Design and synthesis of Atglistatin derivatives as adipose triglyceride lipase inhibitors

Article abstract of DOI:10.1111/cbdd.13029

Adipose triglyceride lipase (ATGL) is a rate-limiting enzyme that mobilizes fatty acids from cellular triglyceride stores. Metabolic syndrome, which refers to a group of abnormalities that occur together and increase the risk of coronary artery disease, stroke, type 2 diabetes, and cachexia, can be treated using ATGL-specific inhibitors. Atglistatin (1) is the first small-molecule inhibitor of ATGL. In this study, we designed and synthesized 29 Atglistatin derivatives and evaluated their inhibition of forskolin-stimulated lipolysis in 3T3-L1 adipocytes as an indicator of their potential to inhibit ATGL in adipose tissues. Among all the tested Atglistatin analogs, we previously found that the thiourea compound 9e showed potent ATGL inhibitory activity in vitro, which was much stronger than that of Atglistatin, and its inhibitory activity in vivo was similar to that of Atglistatin. This tool compound could be used to study the pathophysiology and druggability of ATGL in animal models of metabolic disease and cachexia.

Full text of DOI:10.1111/cbdd.13029

DR. WEI LU (Orcid ID : 0000-0003-3041-9200)
Article type
: Research Article
Design and synthesis of Atglistatin derivatives as adipose triglyceride lipase
inhibitors
Jiyu Jin1,† | Suling Huang2,† | Lei Wang1 | Ying Leng2 | Wei Lu1
1 School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, P. R. China
2 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, China
Adipose triglyceride lipase (ATGL) is a rate-limiting enzyme that mobilizes fatty acids from cellular
triglyceride stores. Metabolic syndrome, which refers to a group of abnormalities that occur together
and increase the risk of coronary artery disease, stroke, type-2 diabetes, and cachexia, can be treated
using ATGL-specific inhibitors. Atglistatin (1) is the first small-molecule inhibitor of ATGL. In this
study, we designed and synthesized 29 Atglistatin derivatives and evaluated their inhibition of
forskolin-stimulated lipolysis in 3T3-L1 adipocytes as an indicator of their potential to inhibit ATGL
in adipose tissues. Among all the tested Atglistatin analogs, we previously found that the thiourea
compound 9e showed potent ATGL inhibitory activity in vitro, which was much stronger than that of
Atglistatin, and its inhibitory activity in vivo was similar to that of Atglistatin. This tool compound
could be used to study the pathophysiology and druggability of ATGL in animal models of metabolic
disease and cachexia.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.13029
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KEYWORDS
Atglistatin, Adipose Triglyceride Lipase Inhibitors, Thiourea, Structure-activity Relationship
Correspondence
Wei Lu, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, P. R. China.
E-mail: wlu@chem.ecnu.edu.cn
and
Ying Leng, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai,
China
E-mail: yleng@simm.ac.cn
† These authors contributed equally to this study.
Triglyceride plays an important role in maintaining the energy metabolism. When energy intake is
insufficient, triglyceride, which is in the form of fat droplets is dissolved into free fatty acids by the
action of lipases. Recent studies show that adipose triglyceride lipase (ATGL) plays a key role in
catalyzing the lipolysis reaction of the whole body, especially the first step. The regulatory
mechanism of ATGL is different from that of hormone-sensitive lipase (HSL) on lipolysis.[1-3] ATGL
research has currently become a hot spot.[4] Although there are numerous studies on the lipolysis
function of ATGL in adipose tissue, whether it plays a similar role and its performance in
non-adipose tissue, as well as its action mechanism, are still unclear
Inhibition of ATGL appears to be a promising strategy for the treatment of metabolic diseases
such as obesity-induced insulin resistance, type-2 diabetes,[5, 6] and cachexia.[7] Chronic ATGL
deficiency can induce cardiomyopathy in mice and humans, which would cause premature death by
excessive accumulation of triacylglycerols and impairment of mitochondrial function in cardiac
tissues.[8-10] However, the inhibition of ATGL does not appear to increase cardiac triacylglycerol
accumulation because ATGL inhibitors such as Atglistatin (1, Figure 1) do not accumulate in cardiac
tissues.[11]
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A few small-molecule inhibitors of adipose triglyceride lipase have been identified.[12, 13]
Atglistatin (1), the first small-molecule inhibitor of ATGL was reported by Mayer et al.[11] It is a
highly potent, selective, and competitive inhibitor of ATGL. The structure–activity relationship
(SAR) of Atglistatin suggests that the electron-rich substituents in the bottom ring and the
1,3-substituted pattern in the top ring are important.[11] Moving the carbamate or the N,N-dimethyl in
the para or meta position resulted in the total loss of ATGL inhibition compared with the inhibitory
activity of Atglistatin (1).[4] In this study, we clarified the process of exploring and developing
Atglistatin derivatives and discussed the SAR data that deviated from that of the urea chemotype.
In this study, we designed and synthesized a series of Atglistatin derivatives including carbamate,
hydrazinecarboxamide, thiourea, carbamothioate, carbamodithioate, and guanidine from the urea
chemotype (Figure 1). These derivatives were evaluated for their inhibition of forskolin-stimulated
lipolysis in 3T3-L1 adipocytes as an indicator of their potential to inhibit ATGL in adipose tissue.
Although thiourea is well-known to produce toxic effects and, therefore, unsuitable for drug
designing,[14] it can be used to study ATGL and validate its potential as a drug target.
FIGURE 1 Schematic modifications of Atglistatin.
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1 | MATERIALS AND METHODS
1.1 | Chemistry
1.1.1
|
Experimental
All reagents are commercially available and were used without further purification. The solvents
used were of analytical grade. Melting points were taken on a Fishere Johns melting point apparatus,
uncorrected and reported in degrees Centigrade. 1H NMR and 13C NMR spectra were scanned on a
Bruker DRX-400 (400 MHz) using tetramethylsilane (TMS) as internal standard and using one or
two of the following solvents, DMSO-d6 and CDCl3. Chemical shifts are given in δ, ppm. Splitting
patterns were designated as follows: s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet. The
mass spectra (MS) were recorded on a Finnigan MAT-95 mass spectrometer. The purity of all tested
compounds was established by HPLC to be >95.0%. HPLC analysis was performed at room
temperature using an Agilent Eclipse XDBC18 (250 mm × 4.6 mm) and as a mobile phase gradient
from 5% MeCN/H2O (1‰ TFA) for 1 min, 5% MeCN/H2O (1‰TFA) to 95% MeCN/H2O
(1‰TFA) for 9 min and 95% MeCN/H2O (1‰TFA) for 5 min more, a flow rate of 1.0 mL/min and
plotted at 254 nm.
1.1.2 | N,N-dimethyl-3'-nitro-[1,1'-biphenyl]-4-amine (4)
In a 1 L round-bottomed flask was added 3-nitrophenylboronic acid (12.51 g, 75.0 mmol),
4-bromo-N,N-dimethylaniline (15 g, 75.0 mmol), Pd(dppf)Cl2DCM (3.06 g, 3.75 mmol) and CsF
(23.92 g, 157 mmol) in anhydrous DME (600 ml). The reaction vessel was purged with nitrogen. The
reaction was heated to 80 °C with stirring on for 18 h. The reaction mixture was filtered through
celite with EA (100 mL). The mixture was concentrated by rotovap and was purified by column
chromatography to give N,N-dimethyl-3'-nitro-[1,1'-biphenyl]-4-amine 4 (15.4 g, 85% yield) as red
solid. mp 153-155 °C; 1H NMR (400 MHz, CDCl3) δ 8.40 (t, J = 1.8 Hz, 1H), 8.07 (dd, J = 8.0, 1.6
Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.53 (t, J = 8.3 Hz, 3H), 6.81 (d, J = 8.8 Hz, 2H), 3.02 (s, 6H). 13C
NMR (101 MHz, CDCl3) δ 150.65, 148.80, 142.85, 131.85, 129.50, 127.73, 126.00, 120.65, 120.50,
112.62, 40.36.
