New Atglistatin closely related analogues: Synthesis and structure-activity relationship towards adipose triglyceride lipase inhibition

Article abstract of DOI:10.1016/j.ejmech.2016.04.021

Adipose Triglyceride Lipase (ATGL) performs the first and rate-limiting step in lipolysis by hydrolyzing triacylglycerols stored in lipid droplets to diacylglycerols. By mediating lipolysis in adipose and non-adipose tissues, ATGL is a major regulator of

Full text of DOI:10.1016/j.ejmech.2016.04.021

Accepted Manuscript
New Atglistatin closely related analogues: synthesis and structure-activity relationship
towards adipose triglyceride lipase inhibition
Pierre-Philippe Roy, Kenneth D'Souza, Miroslava Cuperlovic-Culf, Petra C.
Kienesberger, Mohamed Touaibia
PII:
S0223-5234(16)30305-1
10.1016/j.ejmech.2016.04.021
EJMECH 8536
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 February 2016
Revised Date: 6 April 2016
Accepted Date: 7 April 2016
Please cite this article as: P.-P. Roy, K. D'Souza, M. Cuperlovic-Culf, P.C. Kienesberger, M. Touaibia,
New Atglistatin closely related analogues: synthesis and structure-activity relationship towards
adipose triglyceride lipase inhibition, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.04.021.
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ACCEPTED MANUSCRIPT
Graphical abstract
New Atglistatin closely related analogues: synthesis and structure-activity
relationship towards adipose triglyceride lipase inhibition
Pierre-Philippe Roy, Kenneth D'Souza, Miroslava Cuperlovic-Culf, Petra C. Kienesberger
and Mohamed Touaibia*.
New Atglistatin closely related analogues were synthesized. Their effects on adipose
triglyceride lipase activities were evaluated.
O
N
R1
Series A
R2
R1
NH
R1 or R2 = NHCON(CH3)2 or H; R3 or R4 = N(CH3)2 or H
Series B
R2
R1 or R2 = N(CH3)2 or H; R3 or R4 = N(CH3)2 or H
Series C
R1 or R2 = NHCON(CH3)2 or H; R3 or R4 = NHCON(CH3)2 or H
R3
R4
Series D
R3
R1 or R2 = NHCO2N(CH3)2 or N(CH3)2
R3 = NHCON(CH3)2 or N(CH3)2
N
Aglistatin (1)

ACCEPTED MANUSCRIPT
New Atglistatin closely related analogues: synthesis and structure-activity
relationship towards adipose triglyceride lipase inhibition
Pierre-Philippe Roy1, Kenneth D'Souza2, Miroslava Cuperlovic-Culf3, Petra C. Kienesberger2 and
Mohamed Touaibia1*
1Department of Chemistry and Biochemistry, Université de Moncton, Moncton, New Brunswick,
Canada E1A 3E9.
2Department of Biochemistry and Molecular Biology, Dalhousie University, Saint John, NB E2L
4L5, Canada.
3National Research Council of Canada, 1200 Montreal Road; Ottawa, ON, Canada
* To whom correspondence should be sent at the above address.
Tel: (506) 858-4493; Fax: (506) 858-4541
Email: mohamed.touaibia@umoncton.ca
Abstract
Adipose Triglyceride Lipase (ATGL) performs the first and rate-limiting step in lipolysis by
hydrolyzing triacylglycerols stored in lipid droplets to diacylglycerols. By mediating lipolysis in
adipose and non-adipose tissues, ATGL is a major regulator of overall energy metabolism and
plasma lipid levels. Since chronically high levels of plasma lipids are linked to metabolic
disorders including insulin resistance and type 2 diabetes, ATGL is an interesting therapeutic
target. In the present study, fourteen closely related analogues of Atglistatin (1), a newly
discovered ATGL inhibitor, were synthesized, and their ATGL inhibitory activity was evaluated.
The effect of these analogues on lipolysis in 3T3-L1 adipocytes clearly shows that inhibition of
the enzyme by Atglistatin (1) is due to the presence of the carbamate and N,N-dimethyl moieties
on the biaryl central core at meta and para position, respectively. Mono carbamate-substituted
analogue C2, in which the carbamate group was in the meta position as in Atglistatin (1), showed
slight inhibition. Low dipole moment of Atglistatin (1) compared to the synthesized analogues
possibly explains the lower inhibitory activities.
Keywords: Adipose Triglyceride Lipase (ATGL), Atglistatin, Inhibitors, Structure–activity
relationship, Lipolysis, Adipocytes
1

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1. Introduction
Obesity is associated with increased adipose tissue lipolysis, which is believed to contribute to
obesity-related comorbidities including insulin resistance and type 2 diabetes [1]. In light of the
worldwide obesity pandemic, there has been a growing interest in developing drugs to control
adipose tissue lipolysis and circulating lipid levels. During obesity, chronically elevated levels of
plasma fatty acids (FA) can cause an accumulation of intracellular lipids in non-adipose tissues
such as liver, skeletal muscle and heart, producing a lipotoxic environment that can trigger
insulin resistance, inflammation [2,3] and lipoapoptosis [4]. Besides playing an important role in
obesity and obesity comorbidities, adipose tissue lipolysis has also been shown to contribute to
cancer cachexia, a metabolic disorder which is characterized by the extensive loss of adipose
tissue and skeletal muscle mass and increases morbidity and mortality in cancer patients [5,6].
Hence, adipose tissue lipolysis could possibly be targeted for the treatment of cachexia by
protecting from excessive adipose tissue depletion in cancer patients [6].
The release of FA from adipose tissue into the blood stream requires three lipases, which
sequentially break down triacylglycerol stores within lipid droplets: Adipose Triglyceride Lipase
(ATGL), Hormone-Sensitive Lipase (HSL) and Monoglyceride Lipase (MGL)[7,8]. ATGL
initiates lipolysis, hydrolyzing triacylglycerol to generate FA and diacylglycerol [9]. The
diacylglycerol is then hydrolyzed by HSL, producing a FA and monoacylglycerol, which can be
further hydrolyzed by MGL [7], completing the lipolytic cascade. Since ATGL performs the first
and rate-limiting step in lipolysis, this enzyme is an interesting target for the design of lipolysis
inhibitors with therapeutic potential.
Recently, Mayer et al. [10] have developed a specific inhibitor of ATGL named Atglistatin (1,
Figure 1). Atglistatin (1) inhibits the activity of ATGL (IC50 = 0.7 µM) in a competitive
mechanism [10]. Few other ATGL inhibitors have also been developed, such as a peptide derived
from G0/G1 Switch Gene 2 protein [11] and a hydrazone base compound [12].
While the inhibition of ATGL shows promise in the treatement of metabolic diseases such as
obesity-induced insulin resistance and type-2 diabetes [13,14] as well as cachexia [6], chronic
ATGL deficiency can cause cardiomyopathy in mice and humans, leading to premature death
through severe accumulation of triacylglycerols and impairment of mitochondrial function in
cardiac tissues [15,16,17]. However, inhibition of ATGL does not appear to cause increased
2

