 and
Plasmodium falciparum strain NF54. The screening tests were performed at two concentrations
of 2 and 10 µg/mL. Melarsoprol, benznidazole, miltefosine, and artemisinin were employed as
standard drugs, respectively, for T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum
positive controls. The results are depicted in Fig. 4.
Fig. 4. Percentage of growth inhibition for protozoa parasites: a) T. b. rhodesiense, b) T. cruzi, c) L.
donovani, and d) P. falciparum.
As demonstrated, all the noscapine derivatives and most of the isothiocyanate analogues inhibited
the growth of T. b. rhodesiense with inhibitions greater than 50% at a concentration of 2 μg/mL. Bile
amide derivatives (5f-5j) showed lower growth inhibitions, but their corresponding isothiocyanates
8

(7f-7j) had excellent inhibition activities (Fig. 4a). At 2 μg/mL concentration, just two noscapine
analogues 6b and 7c were active against T. cruzi with 53 and 100% growth inhibition, respectively,
and isothiocyanate compounds did not show notable activity in this case (Fig. 4b). For L. donovani, all
of the bulky synthesized isothiocyanates (7a-7n) showed high percentages of growth inhibition
(>90%), while the small isothiocyanate analogues (7o-7s) and other derivatives did not show any
activity at both concentrations (Fig. 4c). Among all tested compounds, sixteen of them had growth
inhibition more than 50% at 2 μg/mL for P. falciparum (Fig. 4d).
Based on the growth inhibition assay, the most active compounds were selected for determination
of IC50 values. The in vitro potencies of the target derivatives against T. b. rhodesiense, T. cruzi, L.
donovani axenic amastigotes, and P. falciparum along with their cytotoxic activities to rat myoblast
L6 cells are summarized in Table 1. Selectivity indices (SIs) were calculated using the ratio of
cytotoxicity to antiprotozoal IC50 values, and are demonstrated for every four parasites. In continue,
we will discuss more their antiparasitic activities.
2.2.1. Antitrypanosomal activities
Fourteen out of 21 derivatives that investigated against T. b. rhodesiense exhibited IC50 values below
5μM, in which seven of them (6a, 6b, 6e,7b, 7g, 7h, and 7j) were more potent analogs with IC50 values
in the range of 1.0 – 1.7 μM. Two noscapine derivatives with amine group 6b (IC50 = 1.3 μM and SI =
29.3) and 6e (IC50 = 1.0 μM and SI = 24.4) showed the best antitrypanosomal activity and selectivity
indices. Hoewer, other compounds including 6a, 7b, 7g, 7h, and 7j had good IC50 values, but they had
selectivity indices between 0.5 – 8.8. As the results showed, among noscapine derivatives, the
corresponding primary amines (6a-6e) were the most active and selective against T. b. rhodesiense.
Comparison of compounds 6a-6e activities showed that insertion of the bulky group (6d) or
heterocyclic aromatic ring (6c) as R side chain lowered the activity, but fluoro or methyl substitution
on the phenyl side chain did not have a significant effect on antitrypanosomal activity. The primary
amine derivatives of noscapine (6a-6e) showed better activities and SIs than their corresponding
isothiocyanate derivatives against T. b. rhodesiense. So, the amine functional group has an essential
role in the antitrypanosomal activity of these noscapinoids.
9

Table 1
In vitro antiprotozoal, cytotoxic activities and selectivity index of selected derivatives.
T.b. rhodesiense
T. cruzi
IC50
(μM)
L. donovani
P. falciparum
L6 Cell
compound
IC50
IC50a(μM)
SIb
SI
SI
IC50 (μM)
SI
IC50 (μM)
(μM)
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
1.4±0.1
1.3±0.1
3.9±0.3
3.5±0.0
1.0±0.4
2.3±1.0
1.7±0.6
2.7±0.3
6.5±2.6
3.1±0.5
9.7±2.1
1.3±0.1
1.1±0.4
4.5±0.6
1.6±0.2
5.7±0.3
4.0±0.5
22.5±0.7
-
8.8
29.3
3.7
5.6
24.4
8.2
8.0
2.6
0.6
0.5
0.5
2.9
3.3
1.2
2.7
1.8
1.8
3.5
-
-
-
3.7
-
-
-
-
-
3.8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.0±0.2
2.7±0.0
-
6
14.5
-
12.1±0.7
38.8±27.3
14.4±2.5
19.6±9.3
23.3±11.3
18.7±17.0
13.6±12.3
7.1±2.8
3.9±2.2
1.5±0.1
4.5±0.5
3.9±0.6
3.6±0.2
5.4±0.1
4.3±0.3
10.0±0.1
7.1±2.8
78.1±0.8
13.9±5.4
286.9±64.1
325.1±6.4
24.2±7.3
>380
10.6±0.6
-
-
-
-
1.8±0.1
1.8±0.1
3.7±0.1
2.1±0.3
2.5±0.3
2.6±1.1
1.1±0.3
4.1±0.6
5.2±0.1
3.4±1.0
4.9±1.6
-
11.2
12.8
5.07
6.5
2.9
1.5
1.4
1.1
0.7
1.1
1.1
-
9.4
0.7
7.7
-
-
-
-
1.0±0.1
0.8±0.4
0.9±0.0
1.3±0.2
0.8±0.3
5.4±0.1
0.5±0.0
0.4±0.2
1.5±0.1
-
4.2±1.2
1.7±0.7
7.1±0.8
1.0±0.0
-
18.4
17.5
8
3.1
1.7
0.8
7.8
9.9
3.6
-
2.4
4.2
10.9
14.4
-
-
1.9±0.4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1.1±0.1
10.3±3.1
10.2±0.6
-
-
-
-
-
-
-
7m
7n
7q
7r
46.6±8.1
37.1±7.2
0.0075
-
6.2
8.8
3227
-
-
-
-
-
-
-
-
-
-
-
-
-
Melarsoprol
Benznidazole
Miltefosine
Chloroquine
Podophyllotoxin
-
-
-
2.41
>158
-
-
-
-
-
-
-
-
-
0.70
-
-
196
-
-
-
137±35
91±21
0.012h
0.012
-
7592
-
-
a IC50: 50% inhibitory concentration ±SD, b Selectivity Index: IC50 value of cytotoxicity/ IC50 value of the
antiprotozoal activity, c Not tested.
Among the tested isothiocyanate compounds (7a-7r) for T. b. rhodesiense, bile acids
derivatives (7g, 7h, and 7j) had the best IC50 values albeit they showed poor selectivity. Also,
bulky isothiocyanate products (7a-7k) exhibited better antitrypanosomal activity than smaller
ones (7m-7r).
Two synthesized noscapine analogues 6b and 7c showed good IC50 values (10.6 μM and 1.9
μM, respectively) against T. cruzi. Compound 6b was even more potent than standard drug
benznidazole but the activity is probably more due to the cytotoxic effect of the compound (SI =
3.8).
2.2.2. Antileishmanial activities
Most of the synthesized isothiocyanate compounds showed excellent antileishmanial activities
compared to miltefosine as a standard drug. Isothiocyanates 7a, 7b, 7c, 7e, 7g, 7h, and 7n
10

