Synthesis and biological evaluation of novel sulfonamide derivatives of tricyclic thieno[2,3-d]pyrimidin-4(3H)-ones on melanin synthesis in murine B16 cells

Article abstract of DOI:10.1007/s11164-017-3023-3

Phototherapy with psoralens as photosensitizers is the commonly used method for the treatment of vitiligo. Considering the structure similarity between psoralens and thieno[2,3-d]pyrimidinones, a series of novel sulfonamide derivatives containing tricyclic thieno[2,3-d]pyrimidinone were synthesized and evaluated for melanin synthesis in murine B16 cells. All new compounds were characterized by ¹H NMR, ¹³C NMR, IR and HRMS (ESI). Among them, 6 compounds demonstrated excellent activity than positive control (8-methoxylpsoralen, 8-MOP) with more than 1.5-fold potency. Compound 11w with dichloro substitution at meta-positions in the benzenesulfonyl moiety was the most potent one (658.3?±?8.7%), exhibiting 5.0-fold stronger activity?than 8-MOP (130.9?±?8.6%). The difluoro analog compound 11o increased melanin synthesis in murine B16 cells with a 4.35-fold potency as compared to 8-MOP. These compounds may serve as lead compounds for further drug discoveries for the treatment of vitiligo.

Full text of DOI:10.1007/s11164-017-3023-3

Res Chem Intermed
DOI 10.1007/s11164-017-3023-3
Synthesis and biological evaluation of novel sulfonamide
derivatives of tricyclic thieno[2,3-d]pyrimidin-4(3H)-ones
on melanin synthesis in murine B16 cells
Li Fei Nie1,2,3 Khurshed Bozorov1,4 Chao Niu1,2
•
•
•
Guozheng Huang1,2 Haji Akber Aisa1,2
•
Received: 21 April 2017 / Accepted: 26 June 2017
Ó Springer Science+Business Media B.V. 2017
Abstract Phototherapy with psoralens as photosensitizers is the commonly used
method for the treatment of vitiligo. Considering the structure similarity between
psoralens and thieno[2,3-d]pyrimidinones, a series of novel sulfonamide derivatives
containing tricyclic thieno[2,3-d]pyrimidinone were synthesized and evaluated for
melanin synthesis in murine B16 cells. All new compounds were characterized by
1H NMR, 13C NMR, IR and HRMS (ESI). Among them, 6 compounds demon-
strated excellent activity than positive control (8-methoxylpsoralen, 8-MOP) with
more than 1.5-fold potency. Compound 11w with dichloro substitution at meta-
positions in the benzenesulfonyl moiety was the most potent one (658.3 8.7%),
exhibiting 5.0-fold stronger activity than 8-MOP (130.9 8.6%). The difluoro
analog compound 11o increased melanin synthesis in murine B16 cells with a 4.35-
fold potency as compared to 8-MOP. These compounds may serve as lead com-
pounds for further drug discoveries for the treatment of vitiligo.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-017-3023-3)
contains supplementary material, which is available to authorized users.
&
Haji Akber Aisa
haji@ms.xjb.ac.cn
1
Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical Institute
of Physics and Chemistry, Chinese Academy of Sciences, South Beijing Road 40-1,
Urumqi 830011, People’s Republic of China
2
State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization,
Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, South
Beijing Road 40-1, Urumqi 830011, People’s Republic of China
3
4
University of Chinese Academy of Sciences, Yuquan Road 19 A, Beijing 100049,
People’s Republic of China
Institute of the Chemistry of Plant Substances, Academy of Sciences of Uzbekistan, Mirzo
Ulugbekstr. 77, Tashkent, Uzbekistan 100170
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L. F. Nie et al.