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cardiac triacylglycerol accumulation as ATGL inhibitors such as Atglistatin (1) do not
accumulate in cardiac tissues [10].
Since ATGL has not been crystallized the developpement of new ATGL inhibitors must be done
by the traditional struture-activity relationship study (SAR). Studying the relationship between
the structure of a known inhibitor and its capacity to inhibit the target enzyme leads not only to
the optimization of said inhibitor, but also brings new insight into the enzyme structure.
In this paper we describe the design, synthesis, and biological activity of fourteen novel
analogues closely related to Atglistatin (1). These compounds were evaluated for their ability to
inhibit isoproterenol-stimulated lipolysis in 3T3-L1 adipocytes as a readout of their potential to
inhibit ATGL in adipose tissue. Our work represents the first systematic structure-activity
relationship study of such Atglistatin (1) analogues.
2. Results and discussion
2.1. Chemistry
To investigate the structure-activity relationship of Atglistatin (1) as an ATGL inhibitor, closely
related analogues were synthesized. We examined the importance of the carbamate and the N,N-
dimethyl moieties by either removing them or by interchanging the carbamate by the N,N-
dimethyl and vice versa (Figure 1). Then, the effect of the position of these groups was
investigated by the synthesis of analogues with the carbamate and the N,N-dimethyl moieties in
the meta or para positions. The synthesis of analogues having an aryl central core allowed us to
investigate whether the biaryl core of Atglistatin (1) is necessary for ATGL inhibition.
O
N
R1
Series A
R2
R1
NH
R1 or R2 = NHCON(CH3)2 or H; R3 or R4 = N(CH3)2 or H
Series B
R2
R1 or R2 = N(CH3)2 or H; R3 or R4 = N(CH3)2 or H
Series C
R1 or R2 = NHCON(CH3)2 or H; R3 or R4 = NHCON(CH3)2 or H
R3
R4
Series D
R3
R1 or R2 = NHCO2N(CH3)2 or N(CH3)2
R3 = NHCON(CH3)2 or N(CH3)2
N
Aglistatin (1)
Figure 1. Structural analogues based on (1) synthesized in this work.
Analogues of Aglistatin (1) A1-D3 (Figure 1) were obtained in moderate to good yields through
Suzuki-Miyaura [18] cross coupling or copper(I) catalyzed homocoupling conditions [19],
catalytic hydrogenation, N-methylation, and Schotten-Baumann condensation.
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To investigate the necessity of the 3 and 4’ substitution of the biaryl central core of Aglistatin (1),
analogues with the carbamate and the N,N-dimethyl moieties at 3 and 4’ positions were
synthesized (A1-A3, Scheme 1). Intermediates 6, 7, and 8 were synthesized starting from meta
and para nitroboronic acids (2, 3) and bromo-N,N-dimethylaniline (4, 5) under Suzuki-Miyaura
[18] cross coupling conditions. Corresponding amines 9, 10, and 11 were then obtained in a
quantitative yield after a catalytic hydrogenation over Pd/C. The final products A1, A2, and A3
were obtained from amines 9, 10, and 11 through Schotten-Baumann condensation conditions
with dimethylcarbamoyl chloride (Scheme 1).
6 (2+4) R1 = NO2, R2 =H, R3 = N(CH3)2, R4 = H
R1
ii
9 R1 = NH2, R2 =H, R3 = N(CH3)2, R4 = H
R1
R3
R2 iii
A1 R1 = NHCON(CH3)2, R2 = H, R3 = N(CH3)2, R4 = H
R2
R4
7 (3+5) R1= H, R2= NO2, R3 = H, R4 = N(CH3)2
10 R1 = H, R2 = NH2, R3 = H, R4 = N(CH3)2
i
+
ii
iii
B(OH)2
Br
A2 R1 = H, R2 = NHCON(CH3)2, R3 = H, R4 = N(CH3)2
2 R1 = NO2, R2 = H
4 R3 = N(CH3)2, R4 = H R4
3 R1 = H, R2 = NO2 5 R3 = H, R4 = N(CH3)2
8 (2+5) R1= NO2, R2 = H, R3 = H, R4 = N(CH3)2
11 R1 = NH2, R2 = H, R3 = H, R4 = N(CH3)2
A3 R1 = NHCON(CH3)2, R2 = H, R3 = H, R4 = N(CH3)2
R3
ii
iii
A1-A3
Scheme 1. Synthesis of series A : (i): Pd(dppf)Cl2*DCM, CsF, dry DME, 80°C, overnight, 6 (40%), 7
(80%), 8 (85%); (ii): Pd/C 10%, H2, DCM, R.T., overnight, quantitative yield; (iii): ClCON(CH3)2, Et3N,
dry DCM, 0°C-R.T., A1 (46%), A2 (46%), A3 (36%)
.
The effect of the presence of only the N,N-dimethyl or carbamate or moiety as well as their
position on the biaryl core (meta or para) was investigated by the series B and C (Figure 1 and
Scheme 2 and 3). Analogues B1 and B2 bearing one N,N-dimethyl moiety at para and meta
position were obtained from the commercially available 3- and 4-aminobiphenyl (12 and 13)
using dimethylsulfate as methylation agent. Both the disubstituted and trisubstituted products
were isolated. Since disubstituted, trisubstituted, and tetrasubstituted amines were obtained, B1
and B2 were obtained with low yield. Corresponding analogues C1 and C2 were obtained
through the condensation of amines 12 and 13 with dimethylcarbamoyl chloride under basic
conditions.
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R1
R1
R1
R2
R2
R2
C1 R1 = NHCO(CH3)2, R2-R4 = H
C2 R1 = R3 = R4 = H, R2 = NHCO2(CH3)2
C4 R1 = NHCO(CH3)2, R2 = R4 = H, R3 = NH2
C5 R1 = R3 = NHCO(CH3)2, R2 = R4 = H
B1 R1= N(CH3)2, R2-R4 = H
B2 R1= R3 = R4 = H, R2 = N(CH3)2
i
ii
iii
R4
R4
R4
R3
R3
R3
12 R1= NH2, R2-R4 = H
13 R1= R3 = R4 = H, R2 = NH2
14 R1 = R3 = NH2, R2= R4 = H
Scheme 2. Synthesis of series B and C: (i): Me2SO4, K2CO3, THF/H2O, R.T., 24h, B1 (11%), B2 (14%);
(ii): ClCON(CH3)2, Et3N, dry DCM, 0°C-R.T., C1 (47%), C2 (37%), C4 (70%); (iii): ClCON(CH3)2,
piperidine, dry THF, 0°C-R.T., C5 (5%).
Analogues bearing two N,N-dimethyl or carbamate moieties were further investigated. While
4,4’-Bis(dimethylamino)biphenyl (B4, Figure 2) was commercially available, the remaining
analogues (B3, C3, C5) N,N-dimethyl or carbamate moieties were obtained as shown in scheme 2
and 3.
The reaction with benzidine (14) did not produce the disubstituted product C5, only the
monosubstituted product C4 was obtained (Scheme 2). As the first substitution occurred, the
monosubstituted product C4 precipitated due to its poor solubility. Furthermore, the second
amine is not reactive enough to perform the nucleophile attack on the acyl chloride. To resolve
this problem, compound C4 was solubilized in THF and piperidine was used as base. Compound
C5 was obtained with a yield of 5%, demonstrating the poor reactivity of the aniline.
Analog B3 (scheme 3) was synthesized starting from 3-(N,N-Dimethylamino)phenylboronic acid
15, using a copper(I)-catalyzed homocoupling [19]. This reaction is useful for the synthesis of
symmetric biaryl compound with mild reaction condition. Multiple side products were formed
due to the degradation of the boronic acid. Therefore, B3 was obtained only with a yield of 29%.
Analogue C3 (scheme 3) was synthesized in a similar fashion to compounds A1-A3. 3,3'-
biphenyldiamine (18) was obtained through copper(I)-catalyzed homocoupling [19] conditions of
3-Nitrophenylboronic acid (16) followed by a catalytic hydrogenation over Pd/C. Condensation
of amine (18) with dimethylcarbamoyl chloride afford analog C3.
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R1
R2
NO2
NH2
NO2
N
iii
ii
i
i
B(OH)2
16
B(OH)2
R4
O2N
H2N
15
R3
B3 R1 = R3 = H, R2 = R4 = N(CH3)2
C3 R1 = R3 = H, R2 = R4 = NHCO(CH3)2
18
17
Scheme 3. Synthesis of series B and C : (i): CuCl, MeOH, R.T., air, B3 (29%), 17 (34%); (ii): Pd/C 10%,
DCM, H2, R.T., overnight, quantitative yield; (iii): ClCON(CH3)2, Et3N, dry DCM, 0°C-R.T., C3 (36%)
To investigate whether the biaryl central core of Atglistatin (1) is necessary for ATGL inhibition,
analogues bearing only an aryl substituted with N,N-dimethyl or carbamate moieties were
synthesized (D1-D3, scheme 4). Condensation of commercially available amines 19 and 20 with
dimethylcarbamoyl chloride affords analogues D1 and D2. D2 and D1 are the analogues of
Atglistatin (1) and A1, respectively. Analogue D3, bearing two carbamate moieties in para
position, was obtained from amine 21 through condensation with dimethylcarbamoyl chloride
(scheme 4). A pre-treatment with base was required to obtain amine 21 form the corresponding
hydrochloride salt.
R1
R1
R2
R2
ii
R3
R3
19 R1 = NH2, R2 = H, R3 = N(CH3)2
20 R1 = H, R2 = NH2, R3 = N(CH3)2
21 R1 = NH2, R2 = H, R3 = NH2
D1 R1 = NHCON(CH3)2, R2 = H, R3 = N(CH3)2
D2 R1 = H, R2 = NHCON(CH3)2, R3 = N(CH3)2
D3 R1 = NHCON(CH3)2, R2 = H, R3 = NHCON(CH3)2
Scheme 4. Synthesis of series D: (i): ClCON(CH3)2, Et3N, dry DCM, 0°C-R.T., D1 (81%), D2 (67%), D3
(42%).
Even after repeated attempts, we were not able to obtain the corresponding analogue with two
carbamate moieties in meta position. Analogues D4 and D5 (Figure 2) bearing two N,N-dimethyl
moieties at para and meta position were commercially available.
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O
O
HN
N
HN
N
N
N
N
H
H
N
N
N
N
N
N
N
O
O
N
N
N
N
(B2)
(B1)
(B3)
(A3)
(B4)
(A1)
(1)
(A2)
O
O
O
O
O
HN
N
HN
N
HN
HN
N
N
H
H
N
N
N
N
N
HN
N
HN
HN
N
O
N
H
N
O
N
N
O
O
O
N
N
N
N
N
H
N
(D4)
(D2)
NH2
(C4)
(D1)
(D5)
(C2)
(C3)
(C1)
(D3)
(C5)
O
Figure 2. Structures of synthesized Atglistatin (1) closely related analogues.
2.2. ATGL inhibitory activity
The potential of our Atglistatin analogues to inhibit ATGL was examined indirectly by
determining their effect on isoproterenol-stimulated lipolysis in 3T3-L1 adipocytes (Figure 3-5).
Following incubation of adipocytes with the analogues for 1 h, concentrations of released non-
esterified FA (NEFA) and glycerol, products of lipolysis, were measured in the media. The
substitution of the central biaryl core seems to be crucial for inhibition of lipolysis and thus
ATGL activity with Atglistatin (1). In fact, the analogue A1 in which the carbamate group is in
the para position was completely inactive (Figure 3). This change, which seems minor, causes a
total inactivation of compound A1 since levels of NEFA in the media from adipocytes incubated
with A1 were similar when compared to controls. The analogue A2, in which the N,N-dimethyl
moiety is this time in the meta position, also appears to lose inhibitory effectiveness as compared
to Atglistatin (1) (Figure 3). The interconversion of the two groups as shown with analogue A3
compared to Atglistatin (1) causes also a complete inactivation. The total loss of activity with A1,
A2, and A3 clearly shows that the substitution pattern, position 3 and 4', is necessary for
inhibition of ATGL with Atglistatin (1). More specifically, the carbamate moiety must be at
position 3 and the N,N-dimethyl moiety at position 4’. Co-crystallization of Atglistatin (1) with
ATGL would confirm the presence of favorable interactions with these two substituents.
7