exhibited IC50 values below 1.0 μM with selectivity indices between 1.7-18.4. Compound 7h
(IC50 = 0.4 μM, SI=9.9) was two-fold more active than miltefosine and was the most potent of
them. Four derivatives 7a, 7b, 7n, and 7m had selectivity indices above 10. Compounds 7a and
7b (possessing noscapine scaffold) were the most selective among them with selectivity indices
of 18.4 and 17.5, respectively. Among all of the synthesized compounds, phenylalanine
isothiocyanate derivative (7h) exhibited antiprotozoal activity just for L. donovani (IC50 = 1.0
μM and SI = 14.4). The antileishmanial results demonstrated that the bulky isothiocyanate
derivatives (7a-7i) were more active than other ones. Also, these synthesized isothiocyanates
from noscapine and bile acids (7a-7i) showed inhibition activity similar to miltefosine, while
their parents were not active against L. donovani. These results underlined that the introduction
of the isothiocyanate group significantly increased the antileishmanial activities.
2.2.3. Antiplasmodial activities
Thirteen of the investigated compounds exhibited IC50 values below 5.0 μM against P.
falciparum. Two isothiocyanate derivatives 7e (IC50 = 1.1 μM and SI = 9.4) and 7k (IC50 = 1.1
μM and SI = 9.4) were the most potent but did not show good selectivities. 6b bearing noscapine
scaffold with an amine group was the most selective compound (IC50 = 2.7 μM and SI = 14.5).
Comparison between inhibition activities of noscapine derivatives displayed that primary amine
products (6a-6e) had better antiplasmodial activities and selectivities than their isothiocyanate
analogues(7a-7e).
2.3. Molecular docking study
Trypanothione reductase is an essential enzyme in trypanosomatids (T. brucei, T. cruzi, and
leishmaniasis) that have a unique role in trypanothione-based redox metabolism and oxidant
defense. Lack of this enzyme in mammalian host is one of the reasons that turned it to attractive
drug-target molecules.31,32 In our previous paper, we reported the irreversible inhibition of TR by
isothiocyanate compound 1 16. So, the interaction between the more active synthetic compounds
and T. brucei trypanothione reductase (TbTR) was investigated by molecular docking study. The
molecular docking study was carried out in the flavin adenine dinucleotide (FAD) binding site of
TbTR (PDB ID: 2WOW) 33 using Glide application in Maestro 10.2 platform (Schrödinger, LLC
). Docking scores are reported in Table 2.
All investigated compounds except small isothiocyanate derivatives (7k, 7l, 7m, 7n, 7q, and
7r) illustrated good affinity to FAD binding site of TbTR. Compound 7e showed the best affinity
11

to TbTR with docking score of -8.37 kcal/mol (Fig. 5a). Two groups of interactions that
stabilized the binding of 7e, consisted of: hydrophobic interaction between hydrophobe section
of 7e (isoquinoline and R side chain) and PHE367, ALA 365, VAL366, PHE198, MET333, ILE
285, and ALA284 and polar interaction of oxygenated groups with THR335, ASN179, SER178,
and THR51. The similarity of hydrophobic and polar pockets amino acid residues with FAD
binding site represented this compound placed in proper situate. Also, 7e had good inhibition
activity against L. donovani (IC50 = 0.8 μM) that this affinity could explain it.
Table 2
Docking scores (kcal/mol) of the synthesized and reference compounds at FAD
cavity for TbTR.
Docking score
(kcal/mol)
Docking score
(kcal/mol)
Compound
Compound
1
2
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
7f
-7.99
-7.49
-7.97
-7.6
12
13
14
15
16
17
18
19
20
21
22
7g
7h
7i
-7.33
-6.88
-5.77
-5.44
-4.98
-5.93
-5.55
-4.39
-3.28
-1.87
-13.816
3
4
7j
5
-7.4
7k
7l
6
-6.65
-6.13
-7.05
-7.06
-8.37
-5.70
7
7m
7n
7q
7r
8
9
10
11
FAD
All noscapine primary amine derivatives (6a-6e) were active against T. b. rhodesiense
(1.0<IC50<3.9 μM) illustrated good docking scores. These ligands revealed hydrogen bond, Pi-Pi
stacking, Pi-cation, polar and hydrophobe interactions with the active site of TbTR. For example,
compound 6b (IC50 = 1.3 μM and SI = 29.3 for T. b. rhodesiense), the most selective of them, in
complex with TbTR showed docking score value of -7.49 kcal/mol (Fig. 5b). There are three
essential types of interactions with enzyme active site including hydrogen bond, Pi-Pi stacking,
and Pi-cation interactions. The oxygen of the ketal group interacted with the backbone of
ALA365 by a hydrogen bond, and also, ALA365 had a significant role in the binding of FAD to
enzyme active site by a hydrogen bonding. The hydrogen bond interaction was established
12

between NH2 and the side chain of ASP327. LYS60 was responsible for Pi-cation interaction of
6b with the TbTR. The isoquinoline group involved in the Pi-Pi interaction with PHE98. The
amino acid residues containing SER162, THR51, ASN179, and SER178 created a polar pocket
that stabilized the ligand-enzyme complex. Moreover, hydrophobic interactions with CYS52,
LEU334, PRO336, ALA363, PHE367, ALA365, and VAL366 contributed to the binding of 6b
to the enzyme (Fig. 5b). These results showed the importance of amine groups in the binding of
ligands to the active site of the enzyme.
Fig. 5. The 2D and 3D docking the complex of ligands with the binding site of TbTR a) compound 7e, b)
compound 6b, c) compound 7c, and d) overlay of the noscapine derivatives and FAD.
13

The isothiocyanate derivative 7c that displayed the best activity against T. cruzi, placed in the
TbTR active site with docking score of -7.05 kcal/mol (Fig. 5c). Several amino acids participated
in hydrophobic interactions between 7c and TbTR such as CYS52, VAL55, GLY56, PHE182,
ILE199, PRO336, and VAL366. The hydrogen bond interactions of LYS60 (side chain) and
ALA365 (backbone) with the nitrogen atom of amine and isothiocyanate groups had a very
significant role in stabilizing the ligand-enzyme complex. Also, 7c exhibited polar interaction
with ARG287, SER162, and ASP 327.
As the results demonstrated, small isothiocyanate molecules did not show affinity to bind the
active site. Moreover, most of the isothiocyanate derivatives of bile acids had a low docking
score. It can be concluded that the size and conformation of ligands had a severe effect on the
stability of the ligand-enzyme complex. Noscapine moiety of the synthesized derivatives 6a-6e
and 7a-7e was placed precisely in a part of FAD binding site and avoided binding of FAD to the
enzyme (Fig. 5d). Several polar, aromatic, and hydrogen bond acceptor groups existed in this
scaffold that created a variety of interactions with the active site of TR. These reasons can be
explained the high activity of noscapine derivatives.
Evaluation of ADMET properties of active compounds is a useful tool for selection of drug
candidates at first steps. Some pharmacokinetic properties (QPPCaco, QplogBB, and
QPPMDCK) and Lipinski’s rule of five (molecular weight, H acceptor bonds, H donor bonds,
and octanol/water partition coefficient) were calculated through ADMET prediction by Qikprop
4.4. The calculated amounts of these parameters and their permissible ranges are listed in Table
3. All investigated products obeyed the Lipinski’s rule of five and did not have the more than
two violations of these criteria, which indicated their potential to become orally active drugs.
QPPCaco prediction showed good (for 6a-6e primary amine derivatives of noscapine) to
excellent (for isothiocyanate derivatives 7a-7k) values for Caco-2 cells permeability. Prediction
of brain/blood partition coefficient (QPlogBB) and the blood-brain barrier mimic MDCK cell
permeability (QPPMDCK) demonstrated satisfactory values in permissible ranges for all the
tested synthesized derivatives.
14