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1.1.3 | N4',N4'-dimethyl-[1,1'-biphenyl]-3,4'-diamine (5)
In a 500 mL round-bottomed flask was added N,N-dimethyl-3'-nitro-[1,1'-biphenyl]-4-amine 4 (14.5
g, 59.8 mmol) and Raney-Ni (0.513 g, 5.98 mmol) in MeOH (200 ml) to give a yellow solution. The
reaction vessel was purged with hydrogen. The reaction mixture was held at RT with stirring on for
18 h. The reaction mixture was filtered through celite with 100 mL MeOH. The mixture was
concentrated by rotovap to give N4',N4'-dimethyl-[1,1'-biphenyl]-3,4'-diamine 5 (12.5 g, 98% yield)
as bright yellow solid. The product was used to next step without further purification. mp 68-70 °C;
1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 8.8 Hz, 2H), 7.18 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.6 Hz,
1H), 6.87 (s, 1H), 6.81 (d, J = 8.7 Hz, 2H), 6.60 (d, J = 7.3 Hz, 1H), 3.72 (s, 2H), 2.99 (s, 6H). 13C
NMR (101 MHz, CDCl3) δ 150.03, 146.74, 142.45, 129.61, 129.46, 127.72, 116.98, 113.17, 113.08,
112.75, 40.65.
1.1.4 | 4-nitrophenyl(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamate (6a)
In a 100 mL round-bottomed flask was added N4',N4'-dimethyl-[1,1'-biphenyl]-3,4'-diamine 5 (1 g,
4.71 mmol), 4-nitrophenyl carbonochloridate (1.424 g, 7.07 mmol), and pyridine (0.952 ml, 11.78
mmol) in DCM (30 ml) to give a yellow solution. The reaction mixture was held at RT with stirring
on for 1 h. The reaction mixture was washed with H2O, sat.CuSO4 (aq), and sat.NaCl (aq). The
organic was dried Na2SO4, filt and conc. The crude product was recrystallized from PE and EA to
give 4-nitrophenyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamate 6a (1.57 g, 88% yield) as
yellow solid. mp 115-117 °C; 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 9.0 Hz, 2H), 7.64 (s, 1H),
7.50 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 9.0 Hz, 2H), 7.39–7.29 (m, 3H), 7.04 (s, 1H), 6.79 (d, J = 8.4
Hz, 2H), 3.00 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 155.43, 154.85, 150.24, 150.06, 145.89,
142.47, 129.55, 127.70, 126.91, 126.19, 125.56, 125.24, 122.20, 121.68, 112.73, 40.53; HPLC: room
temperature; tR=9.15 min, UV254= 95.5%; HRMS (ESI) m/z calcd for C21H19N3O4[M+H]+: 377.1376,
Found: 377.1385
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1.1.5 | General procedure for the synthesis of 6b-6c
In a 25 mL round-bottomed flask was added 4-nitrophenyl
(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamate (1.0 eq), alcohols (2.0 eq), and pyridine (3.0 eq)
in THF (5 ml) to give a yellow solution. The reaction was heated to 60°C with stirring on for 2 h.
The mixture was concentrated by rotovap and was purified by column chromatography to give
6b-6c.
ethyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamate (6b)
White solid (180 mg, 67% yield). Mp 122-124 °C; 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.49
(d, J = 8.8 Hz, 2H), 7.35–7.27 (m, 2H), 7.26 (d, J = 3.1 Hz, 1H), 6.78 (d, J = 8.8 Hz, 2H), 6.60 (s,
1H), 4.24 (q, J = 7.1 Hz, 2H), 2.99 (s, 6H), 1.32 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
153.73, 150.13, 142.15, 138.33, 129.31, 128.70, 127.74, 121.38, 116.52, 116.38, 112.73, 61.21,
40.58, 14.61; HPLC: room temperature; tR=7.83 min, UV254= 98.6%;HRMS (ESI) m/z calcd for
C17H20N2O2[M+Na]+: 307.1422, Found: 307.1427.
cyclopentyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamate (6c)
White solid (70 mg, 41% yield). mp 106-108 °C; 1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.49
(d, J = 8.7 Hz, 2H), 7.33 – 7.28 (m, 2H), 7.24 (s, 1H), 6.78 (d, J = 8.5 Hz, 2H), 6.56 (s, 1H), 5.22 (s,
1H), 2.99 (s, 6H), 1.97 – 1.85 (m, 2H), 1.80 – 1.70 (m, 4H), 1.67 – 1.60 (m, 2H). 13C NMR (101
MHz, CDCl3) δ 150.11, 142.12, 138.41, 129.29, 128.72, 127.72, 127.35, 126.74, 121.26, 116.29,
112.70, 78.08, 40.58, 32.81, 23.70; HPLC: room temperature; tR=9.23 min, UV254=95.1%; HRMS
(ESI) m/z calcd for C20H24N2O2[M+H]+: 325.1916, Found: 325.1903.
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1.1.6 | General procedure for the synthesis of 7a-7d
In a 25 mL round-bottomed flask was added 4-nitrophenyl
(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamate (1.0 eq), hydrazides (2.0 eq), and DIPEA (3.0
eq) in THF (10 ml) to give a yellow solution. The reaction was held at RT with stirring on for 18 h.
The mixture was concentrated by rotovap and was purified by column chromatography to give
7a-7d.
N-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)hydrazinecarboxamide (7a)
White solid (80 mg, 56% yield). mp 196-198 °C; 1H NMR (400 MHz, DMSO) δ 8.65 (s, 1H), 7.74
(s, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 7.24 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 7.5
Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H), 4.36 (s, 2H), 2.93 (s, 6H). 13C NMR (101 MHz, DMSO) δ 157.38,
149.80, 140.65, 140.34, 128.92, 127.81, 126.98, 118.73, 115.83, 115.31, 112.55, 40.02; HPLC: room
temperature; tR=5.69 min, UV254=97.7%; HRMS (ESI) m/z calcd for C15H18N4O[M+Na]+: 293.1378,
Found: 293.1376.
N-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-2-propionylhydrazine-1-carboxamide (7b)
White solid (150 mg, 87% yield). mp 209-211 °C; 1H NMR (400 MHz, DMSO) δ 9.57 (s, 1H), 8.71
(s, 1H), 7.98 (s, 1H), 7.67 (s, 1H), 7.45 (d, J = 7.7 Hz, 2H), 7.34 (d, J = 6.9 Hz, 1H), 7.26 (t, J = 7.4
Hz, 1H), 7.16 (d, J = 6.6 Hz, 1H), 6.80 (d, J = 7.8 Hz, 2H), 2.93 (s, 6H), 2.16 (d, J = 7.3 Hz, 2H),
1.04 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 172.96, 155.46, 149.83, 140.72, 140.07,
128.99, 127.67, 126.95, 119.20, 116.12, 115.58, 112.55, 40.00, 26.35, 9.42; HPLC: room
temperature; tR=6.04 min, UV254=99.5%; HRMS (ESI) m/z calcd for C18H22N4O2[M+H]+: 327.1821,
Found: 327.1806.