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Figure 3. Effect of Series A compounds on NEFA release. 3T3-L1 adipocytes were incubated in DMEM
containing 2% FA-free BSA in the presence of 2 µM isoproterenol for 1 hour plus the indicated
compounds. NEFA content of incubation media was determined and normalized to cellular protein content.
NEFA release is presented as % of control (incubated with DMSO), which was determined to be 111.83
nmol NEFA/hr · mg protein. Data are means ± SD. N=3, *p < 0.05.
To investigate the importance of the carbamate and the N,N-dimethyl moieties, series B and C, in
which these groups are absent, were synthesized. As shown in Figure 4, the presence of the
carbamate moiety was necessary for the inhibition of ATGL by Atglistatin (1). Analogues B1 and
B2, bearing only N,N-dimethyl at para or meta position lost any inhibitory activity compared to
Atglistatin (1) (Figure 4a). Even the presence of two N,N-dimethyl moieties at para or meta
positions as in analogues B3 and B4 resulted in a complete loss of ATGL inhibition.
The presence of only the carbamate moiety on the biaryl central core of Atglistatin (1) does not
seem to be as beneficial for ATGL inhibition as demonstrated by the series C (Figure 4b). Only
with the mono-substituted analogue C2, in which the carbamate group was in the meta position
as in Atglistatin (1) (see Figure 2), a slight inhibition was observed. Otherwise the di-substituted
analogues with carbamate moiety at para or meta position (C3 and C5) are completely inactive.
Interestingly, the intermediate C4, which served for the preparation of C5, in which there is a free
amine at the para position, results in a slight, but significant activation of lipolysis, suggesting a
moderate increase in ATGL activity.
8