Keywords Thieno[2,3-d]pyrimidinone Á Sulfonamide Á Melanin synthesis Á Vitiligo
Introduction
Vitiligo, the most frequent cause of skin depigmentation, is a chronic disorder
clinically characterized by the development of white macules due to the loss of
cutaneous melanocytes [1]. Although vitiligo is not as fatal as other diseases like
heart and Alzheimer’s disease, it could be of severe cosmetic disfiguration and the
patients usually also suffer from psychological devastation and social stigmati-
zation. Overall, around 0.06–2.28% of the world’s population is affected by
vitiligo [2], but its etiology remains elusive. It is believed that auto-immune or
auto-inflammation is the leading cause, based on the clinical association of vitiligo
with other auto-immune disorders [3]. Several other hypotheses have also been
elaborated, but not universally accepted, including genetic mutation, skin trauma,
neural theory, reactive oxygen species, and melanocytorrhagy hypothesis.
Interacted factors are suggested to trigger and contribute to the development of
vitiligo lesions.
Clinically, several drugs for treating vitiligo have been applied to stimulate
melanocyte proliferation and/or to improve melanin biosynthesis within melano-
cytes, such as topical corticosteroids, calcineurin inhibitors, vitamin D3 analogs, and
psoralens (with UV raidation) [4]. These treatments, although they cannot cure
vitiligo, might adjust melanogenesis, a multistage process stimulated by various
effectors including ultraviolet light, and then help the repigmentation of vitiligo
lesions [5].
Psoralens (also named furocoumarins) are a class of heterocyclic compounds
with a known phototherapeutic activity which have been isolated from several
natural plants such as Ammi majus and Psoralea corylifolia [6]. The most effective
psoralen is 8-methoxylpsoralen (8-MOP, 1, Fig. 1). It is argued that 8-MOP-
mediated phototherapy might stimulate melanocyte proliferation [7] and enhance
enzymatic activity [8], although precise mechanisms still remain unknown.
Psoralens, although frequently used [9], suffer from low water solubility and
carcinogenic hazards in some cases [10]. Therefore, the development of new
photosensitizers is of general interest for vitiligo treatment.
Thieno[2,3-d]pyrimidin-4(3H)-ones have been widely applied in medicinal
chemistry owing to their diverse biological functions [11, 12]. For example,
Fig. 1 The structures of the 8-methoxylpsoralen and thieno[2,3-d]pyrimidinone
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Synthesis and biological evaluation of novel sulfonamide…
eggmaone (2, Fig. 2) has been reported to inhibit Hedgehog signaling pathway for
the treatment of cancers [13]. Endo et al. [14] developed a series of 2-(isopropy-
lamino)thieno[3,2-d]pyrimidin-4(3H)-one derivatives as selective phosphodi-
esterase 7 inhibitors, with compound 3 (Fig. 2) as the most potent one for
treating inflammation disease. ICL-SIRT078 (4, Fig. 2), was found to be a
competitive sirituin 2 inhibitor with a Ki value of 0.62 0.15 lM, and is also
highly neuroprotective and might become a candidate for the treatment of
Parkinson’s disease [15]. Compound 5 containing a 1,2,4-triazole moiety shows
pronounced cytotoxicity on human colorectal cancer cell line HT-29 and cervical
cancer cell HeLa [16], but there are not reports on the synthesis of derivatives of
thieno[2,3-d]pyrimidin-4(3H)-ones as photosensitizers for the treatment of vitiligo.
The development of new therapeutic agents for vitiligo is one of the long-term
research topics of our group. Recently, we have synthesized dozens of isoxazole
chalcones [17] and psoralens derivatives [18, 19] possessing stimulation effects on
tyrosinase and melanin synthesis in murine B16 cells. Considering the structural
similarity between thieno[2,3-d]pyrimidinone and psoralens (Fig. 1), we decided to
synthesize some new sulfonamides containing tricyclic thieno[2,3-d]pyrimidinone
moieties in order to study their effects on melanogenesis in murine B16 cells.