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2-benzoyl-N-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)hydrazine-1-carboxamide (7c)
White solid (150 mg, 76% yield). mp 207-209 °C; 1H NMR (400 MHz, DMSO) δ 10.30 (s, 1H), 8.88
(s, 1H), 8.21 (s, 1H), 7.93 (d, J = 7.3 Hz, 2H), 7.71 (s, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.51 (t, J = 7.4
Hz, 2H), 7.46 (d, J = 8.7 Hz, 2H), 7.37 (d, J = 7.9 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.18 (d, J = 7.6
Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 2.93 (s, 6H). 13C NMR (101 MHz, DMSO) δ 166.42, 155.68,
149.84, 140.74, 140.12, 132.56, 131.74, 129.02, 128.35, 127.68, 127.54, 126.98, 119.27, 116.22,
115.68, 112.56, 40.01; HPLC: room temperature; tR=6.93 min, UV254=98.9%; HRMS (ESI) m/z
calcd for C22H22N4O2[M+H]+: 375.1821, Found: 375.1819.
2-carbamothioyl-N-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)hydrazine-1-carboxamide (7d)
White solid (90 mg, 69% yield). mp 217-219 °C; 1H NMR (400 MHz, DMSO) δ 9.10 (s, 1H), 8.66
(s, 2H), 8.18 (s, 1H), 7.92 (s, 1H), 7.69 (s, 1H), 7.55 (s, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.2
Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 7.7 Hz, 1H), 6.80 (d, J = 8.8 Hz, 2H), 2.93 (s, 6H). 13C
NMR (101 MHz, DMSO) δ 181.11, 154.76, 149.85, 140.70, 139.98, 129.03, 127.62, 126.95, 119.28,
116.15, 115.59, 112.57, 40.02; HPLC: room temperature; tR=5.95 min, UV254=97.8%; HRMS (ESI)
m/z calcd for C16H19N5OS[M+Na]+: 352.1208, Found: 352.1206.
1.1.7 | 3'-isothiocyanato-N,N-dimethyl-[1,1'-biphenyl]-4-amine (8)
In a 250 mL round-bottomed flask was added N4',N4'-dimethyl-[1,1'-biphenyl]-3,4'-diamine (5.7 g,
26.8 mmol) and DMAP (0.656 g, 5.37 mmol) in DCM (150 ml) to give a yellow solution.
thiophosgene (2.161 ml, 28.2 mmol) was added to give yellow suspension. The reaction mixture was
held at RT with stirring on for 1 h. The reaction mixture was washed with H2O, sat. NH4Cl, and sat.
NaCl. The organic was dried Na2SO4, filt and conc. The crude product was purified by column
chromatography to give 3'-isothiocyanato-N,N-dimethyl-[1,1'-biphenyl]-4-amine 8 (5 g, 73% yield)
as light-yellow solid. mp 108-110 °C; 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 8.8 Hz, 3H), 7.37
(t, J = 1.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.07 (dd, J = 7.8, 0.9 Hz, 1H), 6.78 (d, J = 8.9 Hz, 2H),
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2.98 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 150.29, 142.88, 134.94, 131.52, 129.77, 127.67, 127.26,
125.16, 123.31, 123.02, 112.81, 40.57.
1.1.8 | General procedure for the synthesis of 9a-9i
In a 25 mL round-bottomed flask was added 3'-isothiocyanato-N,N-dimethyl-[1,1'-biphenyl]-4-amine
(1.0 eq) and ethanamine (1.5 eq) in DCM (10 ml) to give a yellow solution. The reaction mixture was
held at RT with stirring on for 1 h. The mixture was concentrated by rotovap and was purified by
column chromatography to give 9a-9i.
1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)thiourea (9a)
White solid (210 mg, 79% yield). mp 202-204 °C; 1H NMR (400 MHz, DMSO) δ 9.71 (s, 1H), 7.65
(s, 1H), 7.49 (d, J = 8.8 Hz, 3H), 7.37 – 7.30 (m, 3H), 7.27 – 7.20 (m, 1H), 6.80 (d, J = 8.8 Hz, 2H),
2.94 (s, 6H). 13C NMR (101 MHz, DMSO) δ 180.93, 149.91, 140.80, 139.50, 129.08, 127.25,
127.07, 121.57, 120.43, 120.09, 112.58, 40.00; HPLC: room temperature; tR=6.26 min,
UV254=98.7%; HRMS (ESI) m/z calcd for C15H17N3S[M+Na]+: 294.1041, Found: 294.1048.
1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-3-methylthiourea (9b)
White solid (80 mg, 48% yield). mp 161-163 °C; 1H NMR (400 MHz, DMSO) δ 9.56 (s, 1H), 7.72
(s, 1H), 7.60 (s, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.34 (s, 1H), 7.32 (s, 1H), 7.21 (s, 1H), 6.80 (d, J = 8.8
Hz, 2H), 2.94 (s, 9H). 13C NMR (101 MHz, DMSO) δ 180.99, 149.90, 140.83, 139.52, 129.09,
127.28, 127.06, 121.44, 120.67, 120.33, 112.59, 40.00, 31.25; HPLC: room temperature; tR=6.83
min, UV254=98.2%; HRMS (ESI) m/z calcd for C16H19N3S[M+H]+: 286.1378, Found: 286.1366.
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1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-3-ethylthiourea (9c)
White solid (280 mg, 79% yield). mp 152-154 °C; 1H NMR (400 MHz, DMSO) δ 9.46 (s, 1H), 7.79
(s, 1H), 7.62 (s, 1H), 7.48 (d, J = 8.8 Hz, 2H), 7.35 – 7.28 (m, 2H), 7.21 (s, 1H), 6.80 (d, J = 8.8 Hz,
2H), 3.50 (q, J = 7.2 Hz, 2H), 2.94 (s, 6H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ
179.96, 149.90, 140.74, 139.60, 129.02, 127.31, 127.03, 121.29, 120.55, 120.19, 112.60, 40.00,
38.71, 14.21; HPLC: room temperature; tR=7.62 min, UV254=96.6%; HRMS (ESI) m/z calcd for
C17H21N3S[M+Na]+: 322.1354, Found: 322.1365.
1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-3-isopropylthiourea (9d)
White solid (50 mg, 41% yield). mp 140-142 °C; 1H NMR (400 MHz, DMSO) δ 9.35 (s, 1H), 7.67
(s, 1H), 7.64 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.35 – 7.27 (m, 2H), 7.22 – 7.25 (m, 1H), 6.80 (d, J =
8.8 Hz, 2H), 4.41 (td, J = 13.2, 6.8 Hz, 1H), 2.94 (s, 6H), 1.17 (d, J = 6.5 Hz, 6H). 13C NMR (101
MHz, DMSO) δ 179.10, 149.88, 140.60, 139.83, 128.91, 127.37, 127.01, 121.03, 120.25, 119.93,
112.60, 45.44, 40.00, 21.91; HPLC: room temperature; tR=7.97 min, UV254=99.4%; HRMS (ESI) m/z
calcd for C18H23N3S[M+Na]+: 336.1510, Found: 336.1507.