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A
B
Figure 4. Effect of Series B and C compounds on NEFA release. 3T3-L1 adipocytes were incubated in
DMEM containing 2% FA-free BSA in the presence of 2 µM isoproterenol for 1 hour plus the indicated
compounds. NEFA content of incubation media was determined and normalized to cellular protein content.
NEFA release is presented as % of control (incubated with DMSO), which was determined to be 111.83
nmol NEFA/hr · mg protein. Data are means ± SD. N=3, *p < 0.05.
The presence of a central aryl core instead of a biaryl does not seem to be beneficial for ATGL
inhibition as shown by the series D (Figure 5). Analogues bearing the carbamate and/or N,N-
dimethyl moiety have no effect on lipolysis. Even analogue D2, bearing the same groups as
Atglistatin (1) at meta and para positions, has no effect. Analogues with only two carbamates or
N,N-dimethyl moieties (D3, D4, D5) were not active compared to Atglistatin (1). A decrease of
the lipophilic character and the loss of favorable interactions with the enzyme may explain this
total loss of activity.
Free glycerol quantification gave the same results as the quantification of NEFA; none of the
synthesized analogues showed significant changes in free glycerol release as compared to control
(data not shown). Only the mono-substituted analogue C2, in which the carbamate group was in
the meta position as in Atglistatin (1) (see Figure 2) showed a slight reduction in the free glycerol
concentration in the media.
9

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Figure 5. Effect of Series D compounds on NEFA release. 3T3-L1 adipocytes were incubated in DMEM
containing 2% FA-free BSA in the presence of 2 µM isoproterenol for 1 hour plus the indicated
compounds. NEFA content of incubation media was determined and normalized to cellular protein content.
NEFA release is presented as % of control (incubated with DMSO), which was determined to be 111.83
nmol NEFA/hr · mg protein. Data are means ± SD. N=3, *p < 0.05.
To understand the large difference in activity between Atglistatin (1) and its closest analogues
A1-A3, minimal energy as well as dipole moment of these structures were calculated. Differences
in structure, total minimal energy and dipole moment are apparent from Table 1 and Figure 6.
Although compound A2 has comparable total energy and also has a planar structure like
Atglistatin (1), A2 as well as A1 and A3 have higher dipole moments than Atglistatin (1),
possibly explaining their lower inhibitory potential. It has been shown that dipole moment can
play a key role in drug-receptor interactions [20].
Table 1. Minimal energy and dipole moment of Atglistatin (1) and A1-A3
Compounds
Atglistatin (1)
(A1)
Total energy (a.u.)
-899.7058355
-899.6949831
-899.7051396
-899.6949241
Dipole moment (Debye)
1.6485
4.476
(A2)
4.3191
4.1744
(A3)
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A2
A3
(1)
1
2
A1
Figure 6. Molecules are aligned to have overlapping ring 1 in all for cases. Ring 2 is in the same plane as
ring 1 for Atglistatin (1) and (A2) and is perpendicular to the plane of the ring 1 for (A3) and (A13).
Conclusions
In summary, the present study shows clearly that inhibition of ATGL by Atglistatin (1) is due to
the presence of the carbamate and N,N-dimethyl moieties on the biaryl central core at the meta
and para position, respectively. Moving the carbamate or the N,N-dimethyl in the para or meta
position results in a total loss of ATGL inhibition as compared to Atglistatin (1). As the binding
area of Atglistatin (1) on ATGL was rather sensitive to any modifications made on the structure
of Atglistatin (1), these results may lead to the identification of the pharmacophore groups on
Atglistatin, which brings new insights on the chemical interactions between ATGL and potential
Atglistatin (1)-based inhibitors. Due the lack of a crystal structure of ATGL, the present structure
activity relationship study should assist the development of future ATGL inhibitors.
4. Experimental section
4.1. Chemistry
All chemicals used were purchased from Aldrich (CA). Purification of compounds was carried
out by flash chromatography (CombiFlash®, Separation System SG 100C, ISCO) or by silica gel
circular chromatography (chromatotron®, model 7924, Harrison Research). TLC was run on
silica gel coated aluminium sheets (SiliaPlate TLC, Silicycle®) with detection by UV light (254
nm, UVS-11, Mineralight® shortwave UV lamp). Melting points were obtained using a
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MELTEMP (model 1001D) melting point apparatus. FTIR spectra were recorded on a Nicolet
Impact 400 spectrometer. NMR spectra were recorded on a Bruker Avance III 400 MHz
spectrometer.
General Procedure I: Suzuki-Miyaura coupling
Boronic acid, aryl halide, Pd(dppf)Cl2*DCM, CsF, and dry dimethoxyethane (DME) were
introduced in a 100 ml shlenk tube. The reaction mixture was flushed three times by
vacuum/argon and stirred at 80 °C overnight. The reaction mixture was filtered through celite and
rinsed with EtOAc. The solvent was removed under vacuum and the residue was purified by flash
column chromatography (EtOAc-hexane).
General procedure II: catalytic hydrogenation of nitro analogues
Pd/C (10%) was added to a solution of the appropriate nitro compound in CH2Cl2. The reaction
mixture was stirred under hydrogen atmosphere until completion (verified by TLC). The reaction
mixture was filtered through celite and rinsed with EtOAc. The solvent was removed under
vacuum and the amine structure was confirmed by NMR analysis. The product was used for the
next experiment without any further purification.
General Procedure III: Carbamate from aniline and dimethylcarbamoyl chloride
Triethylamine (1 eq. per amine) was added to a stirred solution of the appropriate amine in dry
dichloromethane. The reaction vessel was flushed with argon and cooled with an ice bath.
Dimethylcarbamoyl chloride (1.3 eq. per amine) was added dropwise and the reaction vessel was
flushed again with argon. The mixture was left to react at room temperature. During the reaction,
additional portions of dimethylcarbamoyl chloride may be added. At the end of the reaction
(verified by TLC), the solution was hydrolyzed with water. The organic layer was separated and
the aqueous layer was extracted twice with EtOAc. The combined organic phase was washed
twice with brine, dried (MgSO4), and concentrated under vacuum. The resulting product was
purified by flash chromatography.
4-Nitro-4'-(dimethylamino)-biphenyl (6)
Following general procedure I with 4-nitrophenylboronic acid (1.28 g, 7.65 mmol), 4-bromo-
N,N-dimethylaniline (1.469 g, 7.34 mmol), Pd(dppf)Cl2*DCM (345.02 mg, 0.42 mmol), CsF
(2.412 g, 15.88 mmol), and DME (10 mL), a reddish solid was obtained after flash
chromatography purification (EtOAc-hexane 0ꢀ 25%). Yield = 370 mg (40%); Rf = 0.65
12