Table 3
Calculated theoretical parameters of Lipinski's rule for the active compounds.
QPPCacoe
(<25 poor,
>500 great)
193.2
QPPMDCKg
(<25 poor,
>500 great)
102.4
186.0
62.5
MWa
(<500 amu)
donorHBb accptHBc QPlogPo/wd Rule of Five
QplogBBf
(_3.0-1.2)
Compound
(<5)
(<10)
(-2 to 6.5)
(<4)
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
504.6
522.6
505.6
592.7
518.6
546.6
564.6
547.6
634.7
560.6
403.6
435.6
419.7
419.6
419.6
327.5
2
2
2
2
2
0
0
0
0
0
1
3
2
2
2
0
8.4
8.4
9.9
8.4
8.4
9.9
9.9
11.4
9.9
9.9
4.2
7.6
5.9
5.9
5.9
2
3.3
3.4
2.3
4.6
3.5
3.8
4
2.6
5.5
3.9
5.5
3.9
4.8
4.5
4.5
5.7
1
1
1
1
1
1
1
1
2
1
1
0
0
0
0
1
0.44
0.58
0.29
0.4
195.8
122.4
193.6
193.4
102.6
102.5
0.45
0.53
0.53
0.24
0.59
0.41
-0.37
-0.91
-0.61
-0.72
-0.7
1775.4
1409.8
888.5
2342.8
1408.2
1959.1
709.1
1242.3
973.8
996.1
2727.5
3412.2
1143.4
2828.5
1901.4
2323.8
1007.3
1855.8
1095.2
1116.9
10000
6867.7
0.37
a MW: molecular weight, b donorHB: hydrogen donor bonds, c accptHB: hydrogen acceptor bonds, d QPlogPo/w:
octanol/water partition coefficient, e QPPCaco: predicating Caco-2 cell permeability in nm s-1, f QplogBB:
predicating brain/blood coefficient, g QPPMDCK: predicating MDCK cell permeability in nm s-1.
3.
Conclusion
In summary, we designed and synthesized several isothiocyanate derivatives as promising
antiprotozoal agents. The investigation of antiprotozoal activity showed that the isothiocyanate
derivatives had excellent activities against L. donovani with IC50 values in the range of 0.4-1.0
μM, as compared to the standard drug miltefosine (IC50 = 0.7 μM). Also, these synthetic
compounds displayed good antitrypanosomal and antiplasmodial activity. Comparison between
antiparasitic activities of isothiocyanate and their parents revealed that the isothiocyanate is an
essential pharmacophore for their activity. The primary amine analogues of noscapine showed
better antitrypanosomal and antiplasmodial activity and selectivity than their isothiocyanate
compounds. The molecular docking study demonstrated the affinity of these compounds to the
active site of TR, and noscapine scaffold had a significant effect in the ligand-enzyme complex
stabilization. Thus, we should investigate the most active compound for antileishmanial in vivo
studies and designed more selective compounds with noscapine scaffold.
4.
Experimental
4.1.
General
15

Miltefosine, artemisinin, chloroquine, and podophyllotoxin were purchased from Sigma, and
benznidazole and melarsoprol were received from WHO (with purity >95% according to the
suppliers). Noscapine was purchased from Faran Shimi Pharmaceutical Co. Other chemicals and
solvents were provided from Sigma-Aldrich, Acros, Alfa Aesar, Merck, and Kimia Exir
chemical companies without further purifications. The reactions were monitored using thin-layer
chromatography (TLC) (pre-coated silica gel F254 sheets from Merck Co.) and were visualized by
UV light or phosphomolybdic acid. Purification of the products was performed by preparative
TLC plates that were coated with silica gel 60 PF254 containing gypsum. Uncorrected melting
points were measured on a Branstead/Electrothermal 9200 apparatus. 1HNMR and 13CNMR
spectra were obtained in CDCl3, CD3OD and DMSO-d6 as solvents on Bruker Avance III
spectrometers at 600, 500 and 300 MHz in 1H NMR and 151, 126 and 75 MHz in 13C NMR by
using TMS as an internal standard. Chemical shifts were recorded as δ by ppm. HRESIMS
spectra were obtained on a Bruker micro TOF ESIMS in positive mode.
4.2. Synthetic protocols
The procedures for the synthesis of compounds 4a-4e and 5a-5e were reported in supporting
information. The esterification of amino acids was performed according to reported protocol.34
4.2.1. General procedure for the synthesis of bile amides 5f-5j
To a solution of 1.0 mmol of bile acid (3a-3e) in acetonitrile (5 ml) was added 1.3 mmol
TBTU, 5.0 mmol NH4Cl, and 2.0 mmol triethylamine. The mixture was stirred in room
temperature. After 1-3 h, the reaction was completed, and the solvent was evaporated under
vacuum. 5 mL of water was added to the residue and precipitates was separated. The precipitates
were dissolved in dichloromethane, then washed with water and dried over anhydrous Na2SO4.
After solvent evaporation, the product was purified using preparative TLC.
4.2.1.1.
4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-Hydroxy-10,13-dimethylhexadecahydro-
1H-cyclopenta[a]phenanthren-17-yl)pentanamide(5f)
1
5f: Yield: 95%; white powder; m.p. 206 °C; H NMR (600 MHz, methanol-d4) δ 3.56 (tt, J =
10.7, 4.6 Hz, 1H), 2.27 (ddd, J = 15.0, 10.3, 5.1 Hz, 1H), 2.13 (ddd, J = 13.8, 9.9, 6.5 Hz, 1H),
2.04 (dd, J = 12.4, 3.3 Hz, 1H), 1.93 (ddt, J = 14.0, 9.7, 5.1 Hz, 2H), 1.74 – 1.87 (m, 3H), 1.63
(tt, J = 10.0, 5.2 Hz, 2H), 1.38 – 1.53 (m, 7H), 1.26 – 1.38 (m, 5H), 1.06 – 1.25 (m, 5H), 1.01 –
1.05 (m, 1H), 0.99 (d, J = 6.5 Hz, 3H), 0.97 (s, 3H), 0.71 (s, 3H) ppm; 13C NMR (151 MHz,
methanol-d4) δ 179.8, 72.4, 57.9, 57.5, 43.9, 43.6, 41.9, 41.6, 37.3, 37.2, 36.9, 36.5, 35.7, 33.5,
16