3-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-1,1-dimethylthiourea (9e)
White solid (250 mg, 89% yield). mp 68-71 °C; 1H NMR (400 MHz, DMSO) δ 9.03 (s, 1H), 7.55 (s,
1H), 7.48 (d, J = 8.9 Hz, 2H), 7.32 (s, 1H), 7.30 (t, J = 5.8 Hz, 1H), 7.21 (dt, J = 6.4, 2.1 Hz, 1H),
6.80 (d, J = 8.9 Hz, 2H), 3.30 (s, 6H), 2.93 (s, 6H). 13C NMR (101 MHz, DMSO) δ 181.12, 149.81,
141.47, 139.97, 128.22, 127.40, 126.91, 123.09, 122.65, 121.39, 112.61, 40.87, 40.02; HPLC: room
temperature; tR=6.99 min, UV254=98.1%; HRMS (ESI) m/z calcd for C17H21N3S[M+Na]+: 322.1354,
Found: 322.1340.
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3-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-1,1-diethylthiourea (9f)
White solid (160 mg, 62% yield). mp 145-147 °C; 1H NMR (400 MHz, DMSO) δ 8.92 (s, 1H), 7.51
(s, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 7.8 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 7.5
Hz, 1H), 6.80 (d, J = 8.8 Hz, 2H), 3.77 (q, J = 7.0 Hz, 4H), 2.94 (s, 6H), 1.18 (t, J = 7.0 Hz, 6H). 13C
NMR (101 MHz, DMSO) δ 179.43, 149.80, 141.39, 139.88, 128.13, 127.37, 126.92, 124.06, 123.58,
121.65, 112.60, 44.76, 40.02, 12.72; HPLC: room temperature; tR=8.11 min, UV254=98.1%; HRMS
(ESI) m/z calcd for C19H25N3S[M+Na]+: 350.1667, Found: 350.1660.
N-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)piperidine-1-carbothioamide (9g)
White solid (56 mg, 18% yield). mp 187-189 °C; 1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 8.8 Hz,
2H), 7.33 (d, J = 6.1 Hz, 2H), 7.25 (s, 1H), 7.21 (s, 1H), 6.98 (d, J = 6.3 Hz, 1H), 6.79 (d, J = 8.9 Hz,
2H), 3.78 (s, 4H), 3.00 (s, 6H), 1.66 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 182.72, 150.15, 142.19,
140.72, 129.30, 128.21, 127.63, 122.65, 120.21, 120.06, 112.67, 51.03, 40.53, 25.53, 24.13; HPLC:
room temperature; tR=8.15 min, UV254=99.1%; HRMS (ESI) m/z calcd for C20H25N3S[M+H]+:
340.1847, Found: 340.1846.
N-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)morpholine-4-carbothioamide (9h)
White solid (150 mg, 47% yield). mp 174-176 °C; 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 8.8 Hz,
2H), 7.35 (d, J = 4.8 Hz, 2H), 7.31 (s, 1H), 7.28 (s, 1H), 7.07–6.93 (m, 1H), 6.78 (d, J = 8.7 Hz, 2H),
3.87–3.80 (m, 4H), 3.75–3.69 (m, 4H), 3.00 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 183.74, 150.22,
142.39, 140.19, 129.46, 127.91, 127.62, 123.14, 120.52, 120.34, 112.67, 66.14, 49.82, 40.50; HPLC:
room temperature; tR=7.08 min, UV254=95.2%; HRMS (ESI) m/z calcd for C19H23N3OS[M+Na]+:
364.1460, Found: 364.1457.
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3-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-1-methyl-1-phenylthiourea (9i)
White solid (220 mg, 65% yield). m.p.109–111°C; 1H NMR (400 MHz, CDCl3) δ 7.53 (t, J = 7.7 Hz,
2H), 7.47 – 7.39 (m, 4H), 7.38 (s, 1H), 7.35 (d, J = 5.1 Hz, 2H), 7.32 (t, J = 7.7 Hz, 1H), 7.23 (d, J =
7.7 Hz, 1H), 7.01 (s, 1H), 6.76 (d, J = 8.8 Hz, 2H), 3.77 (s, 3H), 2.98 (s, 6H). 13C NMR (101 MHz,
CDCl3) δ 181.38, 150.07, 143.02, 141.81, 139.52, 130.79, 128.80, 128.45, 127.73, 127.02, 123.85,
123.45, 123.18, 112.66, 43.59, 40.56; HPLC: room temperature; tR=8.83 min, UV254=100.0%;
HRMS (ESI)m/z calcd for C22H23N3S[M+Na]+: 384.1510, Found: 384.1498.
1.1.9 | General procedure for the synthesis of 10a-10d
In a 50 mL round-bottomed flask was added 1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)thiourea 9a
(1.0 eq) and iodomethane (2.5 eq) in ethanol (20 ml) to give a white suspension. The reaction was
heat to 85°C with stirring on for 6 h. The mixture was concentrated by rotovap. The crude product
and amine (10 eq) was added in sealed tube in 5 mL EtOH. The reaction was heat to 85°C with
stirring on for 16 hr. The mixture was concentrated by rotovap and was purified by column
chromatography to give 10a-10d.
1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)guanidine (10a)
White solid (100 mg, 42% yield). mp 197-199 °C; 1H NMR (400 MHz, DMSO) δ 7.77 (s, 4H), 7.51
(d, J = 8.8 Hz, 2H), 7.41 – 7.34 (m, 2H), 7.29 (s, 1H), 7.07 – 6.91 (m, 1H), 6.80 (d, J = 8.9 Hz, 2H),
2.94 (s, 6H). 13C NMR (101 MHz, DMSO) δ 176.30, 156.16, 150.00, 141.60, 137.80, 129.80,
127.20, 126.95, 122.30, 121.05, 120.60, 112.52, 39.97; HPLC: room temperature; tR=5.69 min,
UV254=98.9%; HRMS (ESI) m/z calcd for C15H18N4[M+H]+: 255.1610, Found: 255.1604.
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1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-3-methylguanidine (10b)
White solid (25 mg, 53% yield). mp 164-166 °C; 1H NMR (400 MHz, DMSO) δ 7.45 (d, J = 8.7 Hz,
2H), 7.19 (t, J = 7.7 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.95 (s, 1H), 6.77 (d, J = 8.8 Hz, 2H), 6.65 (d,
J = 7.8 Hz, 1H), 5.07 (s, 3H), 2.92 (s, 6H), 2.67 (s, 3H). 13C NMR (101 MHz, DMSO) δ 152.13,
151.18, 149.62, 140.90, 129.09, 128.47, 126.98, 120.72, 120.20, 117.69, 112.58, 40.08, 27.70;
HPLC: room temperature; tR=5.78 min, UV254=98.7%; HRMS (ESI) m/z calcd for C16H20N4[M+H]+:
269.31766, Found: 269.1768.
1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-3-ethylguanidine (10c)
White solid (129 mg, 83% yield). mp 167-169 °C; 1H NMR (400 MHz, DMSO) δ 7.46 (d, J = 8.8
Hz, 2H), 7.19 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.7 Hz, 1H), 6.94 (s, 1H), 6.78 (d, J = 8.8 Hz, 2H),
6.65 (d, J = 7.7 Hz, 1H), 5.36 (s, 1H), 4.88 (s, 2H), 3.16 (q, J = 7.2 Hz, 2H), 2.93 (s, 6H), 1.09 (t, J =
7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 151.52, 151.21, 149.61, 140.89, 129.07, 128.52,
126.97, 120.71, 120.19, 117.54, 112.58, 40.08, 35.16, 15.05; HPLC: room temperature; tR=6.06 min,
UV254=98.4%; HRMS (ESI) m/z calcd for C17H22N4[M+H]+: 283.1923, Found: 283.1909.
