ChrisTina Okolo1, Mohamad Akbar Ali2, Matthew Newman1, Steven A Chambers1, Jedidiah Whitt1, Zakeyah A Alsharif1, Victor W Day3, Mohammad A Alam1. 1. Department of Chemistry and Physics, College of Science and Mathematics, Arkansas State University, Jonesboro, Arkansas 72467, United States. 2. Department of Chemistry, Sejong University, Seoul 143-747, Republic of Korea. 3. Department of Chemistry, Integrated Science Building, University of Kansas, Lawrence, Kansas 66046, United States.
Abstract
A cascade reaction of thioamides with 6β-bromoandrostenedione in hexafluoroisopropanol formed substituted thiazolo-androstenones. This is a simple and mild protocol to synthesize novel molecules by using readily available reagents and substrates. Feasibility of the reaction has been rationalized by density functional theory calculations. Moreover, these compounds are potent growth inhibitors of colon, central nervous system, melanoma, ovarian, and renal cancer cell lines with 50% growth inhibition values as low as 1.04 μM.
A cascade reaction of thioamides with 6β-bromoandrostenedione in hexafluoroisopropanol formed substituted thiazolo-androstenones. This is a simple and mild protocol to synthesize novel molecules by using readily available reagents and substrates. Feasibility of the reaction has been rationalized by density functional theory calculations. Moreover, these compounds are potent growth inhibitors of colon, central nervous system, melanoma, ovarian, and renal cancer cell lines with 50% growth inhibition values as low as 1.04 μM.
Several hormones of
the steroidal skeleton are found in biological
signaling in mammals.[1,2] A large number of bioactive steroidal
natural products have been isolated from various plants and microorganisms.[3,4] Numerous synthetic derivatives have also been reported in literature
in a quest of drugs, drug candidates, and other valuable materials
including herbicides.[5−11] Natural and synthetic steroidal derivatives are known to show a
number of useful pharmacological properties such as agonists of cell-surface
G-protein-coupled bile acid receptor,[12] neuroprotective,[13] anticancer,[14] and anti-Alzheimer[15] properties.[7,16] Unnatural steroidal derivatives
are one of the broadest spectra of therapeutic classes of compounds,
which are used to treat different diseases including cancer.[8,17] Thiazole derivatives are another class of important compounds with
several approved drugs such as dasatinib, fanetizole, and nizatidine.[18,19] Several steroidal drugs contain heterocyclic moieties: oxazole in
Emflaza (deflazacort) and pyridine in Zytiga (abiraterone acetate).[20−22] Thiazole-attached progesterone derivatives have been reported as
potent SKOV-3 (ovarian cancer) growth inhibitor.[23] Pyrazole-fused sterone, stanazolol, derivative is known
for potent anabolic activities (Figure ).[24] Not surprisingly, syntheses
of heterocycle-incorporated steroidal derivatives have been reported
in a large number of literature.[25−31] Novel molecules based on the steroidal core structure are synthesized
in a multistep synthesis[7,12,32−34] and using catalyst.[29]
Figure 1
Representative
examples of heterocycle containing steroidal drugs
and pharmacologically active molecules.
Representative
examples of heterocycle containing steroidal drugs
and pharmacologically active molecules.
Results and Discussion
In our quest to synthesize bioactive
molecules[35−37] and to develop
new domino reactions to synthesize heterocycles,[38,39] we planned the synthesis of thiazolino-androstanedione derivatives
by using our recently reported methodology, the synthesis of thiazoline
derivatives (2) by reacting thioamides with γ-bromoenones
(1).[40] Reaction of 6β-bromoandrostenedione
(3) with thiourea derivatives formed aminothiazoloandrostenone
derivative (4) by an unexpected mechanism.[41] Surprisingly, reaction of thioamide derivative
(5) with the electrophile (3) did not form
the product in refluxing ethanol, as we expected from our previous
report (Scheme ).[41]
Scheme 1
Synthesis of Thiazolo-androstenone Derivative
(6)
To our delight, the reaction happened in hexafluoroisopropanol
(HFIP) in 61% yield, and the reaction did not require an anhydrous
solvent and inert atmosphere. The products formed cleanly, and the
pure material was isolated simply by distilling out HFIP followed
by recrystallizing with methanol (Scheme ). Column chromatography was not required
to obtain the pure product (6). After identifying the
product as thiazolo-androstenone in HFIP, we carried out the reaction
in different solvents including different alcohols and polar aprotic
solvents: tetrahydrofuran, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF); however, the
reaction was not successful in any solvent except trifuoroethanol
in moderate yield. Refluxing the reaction mixture in DMF gave the
unidentifiable decomposed products.[42,43] On the basis
of these observations, we can conclude that a very polar protic solvent
is required for the product formation of this domino methodology,
and HFIP has optimum properties for the success of this protocol.
