Attenuation of Mycobacterium species through direct and macrophage mediated pathway by unsymmetrical diaryl urea

Article abstract of DOI:10.1016/j.ejmech.2016.09.083

Tuberculosis is a major threat for mankind and the emergence of resistance strain of Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human civilization. In this study, we have synthesized different diaryl urea derivativ

Full text of DOI:10.1016/j.ejmech.2016.09.083

Accepted Manuscript
Attenuation of Mycobacterium species through direct and macrophage mediated
pathway by unsymmetrical diaryl urea
Anand Babu Velappan, Mamilla R. Charan Raja, Dhrubajyoti Datta, Yi Ting Tsai,
Iman Halloum, Baojie Wan, Laurent Kremer, Hugo Gramajo, Scott G. Franzblau,
Santanu Kar Mahapatra, Joy Debnath
PII:
S0223-5234(16)30817-0
10.1016/j.ejmech.2016.09.083
EJMECH 8949
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 June 2016
Revised Date: 23 September 2016
Accepted Date: 25 September 2016
Please cite this article as: A.B. Velappan, M.R. Charan Raja, D. Datta, Y.T. Tsai, I. Halloum, B. Wan,
L. Kremer, H. Gramajo, S.G. Franzblau, S.K. Mahapatra, J. Debnath, Attenuation of Mycobacterium
species through direct and macrophage mediated pathway by unsymmetrical diaryl urea, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.083.
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ACCEPTED MANUSCRIPT
Attenuation of Mycobacterium species through direct and
macrophage mediated pathway by unsymmetrical diaryl
urea
Anand Babu Velappana, Mamilla R. Charan Raja,b Dhrubajyoti Dattac, Yi Ting Tsaid, Iman
Halloume, Baojie Wanf, Laurent Kremereg, Hugo Gramajod, Scott G. Franzblauf, Santanu Kar
Mahapatrab*, Joy Debnatha*
aDepartment of Chemistry, School of Chemical and Biotechnology, SASTRA University, Tamilnadu 613401, India.
bDepartment of Biotechnology, School of Chemical and Biotechnology, SASTRA University, Tamilnadu 613401,
India.
cDepartment of Chemistry, Indian Institute of Science Education and Research Pune , Maharashtra 411008, India
dInstituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y
Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
eCentre d’étude des Pathogènes pour la Biotechnologie et la Santé CNRS FRE3689, Université de Montpellier,
34293 Montpellier cedex 5, France.
fInstitute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood St.
Chicago, Illinois 60612 USA.
gINSERM, CPBS, 34293 Montpellier Cedex 05, France.
*Corresponding Author
JD: Tel: +91 4362 264101 (Ext: 3757); Fax: +91 4362 264120;
e-mail: joydebnath@scbt.sastra.edu
joydebnathiitkgp@gmail.com
1

ACCEPTED MANUSCRIPT
Highlights:
•
•
Different diaryl ureas are designed and synthesized.
The antimycobacterial activity has been checked against M. smegmatis (mc2155) and M.
tuberculosis (mc26030 and H37Rv strains).
•
The EDG and EWG are found to have a preference for the ortho and meta positions of the first
aromatic ring respectively.
•
•
The cytotoxicity values are in the range of 92-232 µg/ml.
The effective molecules showed selective inhibitory activity on mycolic acid biosynthesis in 14C-
radiolabelled assay.
•
Effective molecules of this series up regulate the expression level of iNOS2, IFN-γ, IL-12 and
down regulate the level of IL-10 to clear the mycobacterium infection.
2

ACCEPTED MANUSCRIPT
Abstract: Tuberculosis is a major threat for mankind and the emergence of resistance strain of
Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human
civilization. In this study, we have synthesized different diaryl urea derivatives targeting the
inhibition of mycolic acid biosynthesis. Among the 39 synthesized molecules, compounds 46,
57, 58 and 86 showed MIC values ≤ 10 µg/ml against H37Rv and mc26030 strains. The best
molecule with a methyl at ortho position of the first aromatic ring and prenyl group at the meta
position of the second aromatic ring showed the MIC value of 5.2 µg/ml and 1 µg/ml against
H37Rv and mc26030 respectively, with mammalian cytotoxicity of 163.4 µg/ml. The effective
compounds showed selective inhibitory effect on mycolic acid (epoxy mycolate) biosynthesis in
14C-radiolabelled assay. At the same time these molecules also executed their potent
immunomodulatory activity by up-regulation of IFN-γ and IL-12 and down-regulation of IL-10.
Key words: Tuberculosis, Diaryl urea, Structure-activity relationship, antimycobacterial activity,
Cytotoxicity, Mycolic acid, cytokines.
3