ACCEPTED MANUSCRIPT
(EtOAc-hexane 1:9); m.p. = 236-239 °C; 1H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 8.27-8.24
(m, 2H, Har), 7.71-7.69 (m, 2H, Har), 7.59-7.57 (m, 2H, Har), 6.83-6.81 (m, 2H, Har), 3.06 (s, 6H,
ArN(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C) δ (ppm): 150.96, 147.63, 145.80, 128.12,
126.08, 125.81, 124.19, 112.50, 40.29.
4-Amino-4'-(dimethylamino)-biphenyl (9)
Following general procedure II with 6 (185 mg, 0.76 mmol), CH2Cl2 (5 mL), and Pd/C (10%, 30
1
mg), a brownish liquid was obtained. Quantitative yield; H NMR (400 MHz, CDCl3, 25°C), δ
(ppm) 7.48-7.46 (m, 2H, Har), 7.41-7.39 (m, 2H, Har), 6.84-6.82 (m, 2H, Har), 6.78-6.75 (m, 2H,
Har), 3.00 (s, 6H, ArN(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C) δ (ppm): 149.43, 144.77,
131.95, 129.73, 127.23, 127.04, 115.52, 113.03, 40.77.
4-(3,3-dimethylureido)-4'-(dimethylamino)-biphenyl (A1)
Following general procedure III with 9 (178 mg, 0.82 mmol), Et3N (120 µL, 0.86 mmol), CH2Cl2
(5 mL), and 150 µL of dimethylcarbamoyl chloride (an additional 150 µl was added during the
reaction, to a total of 3.26 mmol), a beige solid was obtained after purification by flash
chromatography (EtOAc-hexane 0ꢀ 40%). Yield = 105.6 mg (46%); Rf = 0.20 (EtOAc-hexane
1
1:1); m.p. = 228-230°C; H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.51-7.42 (m, 6H, Har),
6.83-6.81 (m, 2H, Har), 6.36 (s, 1H, ArNHCO-), 3.06 (s, 6H, -CON(CH3)2), 3.01 (s, 6H,
ArN(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 155.77, 149.74, 137.38, 136.01,
129.01, 127.33, 126.59, 120.19, 112.89, 40.66, 36.49; HRMS m/s calc. for C17H21N3O + (H+):
284.1757; found: 284.1759.
3-Nitro-3'-(dimethylamino)-biphenyl (7)
Following general procedure I with 3-nitrophenylboronic acid (1.25 g, 7.50 mmol), 3-bromo-
N,N-dimethylaniline (1.07 ml, 7.50 mmol), Pd(dppf)Cl2*DCM (372.36 mg 0.46 mmol), CsF
(2.87 g, 18.89 mmol), and DME (10 mL), an orange solide was obtained after purification by
flash chromatography (EtOAc-hexane 0ꢀ 20%). Yield = 1.44 g (80 %); Rf = 0.65 (EtOAc-
1
hexane 1:9); m.p. = 55-58 °C; H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 8.48-8.47 (m, 1H,
Har), 8.22-8.20 (m, 1H, Har), 7.95-7.92 (m, 1H, Har), 7.63-7.59 (m, 1H, Har), 7.39-7.35 (m, 1H,
Har), 6.98-6.96 (m, 1H, Har), 6.93-6.92 (m, 1H, Har), 6.84-6.81 (m,1H, Har), 3.06 (s, 6H,
ArN(CH3)2), 13C NMR (100 MHz, CDCl3, 25°C) δ (ppm): 151.09, 148.64, 143.96, 139.73,
13

ACCEPTED MANUSCRIPT
133.28, 129.83, 129.49, 122.10, 121.86, 115.46, 112.57, 111.04, 40.63; HRMS m/s calc. for
C14H14N2O2 + (H+): 243.1128; found: 243.1135.
3-Amino-3'-(dimethylamino)-biphenyl (10)
Following general procedure II with 7 (263 mg, 1.08 mmol), CH2Cl2 (5 mL), and Pd/C (10%, 15
1
mg), a brownish liquid was obtained. Quantitative yield; H NMR (400 MHz, CDCl3, 25°C), δ
(ppm): 7.34-7.30 (m, 1H, Har), 7.26-7.22 (m, 1H, Har), 7.04-7.02 (m, 1H, Har), 6.96-6.94 (m, 3H,
Har), 6.78-6.75 (m, 1H, Har), 6.71-6.69 (m, 1H, Har), 3.77 (s, 2H, ArNH2), 3.03 (s, 6H,
ArN(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 150.85, 146.58, 143.51, 142.46,
129.51, 129.29, 117.93, 115.89, 114.16, 113.98, 111.69, 111.64.
3-(3,3-dimethylureido)-3'-(dimethylamino)-biphenyl (A2)
Following general procedure III with 10 (211 mg, 0.97 mmol), Et3N (150 µL, 1.08 mmol),
CH2Cl2 (5 mL), and 150 µL of dimethylcarbamoyl chloride (an additional portion of 150 µL was
added during the reaction, to a total of 3.26 mmol), a white solid was obtained after purification
by flash chromatography (EtOAc-hexane 0ꢀ 40%). Yield = 125 mg (46%); Rf = 0.20 (EtOAc-
1
hexane 1:1); m.p. = 180-181 °C; H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.55 (s, 1H, Har),
7.47-7.45 (m, 1H, Har), 7.38-7.28 (m, 3H, Har), 6.98-6.94 (m, 2H, Har), 6.77-6.74 (m, 1H, Har),
6.42 (s, 1H, ArNHCO-), 3.06 (s, 3H, -CON(CH3)2), 3.02 (s, 3H, ArN(CH3)2); 13C NMR (100
MHz, CDCl3, 25°C), δ (ppm): 155.75, 150;91, 142.99, 142.01, 139.41, 129.31, 129.10, 122.07,
118.72, 115.95, 111.75, 111.60, 40.74, 36.50; HRMS m/s calc. for C17H21N3O + (H+): 284.1757;
found: 284.177.
4-Nitro-3'-(dimethylamino)-biphenyl (8)
Following general procedure I with 4-nitrophenylboronic acid (1.28 g, 7.67 mmol), 3-bromo-
N,N-dimethylaniline (1.07 ml, 7.50 mmol), Pd(dppf)Cl2*DCM (401.39 mg, 0.49mmol), CsF
(2.47 g, 16.26 mmol), and DME (10 mL) a yellow solid was obtained after column purification
(EtOAc-hexane 0ꢀ 10%). Yield = 1.58 g (85%); Rf = 0.38 (EtOAc-hexane 1:9); m.p. = 138-
1
140 °C; H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 8.81-8.29 (m, 2H, Har), 7.77-7.75 (m, 2H,
Har), 7.40-7.36 (m, 1H, Har), 6.98-6.96 (m, 1H, Har), 6.92 (s, 1H, Har), 6.85-6.82 (m, 1H, Har),
3.06 (s, 6H, ArN(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 151.02, 148.78, 146.96,
139.81, 129.81, 127.93, 123.95, 115.67, 112.94, 111.19, 40.59; HRMS m/s calc. for C14H14N2O2
+ (H+): 243.1128; found: 243.112.
14