Scheme 2
Reaction of Thiobenzamide with 6β-Bromoandrostenedione (3)
After establishing
the optimum conditions, we reacted different
thioamides with the electrophile (3), as shown in Scheme . Reaction of thioacetamide
and 2-phenylthioacetamide with 6β-bromoandrostenone (3) formed the products (7 and 8) in 52 and
56% yields, respectively. 2-(2-Methylphenyl)thioacetamide also reacted
with the electrophile (3) to give the benzyl derivative
(9) in 55% yield. Substituted thiobenzamide derivatives
were isolated under the established reaction conditions. m-Methyl- and p-methyl-substituted aryl products
formed (10 and 11) in 59 and 61% yield,
respectively. Similarly, 3-alkoxy-substituted products (12 and 13) formed in an average of 64% yield. A complex
thioamide, 4-[5-(trifluoromethyl)pyrid-2-yloxy]thiobenzamide, did
not hamper the reaction, and the product (14) was obtained
in good yield. 4-Methoxy-substituted product (15) was
obtained in 60% yield. Hydroxy-substituted products (16, 17, and 18) were obtained expectantly.
Scheme 3
Reaction of Thioamides with 6β-Bromoandrostenedione (3)
Furthermore, dihydroxy
thiobenzamide also reacted smoothly to give
the expected product (19) in 58% yield. Thus, the number
and position of the electron-donating groups did not alter the outcome
of the corresponding products. Products containing electron-withdrawing
substituents were obtained under the established reaction conditions.
3-Fluoro-substituted thiazolo-androstenone derivative (20) formed in 60% yield, and 4-fluorophenyl-substituted compound (21) was obtained in 61% yield. 3-Chloro- and 4-chloro-substituted
products (22 and 23) formed in an average
of 50% yield. 3,5-Bischlorophenyl-substituted product (24) also formed accordingly. 4-Bromophenyl product (25) was obtained in 45% yield. 4-(Trifluoromethyl)thiobenzamide reacted
with the electrophile to give the corresponding product (26). Very strong electron-withdrawing group containing aryl ring, such
as 3-nitrothiobenzamide, also reacted with the electrophile (3) to give the corresponding product (27). Last
but not least, pyridine-2-carbothioamide formed the product (28), albeit low yield (38%) was obtained. Thus, the nature
and position of the electron-withdrawing group do effect the product
formation of this methodology. Finally, the limitation we found in
this methodology is that pyridine-4-carbothioamide and pyrimidine-2-carbotioamide
failed to give the corresponding products (29 and 30). Thus, this methodology has the potential to generate
a new class of novel molecules based on the fused thiazolo-androstane
scaffold.These molecules can be further transformed into new
entities by
simple reactions (Scheme ). 17-Hydroxy and 17-aceloxy derivatives of androstane skeleton
are integral parts of drugs, hormones, natural products, and synthetic
bioactive molecules.[44−46] Ketone derivative (20) was reduced with
NaBH4 and led stereoselectively to the corresponding hydroxy
product (31) in an excellent yield. Further acetylation
with Ac2O/pyridine afforded the acetylated product (32) in quantitative yield.
Scheme 4
Reduction and Acetylation of Ketone
Derivative (18)
to Generate a Library of Molecules
The structures of compounds (20, 21, 25, and 32) were confirmed by single-crystal
X-ray diffraction analysis, which has helped to establish the regiochemistry
and stereochemistry of the reactions. Crystal structures (20, 21, and 25) have helped to confirm the
regiospecificity of this methodology. The final product (32) confirmed the formation of substrate-controlled β-hydroxy
product exclusively in NaBH4 reduction (Figure ).
Figure 2
Oak ridge thermal ellipsoid
plot diagrams of 20 (CCDC
1859241), 21 (CCDC 1859222), 25 (CCDC 1861085),
and 32 (CCDC 1859223).
Oak ridge thermal ellipsoid
plot diagrams of 20 (CCDC
1859241), 21 (CCDC 1859222), 25 (CCDC 1861085),
and 32 (CCDC 1859223).
Computational Analysis
All density functional theory
calculations were carried out using Gaussian 09 suite of programs.[46] The hybrid density functional method (M06-2X)/6-311++G(d,p)
+ PCM (solvent = HFIP) has been used to compute the feasibility of
all four pathways. We have used polarizable continuum model (PCM)
using the integral equation formalism variant as the self consistent
reaction field method. This method creates solute cavity via a set
of overlapping spheres. The free energy of all of the species was
calculated using the PCM solvent model i.e., HFIP. The calculations
were done at 1 atm pressure and 298 K.HFIP is a very strong
hydrogen bond donor, which makes hydrogen bonding with the carbonyl
oxygen of the enone of β-bromoandrostenedione (3).[39] This hydrogen bonding makes the enone
carbonyl group a better electrophile for the nucleophilic addition
of thioacetamide, which is the key for the success of this methodology.