Experimental
Reagents and solvents were purchased from Sigma-Aldrich, and used without
further purification. Thin-layer chromatography (TLC) was carried out on glass
plates coated with silica gel (G60F-254; Qingdao Haiyang Chemical) and visualized
by UV light (254 nm). The products were purified by column chromatography over
silica gel (200–300 mesh; Qingdao Haiyang Chemical). Melting points were
determined on a Buchi B-540 apparatus and uncorrected. All the NMR spectra were
recorded with a Varian 400 MHz NMR spectrometer in CDCl3, using TMS as an
Fig. 2 Chemical structure of selected derivatives of thieno [2,3-d]pyrimidin-4(3H)-ones
123

L. F. Nie et al.
internal standard. High-resolution mass spectra (HRMS) were recorded on a AB
SCIEX QSTAR Elite quadrupole time-of-flight mass spectrometer. The IR data
were recorded on a Thermo Fisher Scientific Nilolet 6700 FT-IR infrared
spectrometer (KBr). The detailed synthetic procedure and spectral data of
intermediates 6–10 were recorded in the electronic supplementary material
associated with this article.
General procedure of preparation of (11a–11x)
To a mixture of 3-amino-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]thieno[2,3-
d]pyrimidin-4-one (1.0 mmol) (10) and pyridine (1.2 mmol) in CH2Cl2 (2 mL),
benzenesulfonyl chloride (1.1 mmol) in CH2Cl2 (10 mL) was added dropwise. The
resulting mixture was stirred at room temperature for 12 h. The mixture was
concentrated under reduced pressure, and the residue was diluted with water
(30 mL). The aqueous mixture was neutralized by the addition of aqueous 10% HCl
solution and extracted with CH2Cl2 (2 9 30 mL). The organic phase was washed
with aqueous saturated NH4Cl solution and brine. The organic layer was separated
and dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure
to give the crude product, which was purified by silica gel chromatography to
produce the pure corresponding compounds. The full structual elucidation of
compounds 11a–11x can be found in the electronic supplementary material
associated with this article.
Biological activity
Cell culture
Murine B16 melanoma cell lines (B16F10) were obtained from Chinese Academy
of Sciences, China. The B16F10 cells were grown in DMEM medium (Gibco, USA)
supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL
penicillin and 100 mg/mL streptomycin (Gibco) in a humidified atmosphere with
5% CO2 at 37 °C.
Melanin contents assay
Exponentially growing cells were seeded into 6-well plates at a concentration of
5 9 105 cells per well. After 24 h incubation at 37 °C, the culture medium was
removed and replaced with fresh medium containing the candidate compounds of 50
lM concentration. The cells were incubated for another 48 h, washed with ice-cold
PBS, followed by lysis with RIPA buffer for 40 min on ice, and the lysates were
centrifuged at 10,000 g for 20 min. Supernatants containing protein were subject to
a protein assay and the pellets with intracellular melanin were solubilized in 200 lL
of 1 M NaOH for 2 h at 60 °C. Melanin amounts were determined spectrophoto-
metrically at 405 nm by a multi-plate reader. The themelanin amount was calculated
by normalizing the total melanin values with protein content (abs melanin/L g
protein).
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Synthesis and biological evaluation of novel sulfonamide…
Results and discussion
Synthesis
Chemical preparation of the target compounds is shown in Scheme 1. Diethyl
oxalate was treated with ethylmagnesium bromide to give ethyl 2-oxobutanoate (6).
Condensation of ethyl 2-oxobutanoate (6) with ethyl cyanoacetate and sufur in
ethanol in the presence of trimethylamine yielded 2-aminothiophene (7) [20, 21],
which was further condensed with d-valerolactam to afford the intermediate ethyl
thieno[2,3-d]pyrimidine-3-carboxylate 8 [22]. Hydrolysis of ester 8 was completed
in water and methanol with excessive amounts of lithium hydroxide. Application of
Curtius rearrangement to the acid 9 using diphenylphosphorylazide in tert-butanol
and following acdified hydrolysis resulted in the amine 10.