ACCEPTED MANUSCRIPT
4-Amino-3'-(dimethylamino)-biphenyl (11)
Following general procedure II with 8 (249 mg, 1.03 mmol), CH2Cl2 (8 mL), and Pd/C (10%, 15
1
mg), a brown liquid was obtained. Quantitative yield; H NMR (400 MHz, CDCl3, 25°C), δ
(ppm): 7.46-7.43 (m, 2H, Har), 7.33-7.28 (m, 1H, Har), 6.96-6.95 (m, 2H, Har), 6.79-6.75 (m, 3H,
Har), 3.03 (s, 6H, ArN(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 150.65, 145.75,
142.27, 132.49, 129.36, 128.18, 115.94, 115.28, 111.35, 111.21, 41.03.
4-(3,3-dimethylureido)-3'-(dimethylamino)-biphenyl (A3)
Following general procedure III with 11 (225 mg, 1.03 mmol), Et3N (150 µL, 1.08 mmol),
CH2Cl2 (5mL), and 200 µL of dimethylcarbamoyl chloride (multiple additions during the
reaction to a total of 1.30 ml (14.11 mmol)), a white solid was obtained after purification by flash
chromatography (EtOAc-hexane 0ꢀ 50%). Yield = 106.5 mg (36%); Rf = 0.41 (EtOAc-hexane
1
1:1); m.p. = 180-181 °C; H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.56-7.54 (m, 2H, Har),
7.47-7.45 (m, 2H, Har), 7.33-7.29 (m, 1H, Har), 6.96-6.93 (m, 2H, Har), 6.75-6.73 (m, 1H, Har),
6.39 (s, 1H, ArNHCO-), 3.07 (s, 6H, -CON(CH3)2), 3.02 (s, 6H, ArN(CH3)2); 13C NMR (100
MHz, CDCl3, 25°C), δ (ppm): 155.69, 150.98, 141.76, 138.39, 136.83, 129.36, 127.65, 119.95,
115.54, 111.36, 111.25, 40.75, 36.50; HRMS m/s calc. for C17H21N3O + (H+): 284.1757; found:
284.177.
4-(dimethylamino)-biphenyl (B1)
Dimethyl sulfate (370 µL, 3.96 mmol) was added to a vigorously stirred solution of 4-
aminobiphenyl (500 mg, 2.98 mmol) in THF (6 mL). Afterward, a solution of K2CO3 (540 mg,
3.96 mmol) in water (5 mL) was added and the reaction was left to react at room temperature for
24h. The solution was suspended in EtOAc (30 mL) and the phases were separated. The aqueous
phase was extracted with EtOAc (3 x 30 mL). The combined organic phase were washed with
brine (3 x 15 mL), dried (MgSO4) and concentrated under vacuum. A yellow solid was obtained
after purification by flash chromatography (EtOAc-hexane 0ꢀ 3%). Yield = 66 mg (11%); Rf =
0.63 (EtOAc-hexane 1:9); m.p. = 118-120°C; 1H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.61-
7.59 (m, 2H, Har), 7.56-7.54 (m, 2H, Har), 7.45-7.41 (m, 2H, Har), 7.31-7.27 (m, 1H, Har), 6.86-
13
6.84 (m, 2H, Har), 3.03 (s, 6H, -N(CH3)2); C NMR (100 MHz, CDCl3, 25°C), δ (ppm):150.00,
141.25, 129.29, 128.65, 127.72, 126.31, 126.00, 112.80, 40.61; HRMS m/s calc. for C14H15N +
(H+): 198.1277; found: 198.1279.
15

ACCEPTED MANUSCRIPT
3-(Dimethylamino)-biphenyl (B2)
Dimethyl sulfate (200 µL, 1.95 mmol) was added to a vigorously stirred solution of 3-
aminobiphenyl (250 mg, 1.49 mmol) in THF (6 mL). Afterward, a solution of K2CO3 (270 mg,
1.95 mmol) in water (5 mL) was added and the reaction was left to react at room temperature for
24h. The solution was suspended in EtOAc (30 mL) and the phases were separated. The aqueous
phase was extracted with EtOAc (3 x 30 mL). The combined organic phase was washed with
brine (3 x 15 mL), dried (MgSO4) and concentrated under vacuum. A yellow liquid was obtained
after flash chromatography (EtOAc-hexane 0ꢀ 14%). Yield = 40 mg (14%); Rf = 0.63 (EtOAc-
hexane 1:9); 1H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.65-7.63 (m, 2H, Har), 7.49-7.45 (m,
2H, Har), 7.39-7.28 (m, 2H, Har), 7.00-6.98 (m, 1H, Har), 6.97 (s, 1H, Har), 6.80-6.78 (m, 1H, Har),
3.05 (s, 6H, -N(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 150.97, 142.30, 129.42,
128.60, 127.36, 127.09, 115.87, 111.66, 111.61, 40.74; HRMS m/s calc. for C14H15N + (H+):
198.1277, found: 198.1285.
4-(3,3-dimethylureido)-biphenyl (C1)
Following general procedure III with 4-aminobiphenyl (505 mg, 2.98 mmol), Et3N (420 µL, 2.98
mmol), CH2Cl2 (8 mL), and 420µL of dimethylcarbamoyl chloride (an additional 1 ml was added
during the reaction, to a total of 15.42 mmol), a white solid was obtained after purification by
flash chromatography (EtOAc-hexane 0ꢀ 30%). Yield = 336 mg (47%); m.p. = 176-178°C; Rf =
1
0.19 (EtOAc-hexane 1:1). H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.61-7.54 (m, 4H, Har),
7.50-7.42 (m, 4H, Har), 7.35-7.31 (m, 1H, Har), 6.44 (s, 1H, -NHCO-), 3.07 (s, 6H, -CON(CH3)2);
13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 155.65, 140.75, 138.58, 135.77, 128.72, 127.49,
126.84, 126.76, 120.05, 36.50; HRMS m/s calc. for C15H16N2O + (H+): 241.1335, found:
241.1347.
3-(3,3-dimethylureido)-biphenyl (C2)
Following general procedure III with 3-aminobiphenyl (250 mg, 1.49 mmol), Et3N (210 µL, 1.49
mmol), CH2Cl2 (8 mL), and 201 µL of dimethylcarbamoyl chloride (an additional 500 µL was
added during the reaction, to a total of 9.00 mmol), a white solid after purification by flash
chromatography (EtOAc-hexane 0ꢀ 20%). Yield = 132 mg, 37%; m.p. 152-155°C; RF 0.25
1
(EtOAc-hexane 1:1); H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.68 (s, 1H, Har), 7.63-7.61
(m, 2H, Har), 7.46-7.42 (m, 2H, Har), 7.40-7.28 (m, 4H, Har), 6.42 (s, 1H, -NHCO-), 3.07 (s, 6H, -
CON(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 155.69, 141.98, 140.97, 139.58,
16