Sulfur or nitrogen atom of thioacetamide can undergo nucleophilic
addition to the carbonyl group of β-bromoandrostenedione (3) to form two possible intermediates, hemithioacetal (A1) or hemiaminal (B1), respectively. Both of
these two reactions are endergonic, and the Gibb’s free energy
for the formation of hemithioacetal (A1, +18.72 kcal/mol)
and hemiaminal (B1, +9.41 kcal/mol) is achieved by refluxing
the reaction mixture in HFIP. These reactions for the formation of
hemithioacetal (A1) and hemiaminal (B1)
are reversible under the reaction condition. Hemithioacetal (A1) and hemiaminal (B1) undergo intramolecular
SN2′ reaction to form the thiazoline regioisomers
(A2 and B2). This SN2′
reaction of hemithioacetal (A1) is more favorable than
that of hemiaminal (B1) (−25.57 vs −22.07
kcal/mol). Dehydration, the final step, is more favorable for the
hemithioacetal than the hemiaminal derivative (−28.01 vs −22.52
kcal/mol) to form the final products A3 and 7, respectively. Among the three steps for the formation of possible
products, the last two steps are irreversible and exergonic (Scheme ). Hence, the less-energy
barrier for the formation of hemiaminal (B1) in the first
step is the deciding factor for the formation of the actual product
(7). The other possible pathway (C1 → B2) for the formation of expected product (7)
is the least favorable. The expected product (D3) based
on our previous report[40] is also thermodynamically
not favorable (Figure ). Probable potential energy surface for all of the possible pathways
is shown in Figure .
Scheme 5
Plausible Mechanism for the Formation
of Product (7)
Using M06-2X/6-311++G(d,p) + PCM (Solvent = HFIP) Level of Theory
Figure 3
Probable potential energy surface of formation of actual and expected
products calculated at M06-2X/6-311++G(d,p) + PCM (solvent = HFIP).
Probable potential energy surface of formation of actual and expected
products calculated at M06-2X/6-311++G(d,p) + PCM (solvent = HFIP).
In Vitro Anticancer Studies
After the successful synthesis
of these novel molecules, some of them have been tested against NCI-60
cancer cell lines.[47] Many of these compounds
have shown promising activity against several cancer cell lines at
10 μM concentration. Selected growth inhibition data for compound 20 are shown in Table . This compound has inhibited the growth of 31 of 60 cell
lines with 50% growth inhibition (GI50) value <2.80
μM concentration. The total growth inhibition (TGI) value is
also in the low micromolar range. The compound (20) has
also shown good lethal concentration (LC50) values against
nine cancer cell lines. HCT-116 and SF-539 were inhibited with LC50 values of 7.30 and 5.90 μM concentrations, respectively.
Table 1
Cytotoxic Data of Compound 20 against
NCI-60 Cell Lines
20
cancer panel
cell line
GI50
TGI
LC50
leukemia
HL-60(TB)
2.00
5.35
>10
K-562
1.82
9.08
>10
MOLT-4
1.69
7.89
>10
RPMI-8226
1.91
5.65
>10
NSCLC
HOP-92
2.44
5.63
>10
NCI-H226
1.94
4.39
>10
NCI-H460
2.79
8.27
>10
NCI-H522
2.40
6.35
>10
colon
cancer
COLO 205
2.42
5.46
>10
HCT-116
1.73
3.56
7.33
HCT-15
1.56
4.17
>10
SW-620
2.14
4.50
>10
CNS cancer
SF-295
1.59
4.91
>10
SF-539
1.04
2.48
5.90
melanoma
LOX IMVI
1.74
3.36
6.47
MALME-3M
1.80
4.13
9.48
SK-MEL-28
1.53
4.07
>10
UACC-62
1.90
5.70
>10
ovarian cancer
IGROV1
2.09
4.88
>10
OVCAR-3
1.92
3.79
7.47
SKOV-3
2.91
9.13
>10
renal
cancer
786-0
1.30
2.80
>10
ACHN
1.64
3.01
5.53
CAKI-1
1.87
3.76
7.57
RXF 393
1.41
2.94
6.14
TK-10
2.77
8.29
>10
UO-31
1.61
2.98
5.54
prostate cancer
DU-145
2.01
4.13
>10
breast cancer
MCF7
1.78
4.26
>10
MDA-MB-231/ATCC
2.60
7.34
>10
BT-549
2.20
8.06
>10
T-47D
2.00
5.24
>10
MDA-MB-468
1.95
4.27
>10
Colorectal cancer is the third most common cancer
in men and women
in the US, nevertheless, this cancer is second leading cause of cancer-related
deaths in this country.[47] Central nervous
system (CNS) cancer is one of the most lethal forms of cancer with
very limited treatment options.[48] Our tested
molecule (20) has shown significant activity against
four colon cancer cell lines with GI50 values as low as
1.56 μM concentration. This molecule inhibited the growth of
HCT-116 cell line with TGI and LC50 values 3.56 and 7.33
μM, respectively. Growths of two of the six CNS cancer cell
lines were also inhibited significantly with GI50 values
∼1 μM concentration. In vitro growth inhibition of SF-539
cancer cell line of the CNS panel is very significant with TGI and
LC50 values of 2.48 and 5.90 μM, respectively.Melanoma is the most serious type of skin cancer. It is the 5–7th
most common cancer in the United States, and the incidence of this
cancer is increasing rapidly. Other than the skin, this cancer can
also develop in the eyes and in internal organs such as the intestines.[49] Development of new therapeutic options is urgently
needed to treat this rapidly rising malignancy.[50] Our lead compound (20) has shown promising
growth inhibition activity against four melanoma cell lines with GI50 less than 2 μM concentration. TGI values are also
in low micromolar concentration for these four melanoma cell lines.