3-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-1,1-dimethylguanidine (10d)
White solid (117 mg, 66% yield). mp 119-121 °C; 1H NMR (400 MHz, DMSO) δ 7.48 (d, J = 8.9
Hz, 2H), 7.29 (t, J = 7.8 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 6.78 (d, J = 8.9 Hz, 2H),
6.76 (d, J = 7.8 Hz, 1H), 6.22 (s, 2H), 2.95 (s, 6H), 2.93 (s, 6H). 13C NMR (101 MHz, DMSO) δ
153.13, 150.94, 149.66, 141.06, 129.34, 128.23, 126.96, 120.74, 120.22, 118.02, 112.57, 40.04,
37.36; HPLC: room temperature; tR=5.85 min, UV254=98.8%; HRMS (ESI) m/z calcd for
C17H22N4[M+H]+: 283.1923, Found: 283.1921.
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1.1.10 | General procedure for the synthesis of 11a-11e
In a 50 mL round-bottomed flask was added alcohols (5.0 eq) in anhydrous THF (2 ml) to give a
colorless solution. The reaction vessel was purged with nitrogen. NaH (2.0 eq) was added. The
reaction mixture was held at RT for 0.5 h. 3'-isothiocyanato-N,N-dimethyl-[1,1'-biphenyl]-4-amine 5
(1.0 eq) in 2 mL anhydrous THF was added. The reaction mixture was held at RT for 1 hr. 2 mL
water was added. The mixture was concentrated by rotovap and was purified by column
chromatography to give 11a-11e.
O-methyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamothioate (11a)
White solid (130 mg, 58% yield). mp 125-127 °C; 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 7.46
(d, J = 8.5 Hz, 2H), 7.42 – 7.27 (m, 3H), 7.15 (s, 1H), 6.79 (d, J = 8.8 Hz, 2H), 4.13 (s, 3H), 2.99 (s,
6H). 13C NMR (101 MHz, CDCl3) δ 189.63, 150.24, 142.33, 129.31, 128.12, 127.72, 127.52, 123.60,
119.67, 112.78, 112.72, 40.52, 29.73; HPLC: room temperature; tR=8.42 min, UV254=98.7%; HRMS
(ESI) m/z calcd for C16H18N2OS[M+Na]+: 287.1218, Found: 287.1215.
ethyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamate (11b)
White solid (180 mg, 67% yield). mp 124-126 °C; 1H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 7.46
(d, J = 8.7 Hz, 3H), 7.35– 7.31 (m, 2H), 7.14 (s, 1H), 6.78 (d, J = 8.9 Hz, 2H), 4.64 (q, J = 7.0 Hz,
2H), 2.98 (s, 6H), 1.42 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 188.68, 150.22, 142.25,
137.52, 129.28, 128.17, 127.67, 123.25, 119.29, 119.01, 112.73, 68.79, 40.53, 14.20; HPLC: room
temperature; tR=8.89 min, UV254=99.1%; HRMS (ESI) m/z calcd for C17H20N2OS[M+H]+: 301.1375,
Found: 301.1370.
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O-isopropyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamothioate (11c)
White solid (190 mg, 77% yield). mp 163-165 °C; 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 7.46
(d, J = 8.7 Hz, 3H), 7.39 – 7.28 (m, 2H), 7.13 (s, 1H), 6.79 (d, J = 8.9 Hz, 2H), 5.80 – 5.53 (m, 1H),
2.99 (s, 6H), 1.43 (d, J = 5.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 187.89, 150.20, 142.20,
137.55, 129.26, 128.20, 127.64, 123.04, 119.14, 118.85, 112.79, 112.73, 40.52, 21.79; HPLC: room
temperature; tR=9.62 min, UV254=96.2%; HRMS (ESI) m/z calcd for C18H22N2OS[M+Na]+:
337.1351, Found:337.1364.
Cyclopentyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamate (11d)
White solid (70 mg, 61% yield). mp 157-159 °C; 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 7.46
(d, J = 8.5 Hz, 3H), 7.37 – 7.27 (m, 2H), 7.10 (s, 1H), 6.78 (d, J = 8.8 Hz, 2H), 5.83 (s, 1H), 2.99 (s,
6H), 2.02 – 1.89 (m, 4H), 1.82 – 1.72 (m, 2H), 1.70 – 1.61 (m, 2H). 13C NMR (101 MHz, CDCl3) δ
188.07, 150.20, 142.22, 137.67, 129.26, 128.19, 127.63, 123.01, 118.93, 118.70, 112.70, 86.44,
40.52, 32.82, 23.91; HPLC: room temperature; tR=10.40 min, UV254=98.5%; HRMS (ESI) m/z calcd
for C20H24N2OS[M+Na]+: 363.1507, Found: 363.1523.
O-phenyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamothioate (11e)
Yellow solid (300 mg, 91% yield). mp 114-116 °C; 1H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 8.29
– 7.95 (m, 1H), 7.95 – 7.49 (m, 6H), 7.40 (s, 4H), 7.26 – 7.20 (m, 1H), 7.12 (s, 2H), 3.17 (s, 6H).13C
NMR (101 MHz, CDCl3) δ 187.64, 129.50, 129.42, 129.07, 126.48, 126.39, 124.55, 124.41, 123.03,
122.62, 122.53, 122.03, 121.83, 120.87, 120.81, 46.97; HPLC: room temperature; tR=9.44 min,
UV254=95.6%; HRMS (ESI) m/z calcd for C21H20N2OS[M+H]+: 349.1375, Found: 349.1359.
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1.1.11 | methyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamodithioate (12a)
In a 50 mL round-bottomed flask was added CS2 (0.639 ml, 10.60 mmol) and NaOH (0.459 ml, 9.19
mmol) in DMSO (10 ml) to give a colorless solution. N4',N4'-dimethyl-[1,1'-biphenyl]-3,4'-diamine 5
(1.5 g, 7.07 mmol) was added. The reaction mixture was cooled to 0°C. Dimethyl sulfate (0.743 ml,
7.77 mmol) was added. The reaction mixture was warm to RT with stirring on for 18 h. 30 mL water
was added. The aq layer was extracted with EA. Combined the organic layers and washed with
water, 1M HCl and sat.NaCl. The organic was dried Na2SO4, filt and conc. The crude product was
purified by column chromatography to give methyl
(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamodithioate 12a (1.35 g, 63% yield) as yellow solid.
mp 107-109 °C; 1H NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 7.62 (s, 1H), 7.51 (s, 1H), 7.49 (d, J =
8.8 Hz, 2H), 7.41 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 6.4 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 3.00 (s, 6H),
2.67 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 150.30, 142.55, 138.23, 129.51, 127.69, 125.34, 122.89,
122.47, 112.68, 40.49, 19.21; HPLC: room temperature; tR=8.72 min, UV254=99.2%; HRMS (ESI)
m/z calcd for C16H18N2S2[M+Na]+: 325.0809, Found: 325.0794.