33.2, 31.2, 29.3, 28.4, 27.7, 25.3, 23.9, 21.9, 18.8, 12.5 ppm; HRMS (ESI): [M+Na]+
C24H41NNaO2 calcd 398.3035, found 398.3047.
4.2.1.2.
4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-Trihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (5g)
5g: Yield: 90%; white powder; m.p. 125 °C; 1H NMR (600 MHz, methanol-d4) δ 4.60 (brs,
1H), 3.98 (t, J = 3.1 Hz, 1H), 3.82 (dd, J = 3.1 Hz, 1H), 3.39 (tt, J = 11.0, 4.3 Hz, 1H), 2.23 –
2.35 (m, 3H), 2.10 – 2.20 (m, 1H), 1.72– 2.05 (m, 7H), 1.67 (dt, J = 13.4, 3.4 Hz, 1H), 1.51 –
1.65 (m, 5H), 1.29 – 1. 51 (m, 5H), 1.14 (qd, J = 11.9, 5.7 Hz, 1H), 1.05 (d, J = 6.5 Hz, 3H), 1.01
(td, J = 14.5, 3.7 Hz, 1H), 0.94 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (151 MHz, methanol-d4) δ
179.9, 74.1, 72.9, 69.1, 48.1 , 47.5, 43.2, 43.0, 41.0, 40.5, 36.9, 36.5, 35.9, 35.9, 33.5, 33.2, 31.2,
29.6, 28.7, 27.9, 24.2, 23.2, 17.7, 13.0 ppm; HRMS (ESI): [M+Na]+ C24H41NNaO4 calcd
430.2933, found 430.2978.
4.2.1.3.
Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (5h)
5h: Yield: 83%; white powder; m.p. 101 °C; 1H NMR (600 MHz, methanol-d4) δ 3.98 (t, J = 2.9
Hz, 1H), 3.55 (tt, J = 10.8, 4.6 Hz, 1H), 2.29 (ddd, J = 14.8, 10.3, 5.1 Hz, 1H), 2.14 (ddd, J =
13.9, 9.9, 6.5 Hz, 1H), 1.88 – 1.96 (m, 3H), 1.77 – 1.88 (m, 4H), 1.60 – 1.68 (m, 3H), 1.52 – 1.59
(m, 2H), 1.39 – 1.51 (m, 6H), 1.27 – 1.38 (m, 3H), 1.16 – 1.24 (m, 1H), 1.07 – 1.16 (m, 1H),
1.05 (d, J = 6.6 Hz, 3H), 1.03 – 0.97 (m, 1H), 0.95 (s, 3H), 0.73 (s, 3H).ppm; 13C NMR (151
MHz, methanol-d4) δ 179.9, 74.1, 72. 6, 49.3, 48.1, 47.6, 43.6, 37.5, 37.2, 36.9, 36.5, 35.3, 34.8,
33.5, 33.2, 31.1, 29.9, 28.6, 28.4, 27.5, 24.9, 23.7, 17.6, 13.2 ppm; HRMS (ESI): [M+Na]+
C24H41NNaO3 calcd 414.2984, found 414.3031.
4.2.1.4.
4-((3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-Dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (5i)
5i: Yield: 75%; off white powder; m.p. 114 °C; 1H NMR (600 MHz, methanol-d4) δ 3.82 (s,
1H), 3.39 (tt, J = 8.9, 3.3 Hz, 1H), 2.25 – 2.32 (m, 2H), 2.10 – 2.17 (m, 1H), 1.73 – 2.06 (m, 7H),
1.60 – 1.70 (m, 2H), 1.44 – 1.57 (m, 5H), 1.29 – 1.42 (m, 5H), 1.17 – 1.25 (m, 2H), 1.08 – 1.17
(m, 1H), 1.01 – 1.04 (m, 1H), 1.00 (d, J = 6.6 Hz, 3H), 0.95 (s, 3H), 0.72 (s, 3H) ppm; 13C NMR
(151 MHz, methanol-d4) δ 179.9, 72.9, 69.1, 57.3, 51.6, 43.7, 43.2, 41.1, 40.8, 40.5, 36.9, 36.6,
36.2, 35.9, 34.1, 33.5, 33.4, 31.4, 29.3, 24.6, 23.4, 21.8, 18.9, 12.2 ppm; HRMS (ESI): [M+Na]+
C24H41NNaO3 calcd 414.2984, found 414.3023.
17

4.2.1.5.
4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-Dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (5j)
5j: Yield: 75%; white powder; m.p. 191 °C; 1H NMR (600 MHz, methanol-d4) δ 3.45 – 3.56
(m, 2H), 2.18 – 2. 39 (m, 1H), 2.14 (ddd, J = 14.0, 10.0, 6.5 Hz, 1H), 2.06 (dd, J = 12.7, 3.3 Hz,
1H), 1.88 – 1. 97 (m, 2H), 1.79 – 1.89 (m, 3H), 1.55 – 1.67 (m, 4H), 1.43 – 1.54 (m, 6H), 1.19 –
1.40 (m, 6H), 1.12 (q, J = 9.3 Hz, 1H), 1.05 (td, J = 14.2, 3.2 Hz, 1H), 1.00 (d, J = 6.5 Hz, 3H),
0.99 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (151 MHz, methanol-d4) δ 179.9, 72.1, 71.9, 57.5,
56.5, 44.8, 44.5, 44.1, 41.6, 40.7, 38.6, 38.0, 36.9, 36.1, 35.2, 33.6, 33.3, 31.1, 29.7, 27.9, 23.9,
22.4, 19.0, 12.7 ppm; HRMS (ESI): [M+H]+ C24H42NO3 calcd 392.3165, found 392.3175.
4.2.2. General method for the synthesis of primary amines 6a-6j
1.0 mmol of amide derivatives (5) was dissolved in 19 mmol BF3.Et2O and then added
dropwise to a solution of 5.0 mmol NaBH4 in 20 mL dry THF at -5°C. After 1h, the reaction
mixture was warmed to room temperature and stirred overnight. To quench the reaction, 10 mL
of HCl (10%) was added dropwise in an ice bath. After stirring for 1h, 5ml of NaOH (2M) was
added and pH of reaction was set at 11. The reaction was extracted with CHCl3 (3×20) and the
organic layer was separated, washed with water and dried over anhydrous Na2SO4. The residue
was concentrated under reduced pressure and purified by preparative TLC.
4.2.2.1.
2-((5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-4-methoxy-
5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinolin-6-yl)-2-phenylethanamine (6a)
6a: Yield: 86%; orange powder; m.p. 105 °C; 1H NMR (600 MHz, methanol-d4) δ 7.24 (d, J =
7.5 Hz, 2H), 7.10 – 7.20 (m, 3H), 6.72 (d, J = 8.2 Hz, 1H), 5.99 (s, 1H), 5.89 (s, 1H), 5.87 (s,
1H), 5.79 (d, J = 8.2 Hz, 1H), 5.68 (t, J = 3.7 Hz, 1H), 5.33 (dd, J = 12.3, 2.8 Hz, 1H), 5.16 (d, J
= 12.3 Hz, 1H), 4.81 (d, J = 5.1 Hz, 1H), 4.06 (s, 3H), 4.03 (t, J = 5.7 Hz, 1H), 3.85 (s, 3H), 3.82
(m, 2H), 3.79 (s, 3H), 3.11 (dd, J = 13.2, 5.4 Hz, 1H), 2.85 (dd, J = 14.7, 7.0 Hz, 1H), 2.67 (td, J
= 14.6, 13.7, 5.7 Hz, 1H), 2.18 – 2.26 (m, 1H) ppm; 13C NMR (151 MHz, methanol-d4) δ 153.1,
149.7, 144.3, 140.9, 139.9, 135.1, 133.9, 133.0, 131.7, 129.6, 129.3, 129.0, 118.6, 118.3, 113.9,
103.6, 102.0, 86.6, 72.2, 67.7, 60.6, 60.0, 56.7, 54.3, 47.3, 41.6, 25.9 ppm; HRMS (ESI):
[M+H]+ C29H33N2O6 calcd 505.2339, found 505.2444.
4.2.2.2.
(2R)-2-((5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-4-methoxy-
5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinolin-6-yl)-2-(4-fluorophenyl)ethanamine (6b)
18