The rearrangement of intermediates 9–10 was the crucial step for the preparation
of compounds 11a–11x. A number of rearrangement conditions, such as Lossen
rearrangement, Schmidt rearrangement and Curtius rearrangement were tried to
improve the yield of 10 (Table 1).
Lossen rearrangement of the corresponding acids 9 at the presence of
hydroxylamine hydrochloride in polyphosphoric acid at 150 °C gave compounds
10 in moderate yield (58%, Table 1, Entry 1). Schmidt rearrangement method
Scheme 1 Reagents and conditions: a ethylmagnesium bromide, THF, -10 °C; b ethyl cyanoacetate, S,
EtOH, NEt3, r.t.; c d-valerolactam, phosphorus oxychloride, CH2Cl2, 45 °C; d LiOH, MeOH, r.t.; e tert-
BuOH, DPPA, NEt3, EtOH–HCl, reflux; f aromatic sulfonyl chlorides, pyridine, CH2Cl2, r.t.
123

L. F. Nie et al.
Yield (%)a
Table 1 Rearrangement from 9 to 10 under different conditions
Entry
Substrate
Rearrangement conditions
Product
1
2
3
4
a
9
9
9
9
NH2OHÁHCl, PPA, 150 °C
NaN3, H2SO4, r.t.
10
10
10
10
58
70
59b
91b
DPPA, NEt3, Toluene, 110 °C
DPPA, tert-Butanol, 85 °C
Isolated yields
b
The yield after hydrolysis
(sodium azide and sulfuric acid) gave converted acids 9 into amine 10 in 70% yield
at room temperature (Table 1, Entry 2), slightly better than Lossen rearrangement.
The best yield (91%) was achieved with the application of Curtius rearrangement. In
this case, compound 9 was smoothly transformed to 10 with diphenylphosphory-
lazide in tert-butanol under nitrogen atmosphere followed by deprotection with HCl
in ethanol. Finally, the target sulfonamides 11a–11x were synthesized by
condensation of amine 10 with appropriate aromatic sulfonyl chlorides in
dichloromethane with good to excellent yields (72–92%).
1
All new compounds were characterized by H NMR, 13C NMR, IR and HRMS
(ESI). The structures of compounds 11o and 11w were further confirmed by X-ray
diffraction analysis as shown in Fig. 3 (11o: CCDC No.: 1528441; 11w: CCDC No.
1528442) [23]. These two single crystals were obtained by slow evaporation of
ethanol at room temperature.
Melanin synthesis evaluation
All the thieno[2,3-d]pyrimidine derivatives 11a–11x were screened for their activity
on melanin synthesis in murine B16 cells, using a previously published method [24].
Fig. 3 Crystal structure of compounds 11o (left) and 11w (right)
123

Synthesis and biological evaluation of novel sulfonamide…
According to the results shown in Table 2, in general, the substitution pattern of
the benzene ring of the sulfonamide moiety dramatically influenced the activity on
melanin synthesis. The activities greatly improved when the compounds were
substituted by –NO2, –CF3, –OCF3, –F, and –Cl groups at the benzene ring of the
sulfonamide moiety, which suggested that the electron-withdrawing group might be
favorable to enhance the activity.