1.1.12 | allyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamodithioate (12b)
In a 50 mL round-bottomed flask was N4',N4'-dimethyl-[1,1'-biphenyl]-3,4'-diamine 5 (1.5 g, 7.07
mmol), NaOH (0.424 ml, 8.48 mmol) and CS2 (0.639 ml, 10.60 mmol) in DMSO (10 ml) to give a
yellow solution. The reaction mixture was held at RT for 2 h. Allyl bromide (0.673 ml, 7.77 mmol)
was added. The reaction mixture was held at RT for 5 h. 20 mL water was added. The aq layer was
extracted with EA. Combined the organic layers and washed with water, 1M HCl and sat brine. The
organic was dried Na2SO4, filt and conc. The crude product was purified by column chromatography
to give allyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamodithioate 12b (1.11 g, 48% yield) as
yellow solid. mp 121-123 °C; 1H NMR (400 MHz, CDCl3) δ 9.05 – 8.53 (m, 1H), 7.63 (s, 1H), 7.50
(d, J = 8.4 Hz, 3H), 7.42 (t, J = 7.8 Hz, 1H), 7.33 (s, 1H), 6.82 (s, 2H), 5.93 (ddt, J = 17.1, 10.0, 7.0
Hz, 1H), 5.31 (d, J = 16.9 Hz, 1H), 5.17 (d, J = 10.0 Hz, 1H), 4.00 (d, J = 6.9 Hz, 2H), 3.01 (s, 6H).
13C NMR (101 MHz, CDCl3) δ 150.07, 142.39, 138.26, 132.31, 129.48, 127.75, 125.35, 122.77,
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122.55, 118.94, 112.98, 40.70, 39.44; HPLC: room temperature; tR=9.76 min, UV254=100.0%;
HRMS (ESI) m/z calcd for C18H20N2S2[M+H]+: 329.1146, Found: 329.1152.
1.1.13 | cyclohexyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamodithioate (12c)
In a 50 mL round-bottomed flask was added cyclohexanethiol (2.405 ml, 19.66 mmol) in anhydrous
THF (10 ml) to give a colorless solution. The reaction vessel was purged with nitrogen. Sodium
hydride (0.314 g, 7.86 mmol) was added. The reaction mixture was held at RT with stirring on for 1
h. 3'-isothiocyanato-N,N-dimethyl-[1,1'-biphenyl]-4-amine 8 (1 g, 3.93 mmol) inanhydrous THF (2
mL) was added. The reaction mixture was held at RT with stirring on for 16 h. 20 mL water was
added. The aq layer was extracted with EA. Combined the organic layers and wash with brine. The
organic was dried Na2SO4, filt and conc. The crude product was purified by column chromatography
to give cyclohexyl (4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)carbamodithioate 12c (1.1 g, 76% yield)
as yellow solid. mp 92-94 °C; 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 7.61 (s, 1H), 7.49 (d, J =
8.4 Hz, 3H), 7.40 (t, J = 7.8 Hz, 1H), 7.32 (s, 1H), 6.80 (d, J = 8.5 Hz, 2H), 3.95 (s, 1H), 3.00 (s,
6H), 2.21 – 2.08 (m, 2H), 1.79 – 1.68 (m, 2H), 1.61 – 1.64 (m, 1H), 1.43 – 1.53 (m, 4H), 1.25 – 1.30
(m, 1H). 13C NMR (101 MHz, CDCl3) δ 150.26, 142.39, 138.34, 129.38, 127.76, 127.71, 125.16,
122.85, 122.57, 112.70, 49.81, 40.52, 32.79, 26.19, 25.58; HPLC: room temperature; tR=11.69 min,
UV254=99.4%; HRMS (ESI) m/z calcd for C21H26N2S2[M+H]+: 371.1616, Found: 371.1620.
1.1.14 | N3,N4',N4'-trimethyl-[1,1'-biphenyl]-3,4'-diamine (13)
In a 100 mL round-bottomed flask was added N4',N4'-dimethyl-[1,1'-biphenyl]-3,4'-diamine 5 (260
mg, 1.225 mmol), paraformaldehyde (184 mg, 6.12 mmol), and sodium methanolate (331 mg, 6.12
mmol) in MeOH (25 ml) to give a colorless solution. The reaction was heat to 65°C with stirring on
for 2 h. The reaction mixture was cooled to 0°C with stirring on. Sodium borohydride (232 mg, 6.12
mmol) was added. The reaction was heat to 65°C with stirring on for 1 h. The reaction mixture was
held at RT with stirring on for 16 h. The mixture was concentrated by rotovap. 20 mL water was
added. The aq layer was extracted with DCM. The organic was dried Na2SO4, filt and conc to give
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N3,N4',N4'-trimethyl-[1,1'-biphenyl]-3,4'-diamine 13 (274 mg, 99% yield) as white solid. mp 72-74
°C; 1H NMR (400 MHz, CDCl3) δ 7.53-7.45 (m, 2H), 7.22 (t, J = 7.8 Hz, 1H), 6.91 (d, J = 7.7 Hz,
1H), 6.82-6.75 (m, 3H), 6.54 (dd, J = 8.0, 1.6 Hz, 1H), 3.74 (s, 1H), 2.97 (d, J = 7.5 Hz, 6H), 2.88 (s,
3H).
1.1.15 | 1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-1,3,3-trimethylthiourea (14)
In a 50 mL round-bottomed flask was added N3,N4',N4'-trimethyl-[1,1'-biphenyl]-3,4'-diamine 13
(125 mg, 0.552 mmol), dimethylcarbamothioic chloride (82 mg, 0.663 mmol), and pyridine (0.067
ml, 0.828 mmol) in toluene (10 ml) to give a yellow solution. The reaction vessel was purged with
nitrogen. The reaction was heat to 110°C with stirring on for 48 h. The mixture was concentrated by
rotovap and was purified by column chromatography to give
1-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-1,3,3-trimethylthiourea 14 (20 mg, 12% yield) as white
solid. mp 64-66 °C; 1H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 8.8 Hz, 2H), 7.38 – 7.29 (m, 2H),
7.20 (s, 1H), 6.91 (d, J = 7.2 Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H), 3.59 (s, 3H), 3.00 (s, 6H), 2.99 (s,
6H). 13C NMR (101 MHz, CDCl3) δ 190.45, 150.26, 148.69, 142.86, 129.90, 127.91, 127.62, 122.64,
121.21, 121.09, 112.66, 45.24, 43.06, 40.51; HPLC: room temperature; tR=8.71 min, UV254=100.0%;
HRMS (ESI) m/z calcd for C18H23N3S[M+Na]+: 336.1510, Found: 336.1497.
1.2 | Lipolysis of 3T3-L1 cells in vitro
3T3-L1 fibroblasts were cultivated in DMEM containing 4.5 g/l glucose and L-glutamine
supplemented with 10% FBS and antibiotics under standard conditions. Cells were seeded in 48-well
plates, and grown for 2 days post-confluence and cultured in DMEM/FBS supplemented 5 μg/ml
insulin, 0.5 μM dexamethasone and 0.5 mM isobutylmethylxanthine for 3 days. Medium was
replaced with DMEM/FBS supplemented with only 5 μg/ml insulin for 3 days and then DMEM/FBS
alone for 2 days. 3T3-L1 cells were used at day 9 of differentiation. Cells were preincubated with 1
μM, 10 μM of compounds for 2 h. Then, the medium was replaced by DMEM (Phenol Red-free),
and 1 μM, 10 μM of compounds in the presence of 20 μM forskolin for 1 h. The release of glycerol
in the medium was determined using commercial kits (Applygen, Beijing).
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1.3 | Inhibition of lipolysis in vivo
CD1 mice were maintained under a 12 h light–dark cycle with free access to water and food. Animal
experiments were approved by the Animal Care and Use Committee, Shanghai Institute of Materia
Medica. The inhibitors 9e and Atglistatin (100 µmol/kg, respectively) were dissolved in olive oil and
applied by oral gavage to CD1 mice fasted overnight. Olive oil alone served as control. 8 h after
application, blood was taken retro-orbitally, and serum glycerol was measured using commercial kits
(Applygen, Beijing).