6b: Yield: 95%; off white powder; m.p. 112 °C; 1H NMR (600 MHz, methanol-d4) δ 7.24 (t, J
= 6.9 Hz, 2H), 6.95 (t, J = 8.4 Hz, 2H), 6.81 (d, J = 8.2 Hz, 1H), 6.21 – 6.24 (m, 2H), 5.87 (s,
1H), 5.84 (s, 1H), 5.44 (d, J = 4.2 Hz, 1H), 4.99 (s, 2H), 4.39 (d, J = 4.3 Hz, 1H), 3.83 (s, 3H),
3.79 (s, 3H), 3.73 (t, J = 6.8 Hz, 1H), 3.71 (s, 3H), 3.23 (dd, J = 13.0, 6.9 Hz, 1H), 2.93 – 3.02
(m, 2H), 2.90 (dd, J = 13.0, 6.9 Hz, 1H), 2.56 (ddd, J = 17.6, 10.8, 7.3 Hz, 1H), 2.23 (dd, J =
16.9, 5.0 Hz, 1H) ppm; 13C NMR (151 MHz, methanol-d4) δ 163.7 (d, J = 244.5 Hz), 152.8,
149.5, 144.2, 141.8, 138.3, 135.5, 135.1, 133.3, 131.8, 131.4, 131.3, 119.2, 118.7, 115.9, 115.7,
113.8, 103.7, 101.8, 87.9, 72.1, 68.6, 60.6, 59.6, 57.9, 56.9, 44.6, 44.2, 25.4 ppm; HRMS (ESI):
[M+H]+ C29H32FN2O6 calcd 523.2244, found 523.2356.
4.2.2.3.
(2R)-2-((5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-4-methoxy-
5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinolin-6-yl)-2-(pyridin-3-yl)ethanamine (6c)
6c: Yield: 67%; off white powder; m.p. 88 °C; 1H NMR (600 MHz, methanol-d4) δ 8.45 (d, J
= 2.2 Hz, 1H), 8.33 (d, J = 4.9 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.20 (dd, J = 7.9, 4.9 Hz, 1H),
6.73 (d, J = 8.4 Hz, 1H), 6.01 (s, 1H), 5.89 (s, 2H), 5.81 (d, J = 8.2 Hz, 1H), 5.68 (d, J = 4.8 Hz,
1H), 5.35 (dd, J = 12.3, 3.0 Hz, 1H), 5.17 (d, J = 12.2 Hz, 1H), 4.78 (d, J = 5.2 Hz, 1H), 4.11 (s,
3H), 3.86 (s, 3H), 3.84 (m, 1H), 3.80 (s, 3H), 3.15 (dd, J = 13.1, 5.4 Hz, 1H), 2.87 (dd, J = 14.8,
7.1 Hz, 1H), 2.63 – 2.73 (m, 2H), 2.14 (ddd, J = 18.9, 12.1, 7.4 Hz, 1H), 2.09 (dd, J = 17.2, 6.0
Hz, 1H)ppm; 13C NMR (151 MHz, methanol-d4) δ 153.2, 149.9, 149.5, 144.4, 140.9, 137.6,
136.5, 135.1, 133.8, 133.1, 131.2, 126.0, 124.7, 118.5, 118.0, 114.0, 103.6, 102.1, 86.3, 72.2,
65.7, 60.6, 60.1, 56.7, 54.3, 47.1, 41.2, 25.9 ppm; HRMS (ESI): [M+H]+ C28H32N3O6 calcd
506.2291, found 506.2337.
4.2.2.4.
2-((5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-4-methoxy-
5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinolin-6-yl)-2-(9H-fluoren-2-yl)ethanamine (6d)
6d: Yield: 76 %; orange powder; m.p. 105 °C; 1H NMR (600 MHz, methanol-d4) δ 7.74 (d, J
= 7.5 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.40 (s, 1H), 7.35 (t, J = 7.4 Hz,
1H), 7.29 (t, J = 7.4 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 5.99 (s, 1H),
5.91 (d, J = 8.1 Hz, 1H), 5.82 (s, 1H), 5.72 (s, 1H), 5.66 (t, J = 3.9 Hz, 1H), 5.29 (dd, J = 12.3,
2.8 Hz, 1H), 5.15 (d, J = 12.3 Hz, 1H), 4.80 (d, J = 5.0 Hz, 1H), 4.04 (s, 3H), 3.83 (s, 3H), 3.81
(s, 1H,), 3.80 (s, 3H), 3.77 (s, 1H), 3.67 (s, 1H), 3.15 (dd, J = 13.2, 5.7 Hz, 1H), 2.90 (dd, J =
14.6, 7.1 Hz, 1H), 2.76 (td, J = 14.2, 13.2, 5.7 Hz, 1H), 2.31 (ddd, J = 18.1, 11.9, 7.1 Hz, 1H),
1.99 (dd, J = 18.5, 7.2 Hz, 2H) ppm; 13C NMR (151 MHz, methanol-d4) δ 153.1, 149.6, 144.6,
19

144.5, 144.3, 143.2, 142.3, 141.0, 138.6, 135.0, 134.2, 133.1, 131.8, 128.2, 128.0, 127.9, 126.0,
125.5, 120.9, 120.4, 118.7, 118.6, 113.9, 103.7, 101.9, 86.8, 72.2, 67.9, 60.6, 60.0, 56.7, 54.9,
46.8, 42.1, 37.5, 25.8 ppm; HRMS (ESI): [M+H]+ C36H37N2O6 calcd 593.2652, found 593.2750.
4.2.2.5.
2-((5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-4-methoxy-
5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinolin-6-yl)-2-(4-methylphenyl)ethanamine (6e)
6e: Yield: 88 %; light brown powder; m.p. 98 °C; 1H NMR (600 MHz, methanol-d4) δ 7.11
(d, J = 7.8 Hz, 2H), 6.96 (d, J = 7.7 Hz, 2H), 6.72 (d, J = 8.2 Hz, 1H), 6.02 (s, 1H), 5.90 (s, 1H),
5.88 (s, 1H), 5.80 (d, J = 8.2 Hz, 1H), 5.66 (t, J = 3.7 Hz, 1H), 5.31 (dd, J = 12.3, 2.8 Hz, 1H),
5.15 (d, J = 12.2 Hz, 1H), 4.78 (d, J = 5.1 Hz, 1H), 4.05 (s, 3H), 3.97 (dd, J = 11.2, 5.1 Hz, 1H),
3.84 (s, 3H), 3.82 (t, J = 5.6 Hz, 1H), 3.80 (s, 3H), 3.65 (t, J = 12.3 Hz, 1H), 3.07 (dd, J = 13.2,
5.4 Hz, 1H), 2.83 (dd, J = 14.5, 6.9 Hz, 1H), 2.67 (ddd, J = 14.4, 12.1, 5.6 Hz, 1H), 2.232– 2.31
(m, 1H), 2.23 (s, 3H) ppm; 13C NMR (151 MHz, methanol-d4) δ 153.07, 149.66, 144.30, 140.98,
139.49, 136.93, 135.14, 134.07, 133.07, 131.74, 129.9, 128.9, 118.6, 118.4, 113.9, 103.6, 102.0,
86.6, 72.1, 67.4, 60.6, 60.0, 56.7, 54.5, 47.2, 41.8, 25.9, 21.0 ppm; HRMS (ESI): [M+H]+
C30H35N2O6 calcd 519.2495, found 519.2584.
4.2.2.6.
(3R,5R,8R,9S,10S,13R,14S,17R)-17-(5-Aminopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol (6f)
6f: Yield: 98 %; white powder; m.p. 205 °C; 1H NMR (500 MHz, DMSO-d6) δ 3.27 – 3.46
(m, 1H), 2.52 – 2.91 (m, 2H), 1.89 – 1.96 (m, 1H), 1.73 – 1.83 (m, 3H), 1.67 (dt, J = 13.9, 3.2
Hz, 1H), 1.46 – 1.64 (m, 4H), 1.27 – 1.43 (m, 10H), 1.11 – 1.26 (m, 5H), 1.11 – 0.98 (m, 5H),
0.90 – 0.94 (m, 1H), 0.89 (d, J = 6.7 Hz, 3H, CH3), 0.87 (s, 3H), 0.62 (s, 3H) ppm; 13C NMR
(126 MHz, DMSO) δ 70.3, 56.6, 56.1, 42.7, 41.9, 40.4, 36.7, 35.8, 35.6, 35.3, 34.7, 32.7, 30.8,
28.2, 27.3, 26.6, 24.9, 24.3, 23.7, 20.9, 18.9, 12.3 ppm; ESIMS: [M+H]+ C24H44NO calcd
362.34, found 362.80.
4.2.2.7.
(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-17-(5-Aminopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triol (6g)
1
6g: Yield: 87 %; white powder; m.p. 215 °C; H NMR (500 MHz, DMSO-d6) δ 3.78 (t, J =
2.8 Hz, 1H), 3.60 (q, J = 3.0 Hz, 1H), 3.18 (tt, J = 10.5, 4.2 Hz, 1H), 2.84 (brs, 1H), 2.47 – 2.60
(m, 2H), 2.17 – 2.26 (m, 1H), 2.14 (td, J = 13.4, 12.1, 3.7 Hz, 1H), 1.97 (td, J = 11.9, 7.2 Hz,
1H), 1.57 – 1.84 (m, 5H), 1.18 – 1.49 (m, 13H), 1.13 (q, J = 10.4, 9.9 Hz, 1H), 1.05 – 0.95 (m,
2H), 0.92 (d, J = 6.1 Hz, 3H), 0.85 (dd, J = 14.0, 3.3 Hz, 1H), 0.80 (s, 3H), 0.58 (s, 3H) ppm; 13C
20