ACCEPTED MANUSCRIPT
129.22, 128.65, 127.32, 127.21, 121.80, 118.65, 118,62, 36.49; HRMS m/s calc. for C15H16N2O +
(H+): 241.1335; found: 241.134.
4-Amino-4’-(3,3-dimethylureido)-biphenyl (C4)
Following general procedure III with benzidine (514 mg, 2.79 mmol), Et3N (780 µL, 10.59
mmol), CH2Cl2 (8 mL), and 1.02 ml of dimethylcarbamoyl chloride (an additional 1 ml was
added during the reaction, to a total of 16.11 mmol), a yellow solid was obtained after
purification by flash chromatography (EtOAc-hexane 0ꢀ 100%). There was only a mono-
substitution because the product precipitated during the reaction. Yield = 500 mg (70%); m.p.
>215°C; Rf = 0.56 (MeOH-CH2Cl2 1:20); 1H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.49-7.39
(m, 6H, Har), 6.77-6.75 (m, 2H, Har), 6.35 (s, 1H, ArNHCO-), 3.75 (s, 2H, ArNH2), 3.06 (s, 6H,
ArN(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 155.73, 145.49, 137.59, 135.94,
131.23, 127.63, 126.71, 120.14, 115.41, 36.49; HRMS m/s calc. for C15H17N3O + (H+): 256.1444;
found: 256.1451.
4,4’-Bis-(3,3-dimethylureido)-biphenyl (C5)
Piperidine (563 µL, 5.96 mmol) was added to a vigorously stirred solution of C4 (500 mg, 2.71
mmol) in dry THF (5 mL), The reaction vessel was thoroughly flushed with argon and cooled to
0°C with an ice bath. Dimethylcarbamoyl chloride (502 µL, 6.78 mmol) was added dropwise to
the reaction an ice bath. The mixture was left to react at room temperature. At the end of the
reaction (verified by TLC), the solution is hydrolysed with water (50 mL) and the phases were
separated, the aqueous phase was extract with EtOAc (3 x 30 mL). The combined organic phase
was washed with brine (3 x 15 mL), dried (MgSO4), and concentrated. A brown-red solid was
obtained after purification by chromatography on silica gel coated glass (2 X MeOH/CH2Cl2, 5%).
Yield = 30 mg (5%); m.p. >350°C; Rf = 0.50 (MeOH/CH2Cl2 1:20); 1H NMR (400 MHz, DMSO-
d6, 25°C), δ (ppm): 8.35 (s, 2H, -NHCO-), 7.55-7.53 (m, 4H, Har), 7.51-7.49 (m, 4H, Har), 2.94 (s,
12H, -CON(CH3)2); 13C NMR (100 MHz, DMSO-d6, 25°C), δ (ppm): 156.18, 140.09, 133.62,
126.25, 120.38, 36.74; HRMS m/s calc. for C18H22N4O2 + (H+): 327.1816; found: 327.1817.
3,3'-Dinitrobiphenyl (17)
A solution of 3-nitrophenylboronic acid (1.45 g, 8.69 mmol) and CuCl (56.40 mg, an additionnal
40 mg was added during the reaction, to a total of 0.97 mmol) in methanol (20 mL) was stirred at
25 °C until reaction completion (TLC control). The reaction mixture was filtered through celite
17

ACCEPTED MANUSCRIPT
and washed with EtOAc (30 mL). The combined filtrate was concentrated in vaccum and the
residue was purified by flash chromatography (EtOAc-hexane 0ꢀ 20%). A yellow solid was
1
obtained. Yield 365.8 mg (34%); Rf = 0.56 (EtOAc-hexane 1:3); m.p. = 207-208 °C; H NMR
(400 MHz, CDCl3, 25°C), δ (ppm): 8.53 (s, 2H, Har), 8.34-8.32 (m, 2H, Har), 8.01-7.99 (m, 2H,
13
Har), 7.75-7.71 (m, 2H, Har); C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 148.91, 140.35,
133.06, 130.31, 123.31, 122.12.
3,3'-Diaminobiphenyl (18)
Following general procedure II with 17 (235 mg, 0.96 mmol), CH2Cl2 (8 mL), and Pd/C (10%, 30
mg), a yellow oil was obtained. Quantitative yield; 1H NMR (400 MHz, CDCl3, 25°C), δ (ppm):
7.25-7.21 (m, 2H, Har), 7.00-6.98 (m, 2H, Har), 6.91-6.90 (m, 2H, Har), 6.70-6.68 (m, 2H, Har),
3.74 (s, 4H, ArNH2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 146.60, 142.64, 129.55,
117.68, 114.09, 113.92.
3,3'-Bis-(dimethylamino)-biphenyl (B3)
A solution of 3-(N,N-dimethylamino)phenylboronic acid (192 mg, 1.21 mmol) and CuCl (23.76
mg, 0.24 mmol) in methanol (10 mL) was stirred at 25 °C until reaction completion (verified by
TLC). The reaction mixture was filtered through celite and washed with EtOAc (40 mL). The
combined filtrate was concentrated in vacuum and the residue was purified by circular
chromatography (EtOAc-hexane 0ꢀ 10%). A brownish oil was obtained. Yield = 40.84 mg
(29%); Rf = 0.88 (EtOAc-hexane 1:1); 1H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.37-7.33 (m,
13
2H, Har), 7.01-6.99 (m, 4H, Har), 6.80-6.78 (m, 2H, Har), 3.04 (s, 12H, 2ArN(CH3)2); C NMR
(100 MHz, CDCl3, 25°C), δ (ppm): 150.89, 143.40, 129.25, 116.16, 111.98, 111.58, 40.79;
HRMS m/s calc. for C16H20N2 + (H+): 241.1699; found: 241.1698.
3,3’-Bis-(3,3-dimethylureido)-biphenyl (C3)
Following general procedure III with 18 (195 mg, 1.06 mmol), Et3N (300 µL, 2.15 mmol),
CH2Cl2 (5 mL), and 500 µL of dimethylcarbamoyl chloride (multiple additions during the
reaction to a total of 1.40 ml (15.21 mmol)), a white solid was obtained after purification by flash
chromatography (EtOAc-hexane 0ꢀ 90%). Yield = 124.4 mg (36%) ; Rf = 0.32 (100% EtOAc);
1
m.p. >250 °C; H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.62 (s, 2H, Har), 7.43-7.41 (m, 2H,
Har), 7.36-7.32 (m, 2H, Har), 7.28-7.27 (m, 2H, Har), 6.48 (s, 2H, ArNHCO-), 3.07 (s, 12H, -
CON(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm):155.78, 141.66, 139.52, 129.15,
18