Statistical Analysis
Statistical significance was determined by Student’s unpaired t-test (two-tailed) using Graphpad
Prism software. Values of p<0.05 were considered to be statistically significant.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
The synthesis of all Atglistatin analogues started from the key intermediate
N4',N4'-dimethyl-[1,1'-biphenyl]-3,4'-diamine (5) . We prepared the compound 5 which was illustrated
in Scheme 1. The starting material 4-bromo-N,N-dimethylaniline (2) reacted with (3-nitrophenyl)
boronic acid (3) in order to form compound N,N-dimethyl-3'-nitro-[1,1'-biphenyl]-4-amine (4) in
excellent yield by a Pd(0)-catalyzed Suzuki-Miyaura cross-coupling reaction.[11] Reduction of 4 with
Raney-Ni/H2 produced the key intermediate amine 5 (Scheme 1).
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SCHEME 1 Synthesis of Intermediate 5. Reagents and conditions: (a) Pd(dppf)Cl2DCM, CsF, anhydrous
DME, 80°C; (b) Raney-Ni, H2, MeOH, rt;
The compound 6a was obtained by compound 5 with 4-nitrophenyl chloroformate in the presence
of pyridine. Then the compound 6a reacted with different alcohols and hydrazides using DIPEA to
give the corresponding carbamate and hydrazinecarboxamide compounds 6b-c, 7a-d (Scheme 2).
SCHEME 2 Synthesis of Atglistatin derivatives 6b-c, 7a-d. Reagents and conditions: (a) 4-nitrophenyl
chloroformate, pyridine, anhydrous DCM, rt; (b) R1OH, DIPEA, THF, rt; (c) R2NHNH2, DIPEA, THF, rt;
Compound 5 reacted with thiophosgene to form key intermediate thioisocyanate 8.[15] The
thioisocyanate 8 reacted with nine different amines using DIPEA to produce the thioureas compound
9a-i (Scheme 3). Methylation of 9a and subsequent substitution reactions with various amines
produced the desired guanidine compounds 10a-d (Scheme 3).[16] Addition of appropriate alcohols or
cyclohexanethiol to isothiocyanate 8 with sodium hydride gave the carbamothioate and
carbamodithioate compounds 11a-e, 12c, respectively (Scheme 3).[17] Target carbamodithioate
compounds 12a-b were synthesized from the reaction of compound 5 with carbon disulfide and
dimethyl sulfate or 3-bromoprop-1-ene in one-pot procedure (Scheme 3).[18]
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SCHEME 3 Synthesis of Atglistatin derivatives 9a-i, 10a-d, 11a-e, 12a-b. Reagents and conditions: (a)
thiophosgene, DMAP, DCM, rt; (b) R3NH2, DCM, rt; (c) iodomethane, EtOH, 85°C; (d) R5OH, NaH,
anhydrous THF, rt; (e) R6SH, NaH, anhydrous THF, rt; (f) CS2, NaOH, DMSO, rt;
The compound 5 reacted with paraformaldehyde using NaOCH3 and NaBH4 to form compound
13. [19] Then, the compound 13 was acetylated with dimethylcarbamothioic chloride to give thiourea
14 (Scheme 4).
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SCHEME 4. Synthesis of Atglistatin derivatives 14. Reagents and conditions: (a) paraformaldehyde,
NaOCH3, NaBH4, EtOH, 65°C; (b) dimethylcarbamothioic chloride, pyridine, toluene, reflux.
2.2 | ATGL inhibitory activity
2.2.1 | Inhibition of ATGL on lipolysis in vitro
The inhibitory activity of all the synthesized Atglistatin derivatives against ATGL was determined
based on their effects on forskolin-stimulated lipolysis in 3T3-L1 adipocytes (Figures 2-5), which is
in an indirect pathway. Cells were preincubated with the Atglistatin analogs for 2 h. Then, the
medium was replaced with Dulbecco’s modified Eagle’s medium (DMEM, phenol red-free), and the
Atglistatin analogs were incubated with the cells in the presence of forskolin for 1 hour. Finally, the
glycerol content of the medium was detected using commercial kits.
FIGURE 2 Effect of 7a-7d, 10a-10d on glycerol release in vitro: Differentiated 3T3-L1 adipocytes were firstly
foreincubated for 2 hours with the indicated concentrations of 7a-7d, 10a-10d, or Atglistatin. Then, the medium
was replaced by DMEM (Phenol Red-free), containing 20 μM forskolin and 1 μM, 10μM, of 7a-7d, 10a-10d or
Atglistatin for 1 h. The content of the glycerol in the medium was detected with the aid of commercial kits. 7a-7d
and 10a-10d were dissolved in DMSO. And the separate DMSO was used as the negative control. Data was
presented as mean ± SEM (n=3). * p 0.05, **p 0.01 vs control.
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In conclusion, the carbamate (6a-c), thiourea (9a-i), carbamothioate (11a-e), and carbamodithioate
(12a-c) derivatives inhibited ATGL to different degrees compared with the inhibitory activity of
Atglistatin (Figures 3 and 4). Furthermore, the hydrazinecarboxamide (7a-d) and guanidine (10a-d)
derivatives were completely inactive in inhibiting ATGL (Figure 2). The specific SAR are discussed
below.
In the carbamate compounds, compound 6b, which had an ethyl group as the O-substituent
displayed a weak inhibitory activity compared with that of Atglistatin (Figure 3). When the
O-substitute was an aryl or five-membered cycloaliphatic ring such as in the 6a and 6c derivatives,
the compounds appeared to lose inhibitory effectiveness. Therefore, the small substituent R1 groups
showed higher activity than the large ones did and the carbamate compounds did not exhibit any
improved inhibitory activity compared to that of Atglistatin.
FIGURE 3 Effects of 6a-6c, 11a-11e and 12a-12c on glycerol release in vitro: Differentiated 3T3-L1 adipocytes
were treated as described in figure 2 with the indicated concentrations of 6a-6c, 11a-11e and 12a-12c or Atglistatin.
Data were presented as mean ± SEM (n=3). * p 0.05, **p 0.01 vs control.
The carbamothioate compounds (11a-c) exhibited activities that were in the following order: ethyl >
methyl > isopropyl (Figure 3). The carbamodithioate Atglistatin derivative (11b) showed better
inhibitory effects than Atglistatin did on lipolysis in 3T3-L1 adipocytes in vitro. By comparing the
glycerol release induced by compound 6b with that of compound 11b, we found that the
carbamodithioate was slightly stronger than the carbamate was in ATGL inhibition.
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The carbamodithioate analogs displayed a weaker inhibitory activity than Atglistatin did (Figure
3). The 12a-b analogs, which had methyl or allyl groups as the R6 group showed slight inhibitory
effect, and the cyclohexyl-substituted analog 12c was completely inactive.
The thiourea compounds with an N-substituent, an ethyl (9c) or dimethyl (9e), showed inhibitory
effects (Figure 4) while the non-substituted (9a) or those with bulky steric groups (9f-i) showed
decreased or no lipolysis inhibition. Among all the compounds, the N-dimethyl-substituted thiourea
compound 9e exhibited a more potent inhibitory activity against ATGL than Atglistatin did. Notably,
at a concentration of 10 μM, compound 9e displayed approximately a 2-fold more potent inhibitory
activity than that of Atglistatin. Furthermore, the methylation of arylamine in compound 9e
inadvertently abrogated the activity of the resultant compound 14. Therefore, we concluded that the
N-H in arylamine played a crucial role in inhibiting glycerol release.