This compound also inhibited the two melanoma cell lines: LOX IMVI
and MALME-3M cell lines with LC50 values of 6.47 and 9.48
μM, respectively. We have found several lead molecules such
as 20 to generate a library of molecules to develop potent
antimelanoma agents.In addition, compound 20 has
shown promising activity
against four ovarian cancer cell lines with GI50 values
at low micromolar concentration. The LC50 value for ovarian
cancer cell line, OVCAR-3, is less than 10 μM. Renal cancer
is the most common type of kidney cancer.[51] The fluorophenyl derivative (20) has shown potent activity
against six cancer cell lines with GI50 and TGI values
as low as 1.30 and 2.80 μM, respectively. Four renal cancer
cell lines were inhibited with LC50 values less than 10
μM. Significant growth inhibition of prostate and breast cancer
cell lines was also observed by this lead molecule (20). Complete data are shown in the Supporting Information. Detailed findings and mode of action of potent
molecules will be reported soon.
Conclusions
In
summary, we have discovered a one-pot protocol to synthesize
novel thiazolo-androstenones by using readily available starting materials
and benign reaction conditions. On the basis of the availability of
a number of thioamide starting materials and ease of reaction conditions,
a large number of novel molecules as potent anticancer agents can
be synthesized. Furthermore, these new scaffolds can be easily transformed
into a variety of potential bioactive molecules. Further derivatization,
structure–activity relationship, anticancer, and toxicity studies
of these novel compounds are in progress and will be reported soon.
Experimental
Section
General Consideration
All of the reactions were carried
out under air atmosphere in round-bottom flasks. Solvents, reagents,
and the substrate were bought from Fisher Scientific and Oakwood chemical.
Characterization
1H NMR and 13C NMR
spectra were recorded with a Varian Mercury-300 MHz and Varian
Mercury-75 MHz, respectively, with tetramethylsilane (TMS) as internal
standard. CDCl3 (>99.9%), DMSO-d6 (>99.8%), or mixture of both were used to record NMR spectra.
In
some spectra, trifluoroacetic acid-d was also used
to increase the solubility of the samples. The electrospray ionization-Fourier
transform mass spectra (ESI-FTMS) were recorded using Bruker ApexII-FTMS
system.Crystals were grown in chloroform–methanol mixture
for single-crystal diffraction.
General Procedure for the
Synthesis of Thiazole-androstenones
(6–28)
A mixture of β-bromoandrostenedione
(1 mmol), thioamide derivative (1.1 mmol), and sodium acetate (82
mg, 1.0 mmol) in 10 mL of hexafluoroisopropanol was refluxed for 12
h to complete the reaction. Progress of the reaction was monitored
by thin-layer chromatography. After the completion of the reaction,
HFIP was distilled out and methanol (10 mL) was added. The solid precipitate
was filtered followed by washing with ∼10 mL of methanol and
∼20 mL of water under vacuum to afford the pure product.
Authors: Rawan Alnufaie; Mohamad Akbar Ali; Ibrahim S Alkhaibari; Subrata Roy; Victor W Day; Mohammad A Alam Journal: New J Chem Date: 2021-03-02 Impact factor: 3.591
Authors: Ibrahim S Alkhaibari; Hansa Raj K C; Rawan Alnufaie; David Gilmore; Mohammad A Alam Journal: ChemMedChem Date: 2021-06-09 Impact factor: 3.466
Authors: Jedidiah Whitt; Cameron Duke; Mohamad Akbar Ali; Steven A Chambers; Md Mahbub Kabir Khan; David Gilmore; Mohammad A Alam Journal: ACS Omega Date: 2019-08-21
Figure 1
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Figure 2
Scheme 5
Figure 3