NMR (126 MHz, DMSO) δ 71.5, 70.9, 66.7, 46.7, 41.9, 41.8, 40.0, 35.8, 35.3, 34.8, 33.3, 30.8,
29.0, 27.87, 26.7, 23.3, 23.1, 17.8, 12.8 ppm; ESIMS: [M+H]+ C24H44NO3 calcd 394.33, found
394.93.
4.2.2.8.
(3R,5R,8R,9S,10S,12S,13R,14S,17R)-17-(5-Aminopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (6h)
6h: Yield: 75 %; white powder; m.p. 208 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.48 (brs,
1H), 4.20 (s, 1H), 3.78 (s, 1H), 3.34 – 3.42 (m, 1H), 2.52 – 2.55 (m, 2H), 1.68 – 1.84 (m, 7H),
1.58 – 1.66 (m, 2H), 1.56 (d, J = 8.5 Hz, 1H), 1.41 – 1.53 (m, 3H), 1.25 – 1.40 (m, 8H), 1.20 –
1.25 (m, 3H), 1.16 (d, J = 13.0 Hz, 2H), 1.04 (d, J = 12.5 Hz, 1H), 0.86 - 1.01 (m, 5H), 0.84 (s,
3H), 0.59 (s, 3H) ppm; 13C NMR (126 MHz, DMSO) δ 71.5, 70.4, 52.5, 47.9, 46.8, 46.4, 44.5,
42.1, 36.8, 36.1, 35.6, 34.3, 33.39, 30.7, 29.1, 27.5, 26.6, 23.5, 12.9, 7.6 ppm; ESIMS: [M+H]+
C24H44NO2 calcd 378.34, found 378.93 .
4.2.2.9.
(3R,5S,7R,8R,9S,10S,13R,14S,17R)-17-(5-Aminopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7-diol (6i)
6i: Yield: 75 %; white powder; m.p. 250 °C; 1H NMR (500 MHz, DMSO-d6) δ 2.62 – 2.79
(m, 2H), 1.92 (d, J = 10.0 Hz, 1H), 1.79 (d, J = 12.7 Hz, 2H), 1.45 – 1.71 (m, 6H), 1.25 – 1.44
(m, 10H), 1.11 – 1.22 (m, 6H),0.96 – 1.08 (m, 5H), 0.80 – 0.96 (m, 7H), 0.61 (s, 3H) ppm; 13C
NMR (126 MHz, DMSO) δ 70.3, 56.5, 56.1, 42.7, 41.9, 40.4, 40.2, 39.9, 36.7, 35.8, 35.6, 35.3,
34.6, 32.6, 30.8, 28.2, 27.3, 26.6, 24.3, 23.7, 20.9, 18.8, 12.3 ppm; ESIMS: [M+H]+ C24H44NO2
calcd 378.3372, found 378.93.
4.2.2.10.
(3R,5S,7S,8R,9S,10S,13R,14S,17R)-17-(5-Aminopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7-diol (6j)
6j: Yield: 90 %; white powder; m.p. 238 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.49 (brs, 1H),
4.13 (brs, 1H), 3.89 (brs, 1H), 3.16 (s, 1H), 2.60-2.76 (m, 1H), 2.48 – 2.51 (m, 2H), 1.93 (d, J =
11.6 Hz, 1H), 1.85 (d, J = 11.1 Hz, 1H), 1.55 – 1.77 (m, 4H), 1.42 – 1.51 (m, 4H), 1.26 – 1.41
(m, 8H), 1.05 – 1.22 (m, 6H),0.96 – 1.03 (m, 1H), 0.91 – 0.95 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H),
0.87 (s, 3H), 0.62 (s, 3H, CH3) ppm; 13C NMR (126 MHz, DMSO) δ 70.2, 69.9, 63.3, 56.3, 55.2,
43.5, 43.5, 42.6, 39.2, 38.2, 37.7, 35.3, 34.2, 30.7, 28.7; 27.2, 23.8, 21.3, 19.0, 12.5 ppm;
ESIMS: [M+H]+ C24H44NO2 calcd 378.34, found 378.87.
4.2.3. General procedure for the synthesis of isothiocyanate derivatives 7a-7s
21

To a solution of primary amine compounds (1.0 mmol) in absolute ethanol was added 10.0
mmol CS2 and 10.0 mmol triethyl amine. The mixture was stirred at room temperature until
dithiocarbamate intermediate was formed. Then, the reaction was cooled in an ice bath at -5°C.
1.0 mmol di-tert-butyl dicarbonate (Boc2O) and 3% mol of DMAP was dissolved in 0.5 mL
absolute ethanol, separately. At first, di-tert-butyl dicarbonate solution was added to the reaction
mixture, and then the addition of DMAP solution was performed immediately. The reaction was
kept in an ice bath for 5 min and then was allowed to reach room temperature. The reaction was
monitored by TLC. After 1-2 h, the reaction mixture was concentrated under vacuo and 10 mL
water was added to residue. The mixture was extracted with chloroform (3×20 mL) and organic
phases were collected, dried over anhydrous Na2SO4 and concentrated under vacuum. The
products were purified by preparative TLC.
4.2.3.1.
(5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-6-(2-isothiocyanato-
1-phenylethyl)-4-methoxy-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline (7a)
1
7a: Yield: 62%; off white powder; m.p. 69 °C; H NMR (600 MHz, chloroform-d) δ 7.19 –
7.25 (m, 5H), 6.68 (d, J = 8.2 Hz, 1H), 6.30 (s, 1H), 6.19 (d, J = 8.0 Hz, 1H), 5.88 (s, 1H), 5.85
(s, 1H), 5.38 (brs, 1H), 4.98 (s. 2H), 4.45 (d, J = 3.4 Hz, 1H), 4.23 (t, J = 4.8 Hz, 1H), 3.98 (dd, J
= 14.5, 5.8 Hz, 1H), 3.82 (s, 3H), 3.78 (dd, J = 14.5, 5.2 Hz, 1H), 3.76 (s, 3H), 3.71 (s, 3H), 2.91
– 2.96 (m, 1H), 2.76 – 2.79 (m, 1H), 2.54 – 2.59 (m, 1H), 2.35 – 2.37 (m, 1H) ppm; 13C NMR
(151 MHz, chloroform-d) δ 151.0, 147.9, 142.8, 140.8, 139.6, 134.1, 132.3, 130.7, 128.4, 127.8,
127.7, 118.6, 117.5, 111.9, 102.5, 100.5, 87.1, 71.5, 65.2, 59.9, 59.1, 58.8, 56.3, 47.2, 41.7, 26.4
ppm; HRMS (ESI): [M+H]+ C30H31N2O6S calcd 547.1903, found 547.1979.
4.2.3.2.
(5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-6-((1R)-1-(4-
fluorophenyl)-2-isothiocyanatoethyl)-4-methoxy-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-
g]isoquinoline (7b)
1
7b: Yield: 58%; light yellow powder; m.p. 64 °C; H NMR (600 MHz, chloroform-d) δ 7.18 –
7.22 (m, 2H), 6.90 – 6.95 (m, 2H), 6.69 (d, J = 8.1 Hz, 1H), 6.30 (s, 1H), 6.21 (d, J = 6.8 Hz,
1H), 5.87 (s, 1H), 5.85 (s, 1H), 5.38 (brs, 1H), 4.99 (s. 2H), 4.37 (brs, 1H), 4.15 (s, 1H), 3.93 –
3.95 (m, 1H), 3.82 (s, 3H), 3.74 – 3.77 (m, 4H), 3.71 (s, 3H), 2.91 – 2.94 (m, 1H), 2.76 – 2.79
(m, 1H), 2.55 – 2.59 (m, 1H), 2.34 – 2.36 (m, 1H) ppm; 13C NMR (151 MHz, chloroform-d) δ
184.1, 152.9, 149.2, 142.9, 140.6, 136.8, 134.4, 134.1, 132.3, 131.7, 130.4, 129.5, 118.6,117.4,
22