FIGURE 4 Effects of 9a-9i and 14 on glycerol release in vitro. Differentiated 3T3-L1 adipocytes were treated as
described in figure 2 with the indicated concentrations of 9a-9i and 14 or Atglistatin. Data were presented as mean
± SEM (n=3). * p 0.05, **p 0.01 vs control.
Furthermore, we determined the half-maximal inhibitory concentration (IC50) values of the
compounds using at least eight different concentrations. Furthermore, 9e and Atglistatin inhibited
glycerol release in the 3T3-L1 cells in response to 20 μM forskolin, with IC50 values of 0.9 and 3.3
μM, respectively (Figure 5). This result indicates that the IC50 of 9e significantly improved compared
with that of Atglistatin by nearly 3.5-fold. Moreover, 9e had no effects on cell viability and
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proliferation assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (Supporting information).
FIGURE 5 3T3-L1 cells were used at day 8 of differentiation. Cells were preincubated with 0 μM, 0.001
μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 50 μM, 100 μM of 9e or Atglistatin for 2 h. Then, the medium was
replaced by DMEM (Phenol Red-free), containing 20 µM forskolin and 0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1
μM, 10 μM, 50 μM, 100 μM of 9e or Atglistatin for 1 h. The release of glycerol in the medium was
determined using commercial kits. Data was presented as mean ± SEM (n=3).
2.2.2 | Inhibition of ATGL on lipolysis in vivo of 9e.
Finally, we evaluated the in vivo potential of 9e to inhibit lipolysis in fasted CD1 mice and
compared with that of Atglistatin. We administered 9e and Atglistatin dissolved in olive oil via oral
gavage to overnight fasted CD1 mice.[11] Eight hours after treatment, the glycerol release in the 9e-
and Atglistatin-treated mice decreased to 51% and 45%, respectively (Figure 6). Compound 9e
showed inhibition of lipolysis that was comparable to that by Atglistatin in vivo at 100 μmol/kg.
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FIGURE 6 Effects of 9e and Atglistatin on glycerol release in vivo. Compound 9e (100 µmol/kg) and
Atglistatin (100 µmol/kg) were dissolved in olive oil and applied by oral gavage to CD1 mice fasted
overnight. Application of olive oil alone served as a control. 8 h after application, blood was taken
retro-orbitally, and serum glycerol was measured using commercial kits. Data are presented as mean ± SEM
(n=6-7). * p 0.05, **p 0.01 vs control.
3 | CONCLUSION
In summary, a series of novel Atglistatin derivatives were designed and synthesized to explore
their biological activity and SAR with the lead compound, Atglistatin. SAR analysis indicated that
the carbamate, thiourea, carbamothioate, and carbamodithioate derivatives showed different degrees
of inhibitory effects on ATGL compared with that of Atglistatin. Hydrazinecarboxamide and
guanidine derivatives were inactive in inhibiting ATGL. Among the selected substituent groups, that
in the R position was crucial for the inhibition of ATGL, and the preferred sizes were those of the
ethyl and dimethyl groups. Among all the compounds, the novel thiourea Atglistatin derivative (9e)
showed the highest inhibition of ATGL activity on lipolysis in vitro. In addition, the in vitro effect of
9e was much stronger than that of Atglistatin, whereas its in vivo inhibitory activity was similar to
that of Atglistatin. Furthermore, 9e had no effects on the cell viability in the MTT assay (Supporting
information). In conclusion, in this study, we investigated ATGL antagonists and demonstrated 9e as
a useful in vitro and in vivo pharmacological tool for studying ATGL and validating its potential as a
drug target.
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CINOTNEFRLEISCTT OF
Tnfionhaecnoacmuiatphleoitnristnegdrecstl.are
CONFLICT OF INTEREST
The authors declare no competing financial interest.
FIGURE 1 Schematic modifications of Atglistatin.
SCHEME 1 Synthesis of Intermediate 5. Reagents and conditions: (a) Pd(dppf)Cl2DCM, CsF, anhydrous
DME, 80°C; (b) Raney-Ni, H2, MeOH, rt;
SCHEME 2 Synthesis of Atglistatin derivatives 6b-c, 7a-d. Reagents and conditions: (a) 4-nitrophenyl
chloroformate, pyridine, anhydrous DCM, rt; (b) R1OH, DIPEA, THF, rt; (c) R2NHNH2, DIPEA, THF, rt;
SCHEME 3 Synthesis of Atglistatin derivatives 9a-i, 10a-d, 11a-e, 12a-b. Reagents and conditions: (a)
thiophosgene, DMAP, DCM, rt; (b) R3NH2, DCM, rt; (c) iodomethane, EtOH, 85°C; (d) R5OH, NaH,
anhydrous THF, rt; (e) R6SH, NaH, anhydrous THF, rt; (f) CS2, NaOH, DMSO, rt;
Scheme 4. Synthesis of Atglistatin derivatives 14. Reagents and conditions: (a) paraformaldehyde, NaOCH3,
NaBH4, EtOH, 65°C; (b) dimethylcarbamothioic chloride, pyridine, toluene, reflux.
FIGURE 2 Effect of 7a-7d, 10a-10d on glycerol release in vitro: Differentiated 3T3-L1 adipocytes were firstly
foreincubated for 2 hours with the indicated concentrations of 7a-7d, 10a-10d, or Atglistatin. Then, the medium
was replaced by DMEM (Phenol Red-free), containing 20 μM forskolin and 1 μM, 10μM, of 7a-7d, 10a-10d or
Atglistatin for 1 h. The content of the glycerol in the medium was detected with the aid of commercial kits. 7a-7d
and 10a-10d were dissolved in DMSO. And the separate DMSO was used as the negative control. Data was
presented as mean ± SEM (n=3). * p 0.05, **p 0.01 vs control.
FIGURE 3 Effects of 6a-6c, 11a-11e and 12a-12c on glycerol release in vitro: Differentiated 3T3-L1 adipocytes
were treated as described in figure 2 with the indicated concentrations of 6a-6c, 11a-11e and 12a-12c or Atglistatin.
Data were presented as mean ± SEM (n=3). * p 0.05, **p 0.01 vs control.
FIGURE 4 Effects of 9a-9i and 14 on glycerol release in vitro. Differentiated 3T3-L1 adipocytes were treated as
described in figure 2 with the indicated concentrations of 9a-9i and 14 or Atglistatin. Data were presented as mean
± SEM (n=3). * p 0.05, **p 0.01 vs control.
FIGURE 5 3T3-L1 cells were used at day 8 of differentiation. Cells were preincubated with 0 μM, 0.001
μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 50 μM, 100 μM of 9e or Atglistatin for 2 h. Then, the medium was
replaced by DMEM (Phenol Red-free), containing 20 µM forskolin and 0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1
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μM, 10 μM, 50 μM, 100 μM of 9e or Atglistatin for 1 h. The release of glycerol in the medium was
determined using commercial kits. Data was presented as mean ± SEM (n=3).
FIGURE 6 Effects of 9e and Atglistatin on glycerol release in vivo. Compound 9e (100 µmol/kg) and
Atglistatin (100 µmol/kg) were dissolved in olive oil and applied by oral gavage to CD1 mice fasted
overnight. Application of olive oil alone served as a control. 8 h after application, blood was taken
retro-orbitally, and serum glycerol was measured using commercial kits. Data are presented as mean ± SEM
(n=6-7). * p 0.05, **p 0.01 vs control.
CINOTNEFRLEISCTT OF
Tnfionhaecnoacmuiatphleoitnristnegdrecstlare
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supporting information tab for
this article.
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