ACCEPTED MANUSCRIPT
121.87, 118,82, 118.58, 36.51; HRMS m/s calc. for C18H22N4O2 + (H+): 327.1816; found:
327.1829.
1-(3,3-Dimethylureido)-4-(dimethylamino)benzene (D1)
Following general procedure III with 500 mg of p-(dimethylamino)benzamine (3.67 mmol), Et3N
(1.51 mL, 20.57 mmol), CH2Cl2 (8 mL), and dimethylcarbamoyl chloride (1.340 mL, 10.65
mmol), a white solid was obtained after flash chromatography (EtOAc-hexane 0ꢀ 60%). Yield =
618.9 mg (81%); Rf = 0.28 (EtOAc 100%); m.p. = 170°C; 1H NMR (400 MHz, CDCl3, 25°C), δ
(ppm): 7.25-7.21 (m, 2H, Har), 6.73-6.71 (m, 2H, Har), 6.16 (s, 1H, -CHCO-), 3.02 (s, 6H, -
CON(CH3)2), 2.92 (s, 6H, -N(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ (ppm): 156.48,
147.58, 129.16, 122.48, 113.47, 41.19, 36.44; HRMS m/s calc. for C11H17N3O + (H+): 208.1444;
found: 208.1455.
1-(3,3-Dimethylureido)-3-(dimethylamino)benzene (D2)
N,N-Dimethyl-1,3-phenylenediamine dihydrochloride (700 mg, 3.34 mmol) was treated with
K2CO3 (4 g, 28.9 mmol) in water (5 mL). After 2 hour, the m-(dimethylamino)benzenamine was
extracted from the aqueous phase with dichloromethane (3 X 20 mL). The organic phase was
dried (MgSO4) and concentrated under vacuum. m-(Dimethylamino)benzenamine was obtained
with a yield of 45% (440 mg). Following general procedure III with 440 mg m-
(dimethylamino)benzenamine (3.23 mmol), 470 µL of Et3N (3.34 mmol) and 460 µL of
dimethylcarbamoyl chloride (5.01 mmol), compound D2 was obtained as a beige solid after flash
chromatography (EtOAc-hexane 0ꢀ 40%). Yield = 448.2 mg (67%); Rf = 0.13 (EtOAc-hexane
1
1:1); m.p. = 124-126°C; H NMR (400 MHz, CDCl3, 25°C), δ (ppm): 7.16-7.12 (m, 1H, Har),
7.05-7.03 (m, 1H, Har), 6.62-6.60 (m, 1H, Har), 6.46-6.43 (m, 1H, Har), 6.30 (s, 1H, -NHCO-),
3.04, (s, 6H, -CON(CH3)2), 2.96 (s, 6H, -N(CH3)2); 13C NMR (100 MHz, CDCl3, 25°C), δ
(ppm):155.80, 151.37, 140.11, 129.21, 107.96, 107.54, 104.09, 40.65, 36.48; HRMS m/s calc. for
C11H17N3O + (H+): 208.1444; found: 208.1451.
1,4-Bis-(3,3-dimethylureido)benzene (D3)
Following general procedure III with p-benzenediamine (260 mg, 2.40 mmol), Et3N (800 µL 5.74
mmol), CH2Cl2 (5 mL), and dimethylcarbamoyl chloride (1 ml, 10.86 mmol), a white solid was
obtained without purification. Yield = 230 mg (42%); Rf = 0.39 (MeOH/CH2Cl2 1:20); m.p.
1
>290°C; H NMR (400 MHz, DMSO-d6, 25°C), δ (ppm): 8.10 (s, 2H, -NHCO-), 7.29 (s, 4H,
19

ACCEPTED MANUSCRIPT
Har), 2.91 (s, 12H, -CON(CH3)2); 13C NMR (100 MHz, DMSO-d6, 25°C), δ (ppm):156.41,
135.36, 120.64, 36.65; HRMS m/s calc. for C12H18N4O2 + (H+): 251.1503; found: 251.1500.
4.2. Biological evaluation
4.2.1. Cell Culture
3T3-L1 cells (ATCC) were grown and differentiated to mature adipocytes, as previously
described, with minor modifications (Kershaw et al., 2006). Briefly 3 × 105 3T3-L1 cells were
seeded in 35 mm dishes and maintained in Dulbecco’s modified Eagle’s medium containing high
glucose concentrations (DMEM-HG; 4.5 g/L glucose) supplemented with 10% fetal bovine
serum (FBS) (Gibco). Two days post-confluence (Day 0), cells were differentiated in DMEM-
HG media containing 10% FBS, 10 µg/ml insulin from bovine pancreas, 0.4 µg/ml
dexamethasone and 0.5 mM 3-Isobutyl-1-methylxanthine. After two days (Day 2), the media was
changed to DMEM-HG supplemented with 10% FBS and 10 µg/ml insulin. At Day 4, the media
was changed to DMEM-HG containing 10% FBS and 0.5 µg/ml insulin. After Day 6, cells were
maintained in DMEM-HG containing 10% FBS. Experiments were performed with adipocytes at
Day 8.
4.2.2. Lipolysis Assay
Adipocytes were washed twice with DMEM (1 g/L glucose) and pre-incubated with DMEM
containing 2% (w/v) fatty acid-free bovine serum albumin (BSA) and 20 µM of indicated
compound for 4 h. Lipolysis was stimulated by incubating cells with DMEM containing 2%
FFA-BSA, 20 µM of indicated compound, and 5 µM of triacsin C (Sigma) in the presence of 2
µM isoproterenol for 1 h. Media aliquots were stored at -80ºC until analysis. Free glycerol and
non-esterified fatty acids (NEFA) in the media were determined using colorimetric kit assays
(free glycerol reagent from Sigma and HR Series NEFA-HR (2) kit from Wako Chemicals).
Cells were pelleted through centrifugation at 10,000 ×g for 10 min at 4°C, flash frozen in liquid
N2 and stored at -80°C until further use. 3T3-L1 cells were lysed in 100 µL of lysis buffer (20
mM Tris-HCl pH 7.5, 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 1% NP-40) containing 2
mM sodium orthovanadate, 2 mM protease inhibitor cocktail (Sigma) and 100 µg/mL
phosphatase inhibitor cocktail (Millipore). Protein concentrations in cell lysates were quantified
colorimetrically using a bicinchoninic acid protein assay kit (Thermo Scientific) and BSA as
standard.
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4.3. Structure optimization and energy calculations
Calculations were performed using Gaussian 97 software. Structure optimization and energy
calculations used DFT calculation at B3LYP/6-31G level of theory.
Statistical Analysis
Where necessary, statistical significance was determined using a One-way ANOVA with post-
hoc Tukey HSD using Graphpad Prism software.
Acknowledgments
M.T. would like to acknowledge the contribution of the New Brunswick Innovation Foundation
(NBIF), the Canadian Foundation for Innovation (CFI), and Université de Moncton. P.K. would
like to acknowledge the contribution of the Natural Sciences and Engineering Research Council
of Canada (NSERC).
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Research highlight
► Novel Atglistatin closely related analogues were synthesized.
► Structure-activity relationships were established based on adipose triglyceride lipase
inhibition.
►The meta-relationship of the substituents was critial for adipose triglyceride lipase
inhibition.