Compounds 11d (448.2 0.8%), 11o (570.5 10.1%) and 11w
(658.3 8.7%) of the derivatives with a six-membered ring displayed the best
activities. Among the mono- and difluoro-substituted derivatives (11c–11e, 11o–
11s), the most important factor in their efficacy was the location of the group. The
shift of the fluorine atom from the meta (11d, 448.2 0.8%) into the ortho and
Table 2 Stimulation of melanin content of B16 cells by compounds 11a–x
Compounds
R1, R2
Increased melanin content (%)
11a
11b
11c
11d
11e
11f
R1 = H, R2 = H
92.4 1.9
86.9 7.2
R1 = H, R2 = 4-CH3
R1 = H, R2 = 2-F
R1 = H, R2 = 3-F
R1 = H, R2 = 4-F
R1 = H, R2 = 4-Br
R1 = H, R2 = 4-NO2
R1 = H, R2 = 4-OCH3
R1 = H, R2 = 2-CF3
R1 = H, R2 = 3-CF3
R1 = H, R2 = 4-CF3
R1 = H, R2 = 2-OCF3
R1 = H, R2 = 3-OCF3
R1 = H, R2 = 4-OCF3
R1 = 3-F, R2 = 5-F
R1 = 3-F, R2 = 4-F
R1 = 2-F, R2 = 6-F
R1 = 2-F, R2 = 5-F
R1 = 2-F, R2 = 4-F
R1 = 3-F, R2 = 4-CH3
R1 = 2-CH3, R2 = 4-F
R1 = 3-NO2, R2 = 4-Cl
R1 = 3-Cl, R2 = 5-Cl
R1 = H, R2 = 4-Ph
–
107.1 7.4
448.2 0.8
105.2 7.2
104.0 0.9
142.3 7.1
132.0 9.9
93.8 10.5
207.5 8.2
144.1 5.9
99.1 3.6
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
11r
11s
104.9 11.6
199.5 3.9
570.5 10.1
227.8 6.2
129.9 9.2
171.5 5.2
85.7 5.9
11t
112.8 13.0
96.6 6.2
11u
11v
11w
11x
8-MOP
160.4 8.1
658.3 8.7
121.3 7.9
130.9 8.6
B16 cells were treated with 50 lM of compounds for 48 h. After that, melanin content was measured
directly. The data are recorded as the mean SD % of three experiments in duplicate, as compared to the
blank control
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L. F. Nie et al.
para positions led to 11c (107.1 7.4%) and 11e (105.2 7.2%), respectively,
which had a 4.2-fold lower activity. The introduction of a second fluorine atom to
the meta position of the benzene ring strongly increased the activity, exemplified by
11d (448.2 0.8%), 11e (105.2 7.2%) and 11c (107.1 7.4%), as compared
with 11o (570.5 10.1%), 11p (227.8 6.2%) and 11r (171.5 5.2%). 3.5-
Dichloro-substituted 11w (658.3 8.7%) exhibited the best activity compared with
all the others, being even more potent than 3,5-difluoro 11o (570.5 10.1%). From
the above results, it can be concluded that the presence of halogen (–F or –Cl) on
meta-position of benzene is crucial for maintaining the good activity.
Conclusion
In summary, a novel series of sulfonamides containing tricyclic thieno[2,3-
d]pyrimidinone were synthesized and evaluated for their ability of stimulation on
melanin synthesis in murine B16 cells. Preliminary SARs indicated that compounds
with –F and –Cl groups at the meta-position of the benzensulfonyl fragment
demonstrated better activity than the other prepared compounds. In addition, the
most important factor in their efficacy was the location of the group among these
mono- and di-halogenated compounds. It was of note that the activity of 11o
(570.5 10.1%) and 11w (658.3 8.7%) were nearly 4.4- and 5.0-fold stronger
than 8-MOP (130.9 8.6%) on melanin synthesis in murine B16 cells. The
investigated results suggest that several 5-sulfonamide-containing tri-
cyclic thieno[2,3-d]pyrimidinones revealed promising activity on melanin synthesis
in murine B16 cells and may serve as lead compounds for further drug discoveries
and the treatment of vitiligo.
Further preparation and evaluation of other tricyclic thieno[2,3-d]pyrimidinone
derivatives is ongoing and will be reported in due course.
Acknowledgements This work was supported by the National Natural Science Foundation of China
(NSFC) (Grant No. 21550110495) and the Chinese Academy of Sciences President’s International
Fellowship Initiative (Grant No. 2016 PT014).
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