115.4, 115.3, 112.0, 102.5, 100.6, 86.9, 71.4, 64.4, 59.9, 59.0, 58.6, 56.3, 47.3, 41.7, 25.9 ppm;
HRMS (ESI): [M+H]+ C30H30FN2O6S calcd 565.1809, found 565.1866.
4.2.3.3.
(5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-6-(2-isothiocyanato-
1-(pyridin-3-yl)ethyl)-4-methoxy-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline (7c)
1
7c: Yield: 67%; yellow powder; m.p. 79 °C; H NMR (600 MHz, chloroform-d) δ 8.54 (d, J =
3.8 Hz, 1H), 8.46 (s, 1H), 7.69 (brs, 1H), 7.28 (d, J = 3.6 Hz, 1H), 6.68 (d, J = 8.1 Hz, 1H), 6.35
(s, 1H), 6.19 (d, J = 8.0 Hz, 1H), 5.93 (s, 1H), 5.91 (s, 1H), 5.46 (brs, 1H), 5.05 (s. 2H), 4.37 (d,
J = 1.3 Hz, 1H), 4.35 (brs, 1H), 4.04 – 4.09 (m, 1H), 3.84 – 3.89 (m, 4H), 3.82 (s, 6H), 2.80 –
2.92 (m, 2H), 2.58 – 2.62 (m, 1H), 2.38 – 2.42 (m, 1H) ppm; 13C NMR (151 MHz, chloroform-d)
δ 151.2, 149.2, 142.9, 141.9, 140.7, 137.1, 135.8, 134.1, 132.2, 123.5, 117.4, 112.0, 102.5, 100.6,
86.3, 71.5, 63.1, 59.9, 59.1, 58.9, 56.3, 46.7, 41.6, 26.1 ppm; HRMS (ESI): [M+H]+
C29H30N3O6S calcd 548.1855, found 548.1917.
4.2.3.4.
(5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-6-(1-(9H-fluoren-2-
yl)-2-isothiocyanatoethyl)-4-methoxy-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline (7d)
1
7d: Yield: 65%; off white powder; m.p. 93 °C; H NMR (600 MHz, chloroform-d) δ 7.73 (d, J =
7.4 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.51 (d, J = 7.1 Hz, 1H), 7.33 – 7.35 (m, 2H), 7.27 – 7.28
(1, 2H), 7.20 (d, J = 7.4 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.32 (d, J = 8.1 Hz, 1H), 6.31 (s, 1H),
5.84 (s, 1H), 5.83 (s, 1H), 5.40 (brs, 1H), 4.86 – 4.89 (m. 2H), 4.42 (brs, 1H), 4.20 (brs, 1H),
4.01 (dd, J = 14.7, 5.4 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 2H), 3.73 (s, 4H), 3.63 (s, 3H), 3.07 – 3.09
(m, 1H), 2.86 – 2.89 (m, 1H), 2.62 – 2.66 (m, 1H), 2.40 – 2.43 (m, 1H) ppm; 13C NMR (151
MHz, chloroform-d) δ 151.0, 147.9, 143.5, 142.8, 141.4, 141.0, 138.3, 134.4, 134.1, 132.1,
130.3, 126.7, 126.6, 125.1, 124.6, 119.9, 119.7, 118.7, 117.6, 111.9, 102.6, 100.5, 87.1, 71.4,
65.1, 59.8, 59.0, 58.8, 56.3, 47.7, 41.6, 36.8, 26.4 ppm; HRMS (ESI): [M+H]+ C37H35N2O6S
calcd 635.2216, found 635.2259.
4.2.3.5.
(5R)-5-((1S)-4,5-Dimethoxy-1,3-dihydro-2-benzofuran-1-yl)-6-(2-isothiocyanato-
1-(4-methylphenyl)ethyl)-4-methoxy-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline (7e)
1
7e: Yield: 72%; off white powder; m.p. 68 °C; H NMR (600 MHz, chloroform-d) δ 7.09 (d, J =
8.0 Hz, 2H), 7.05 (d, J = 7.9 Hz, 2H), 6.69 (d, J = 8.2 Hz, 1H), 6.30 (s, 1H), 6.19 (d, J = 8.2 Hz,
1H), 5.87 (s, 1H), 5.85 (s, 1H), 5.38 (brs, 1H), 5.02 (d, J = 14.2 Hz, 1H), 4.99 (d, J = 12.2 Hz,
1H), 4.45 (d, J = 4.1 Hz, 1H), 4.19 (t, J = 5.2 Hz, 1H), 3.95 (dd, J = 14.6, 5.9 Hz, 1H), 3.83 (s,
3H), 3.77 (s, 3H), 3.75 (dd, J = 14.5, 4.9 Hz, 1H), 3.72 (s, 3H), 2.87 – 2.92 (m, 1H), 2.73 – 2.77
23

(m, 1H), 2.53 – 2.58 (m, 1H), 2.32 – 2.36 (m, 1H), 2.29 (s, 3H) ppm; 13C NMR (151 MHz,
chloroform-d) δ 151.0, 147.9, 142.8, 140.8, 137.3, 136.5, 134.2, 134.1, 132.3, 130.7, 129.1,
127.7, 118.6, 117.5, 111.9, 102.5, 100.5, 87.0, 71.5, 64.9, 59.9, 59.1, 58.7, 56.3, 47.2, 41.6, 26.4,
21.1 ppm; HRMS (ESI): [M+H]+ C31H33N2O6S calcd 561.2059, found 561.2111.
4.2.3.6.
(3R,5R,8R,9S,10S,13R,14S,17R)-17-(5-Isothiocyanatopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol (7f)
1
7f: Yield: 64%; white powder; m.p. 91 °C; H NMR (600 MHz, chloroform-d) δ 3.60 – 3.68 (m,
1H), 3.46 – 3.52 (m, 2H), 1.96 – 1.98 (m, 1H), 1.77 – 1.87 (m, 4H), 1.67 – 1.69 (m, 1H), 1.57 –
1.62 (m, 4H), 1.51 – 1.53 (m, 2H), 1.35 – 1.46 (m, 6H), 1.33 – 1.35 (m, 1H), 1.23 – 1.31 (m,
3H), 1.08 – 1.18 (m, 5H), 0.98 – 1.07 (m, 1H), 0.98 (d, J = 6.5 Hz, 3H), 0.93 (s, 3H), 0.66 (s,
3H) ppm; 13C NMR (151 MHz, chloroform-d) δ 71.9, 56.5, 55.9, 45.5, 42.7, 42.1, 40.4, 40.2,
36.4, 35.8, 35.3, 35.2, 32.8, 30.5, 28.3, 27.2, 26.8, 26.4, 24.2, 23.4, 20.8, 18.6, 12.0 ppm; HRMS
(ESI): [M+H]+ C25H42NOS calcd 404.2987, found 404.3349.
4.2.3.7.
(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-17-(5-Isothiocyanatopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triol (7g)
7g: Yield: 58%; white powder; m.p. 130 °C; 1H NMR (600 MHz, methanol-d4) δ 3.97 (brs, 1H),
3.84 (brs, 1H), 3.72 (brs, 1H),3.38 – 3.47 (m, 5H), 2.17 – 2.21 (m, 5H), 1.91 – 1.94 (m, 1H), 1.86
– 1.87 (m, 2H), 1.74 – 1.76 (m, 4H), 1.64 – 1.66 (m, 2H), 1.49 – 1.51 (m, 4H), 1.39 – 1.41 (m,
3H), 1.23 – 1.26 (m, 1H), 1.09 – 1.18 (m, 2H), 1.05 (d, J = 6.5 Hz, 3H), 0.88 (s, 3H), 0.69 (s,
3H) ppm; 13C NMR (151 MHz, methanol-d4) δ 73.1, 72.0, 68.5, 47.1, 46.5, 45.5, 41.8, 41.4,
39.5, 39.4, 35.2, 35.1, 34.7, 34.5, 32.7, 30.3, 28.2, 27.5, 26.7, 26.5, 23.2, 22.5, 17.6, 12.5 ppm;
HRMS (ESI): [M+Na]+ C25H41NNaO3S calcd 458.2705, found 458.2726.
4.2.3.8.
(3R,5R,8R,9S,10S,12S,13R,14S,17R)-17-(5-Isothiocyanatopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (7h)
7h: Yield: 74%; white powder; m.p. 129 °C; 1H NMR (600 MHz, methanol-d4) δ 3.96 (brs, 1H),
3.59 – 3.64 (m, 1H), 3.62 (t, J= 6.4 Hz, 2H), 3.39 (brs, 1H),1.73 – 1.84 (m, 6H), 1.68 – 1.71 (m,
2H), 1.61 – 1.67 (m, 1H), 1.53 – 1.57 (m, 2H), 1.41 – 1.55 (m, 4H), 1.37 – 1.40 (m, 4H), 1.32 –
1.34 (m, 1H), 1.21 – 1.25 (m, 2H), 1.05 – 1.21 (m, 3H), 0.99 – 1.05 (m, 1H), 0.98 (d, J = 6.5 Hz,
3H), 0.89 (s, 3H), 0.66 (s, 3H) ppm; 13C NMR (151 MHz, methanol-d4) δ 73.2, 71.8, 48.3, 47.3,
46.5, 45.5, 42.1, 36.4, 36.0, 35.2, 34.9, 34.1, 33.7, 32.7, 30.5, 28.7, 27.5, 27.1, 26.7, 26.1, 23.6,
23.1, 17.6, 12.7 ppm; HRMS (ESI): [M+Na]+ C25H41NNaO2S calcd 422.2756, found 442.2760.
24

4.2.3.9.
(3R,5S,7R,8R,9S,10S,13R,14S,17R)-17-(5-Isothiocyanatopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7-diol (7i)
7i: Yield: 84%; white powder; m.p. 66 °C; 1H NMR (600 MHz, methanol-d4) δ 3.55 – 3.57 (m,
2H), 3.44 – 3.47 (m, 2H), 3.39 (brs, 2H), 1.96 – 1.99 (d, J = 1.9Hz, 1H),1.85 – 1.89 (m, 1H),
1.73 – 1.79 (m, 4H), 1.64 – 1.66 (m, 3H), 1.45 – 1.52 (m, 3H), 1.37 – 1.45 (m, 5H), 1.17 – 1.33
(m, 5H), 1.10 – 1.15 (m, 2H), 1.00 – 1.06 (m, 2H), 0.89 – 0.98 (m, 6H), 0.66 (s, 3H) ppm; 13C
NMR (151 MHz, methanol-d4) δ 71.5, 71.4, 55.7, 54.9, 45.5, 43.8, 43.7, 42.4, 40.1, 39.2, 37.3,
36.8, 35.1, 34.9, 34.1, 32.8, 30.3, 28.7, 26.9, 26.8, 23.4, 21.2, 18.7, 12.1 ppm; HRMS (ESI):
[M+Na]+ C25H41NNaO2S calcd 422.2756, found 442.2731.
4.2.3.10.
(3R,5S,7S,8R,9S,10S,13R,14S,17R)-17-(5-Isothiocyanatopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7-diol (7j)
7j: Yield: 60%; yellow oil; 1H NMR (600 MHz, methanol-d4) δ 3.83 (brs, 1H), 3.44 – 3.48 (m,
4H), 2.15 – 2.19 (m, 1H), 1.93 – 1.98 (m, 2H),1.85 – 1.89 (m, 1H), 1.79 – 1.81 (m, 2H), 1.70 –
1.78 (m, 2H), 1.50 – 1.62 (m, 3H), 1.45 – 1.49 (m, 4H), 1.35 – 1.44 (m, 3H), 1.20 – 1.34 (m,
3H), 1.09 – 1.11 (m, 4H), 0.95 – 1.00 (m, 1H), 0.92 (d, J = 6.5 Hz, 3H), 0.88 (s, 3H), 0.66 (s,
3H) ppm; 13C NMR (151 MHz, methanol-d4) δ 72.0, 68.5, 55.8, 50.4, 45.5, 42.7, 41.4, 39.8,
39.6, 39.4, 35.3, 35.2, 35.0, 34.6, 32.8, 32.7, 30.6, 28.3, 26.7, 23.7, 22.8, 20.6, 18.6, 11.8 ppm;
HRMS (ESI): [M+Na]+ C25H41NNaO2S calcd 422.2756, found 442.2757.
4.3.
Antitrypanosomal and Cytotoxicity Assays
In vitro assays against the protozoan parasites T. b. rhodesiense (STIB900) trypomastigotes, L.
donovani (MHOM‑ET‑67/L82) amastigotes, P. falciparum (NF54) IEF stage, and T. cruzi
(Tulahuen C4) amastigotes, and cytotoxicity in the L6 cells (rat skeletal myoblasts) were
performed as previously reported.35,36 The growth inhibition screening of derivatives wascarried
out at two concentrations 2 and 10 µg/mL. For determination of IC50s, Alamar Blue assay was
used according to that reported procedure.16 All tests were performed in two independent
experiments. The selectivity index was calculated as the ratio of IC50 for the L6-cells to IC50 for
parasites.
4.4. Molecular docking studies and ADMET prediction
2D structures of the derivatives were created by ChemDraw Professional 15.0 and 3D structures
were generated by Chem3D suite. The SDF format of structures were saved and used for docking
study. The energy minimization of ligand structures were performed by Lig Prep module
25

implemented in Schrodinger 2015-2, using Maestro 10.2 platform. The TbTR crystal structure
(PDB: 2WOW) with a resolution of 2.2 Å was picked out from previous publications 33,37 and
downloaded from protein data bank (www.rcsb.org). Proteins preparation for docking was
performed using Protein Preparation Wizard on Maestro 10.2. The proteins preparation included
deleting of water molecules, assigning formal charge and bond order of protein, adding all
missing hydrogen atoms, loops and side chains to crystal structures. Then the hydrogen network
was refined and hydrogen minimization was carried out by OPLS3 force field parameter. The
native ligand in the crystal structure (FAD for TbTR) was selected to receptor grid box
generation as a 25 Å ×25 Å ×25 Å box with the Grid generation application. All active
compounds and native ligand conformers were docked to grid files of proteins by Glide extra
precision (XP) mode. The refinement and scoring were performed by default. The ADMET
properties prediction of the most active compounds were exertwd by using Qikprop 4.4.
Author contribution
The manuscript was written through the contributions of all authors. All authors have approved
the final version of the manuscript.
Declarations of interest
The authors declare no conflict of interest.
Funding sources
The authors acknowledge the support of the research deputy of Shahid Beheshti University for
financial support of this project.
Acknowledgments
We thank Dr. Martin Smieško, Department of Pharmaceutical Sciences, the University of Basel
in assisting on molecular modeling of current work. The authors thanks also M. Cal, S. Keller-
Märki and R. Rocchetti for their help with parasite assays.
Appendix A. Supplementary Data.synthesis methods of 4a-4e and 5a-5e. 1H & 13C NMR, and
HRMS FTIR spectra of compounds 4a-4e, 5a-5j and 6a-6j and 7a-7j.
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