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1. Introduction: Mycobacterium genus of pathogenic species are responsible for a
variety of infectious diseases, e. g., tuberculosis and leprosy which are caused by M.
tuberculosis (
Mtb) and M. leprae respectively.1 Altogether, infections caused by this
group of species are primarily accountable for millions of death annually among different
life forms. In particular, according to the World Health Organization (WHO) report
(2015), in the year of 2014, 1.5 million deaths were caused by tuberculosis (TB) along
with an estimated 9.6 million new TB incidences.2 Among the 9.6 million new TB cases
12% people were found to be co-infected with HIV, which brings new challenges for
conventional treatment methods that is mostly dependent on antibiotics. The AIDS
patients co-infected with TB, are needed to be completely cure from TB in order to avoid
the interaction of first line antibiotics (e. g., Rifampacin and Isoniazid) with anti-HIV
drugs such as protease inhibitors.3,4 However, the high tolerance level of Mtb towards first
line antibiotics along with their ability to survive in the dormant (non-replicating) state is
a major concern to provide a complete cure against TB.
There are several targets for the antituberculosis drugs; arguably, the most
important one is the cell wall biosynthesis. Early in 1970, Isoxyl or thiocarlide was used
as an antituberculosis drug which inhibits the synthesis of mycolic and oleic acids. The
∆
9-stearoyl desaturase was identified as the primary target of isoxyl.5-7 However, the use
of isoxyl was restricted due to its interference with mammalian monoamine oxidase.8 It is
also reported that isoxyl get converted to an active form by the monooxygenase EthA.9
During this bioactivation process, the thiourea gets converted to the urea one.10 Therefore,
different urea derivatives can represent as a suitable scaffold for the development of
potent antitubercular and immunomodulatory molecule. Several attempts are also made to
achieve this particular purpose.11-15
Most TB infections are commenced through the respiratory route. They first get
implanted in the alveoli to form the Ghon complex.16,17 During the progression of TB they
thrive inside the host alveolar macrophages and impaired the host protective response.
The virulent Mtb stain (H37Rv) modulates the host immune response in their favour by
up-regulation of the anti-inflammatory cytokines e. g., IL-10, arginase and down-
regulation of the proinflammatory cytokines, e. g., iNOS, IL-12, IFN-γ 18
.
Therefore,
4

ACCEPTED MANUSCRIPT
designing of immunomodulatory molecules also has a great possibility to cure against
Mtb by re-educating the host immune response.
In this work, we have synthesized several unsymmetrical diaryl ureas to
understand the relation between activity and cytotoxicity despite knowing the fact that the
minimum inhibitory concentration (MIC) value of the urea analogue of isoxyl is ~100
times higher than isoxyl itself.19 The key reasons for substituting the thiourea moiety with
its bioisostere urea20-22 are (i) to reduce the possibility of interaction with unspecified
target through the sulphur atom and (ii) to find out a cost effective synthetic method for
the low-income TB patients.
The first aromatic ring of this series of molecules is substituted with different
substitutions having disparate inductive (I) and mesomaric (M) effects, whereas, the
second aromatic ring is substituted with three different aliphatic chains at the meta and
para positions (Fig 1). Different combinations of the substitution on both aromatic rings
have a great scope to correlate their efficacy with cytotoxicity.
O
O
S
N
N
H
H
Isoxyl
Symmetrical molecule
Unsymmetrical molecule
Substituted with
aliphatic chain
Substituted with
I & M groups
O
R1
OR2
N
N
H
H
1st ring
2nd ring
Figure 1: Site of modification at two aromatic rings.
The synthesized molecules were first tested for their direct killing effect on Mtb and M.
Smagmatis, followed by the evaluation of their ramification on fatty acid and mycolic acid
14
biosynthesis by the C-radio-labeled assay and it was done with the selected compounds from
each different effective prototypes. Finally, the best three molecules of this entire series were
investigated for their possible immunomodulatory effect upon proinflammatory and anti-
inflammatory cytokines by measuring their mRNA expression level with the help of RT-PCR.
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2. Results and Discussion:
2.1 Synthesis of Urea analogues:
In an effort to trounce the involvement of mammalian monoamine oxidase, we have
prepared several diaryl ureas. A diverse set of molecules have been synthesized in order to
determine the relationship between antitubercular efficiency with its different substitutions. To
obtain this specific goal we had introduced several functionalities (with inductive and/or
mesomeric effect) in the first aromatic ring, whereas, second aromatic was substituted with three
different aliphatic substituents.
To introduce +I, -I, -I & -M and –I & +M effect in the first aromatic ring at its
ortho/meta/para position six groups methyl (+I), trifluoromethyl (-I), nitro, halides, hydroxyl
and amino (-I & -M) were primarily selected. However, the hydroxyl substitution was ruled out
because of its potential participation in Mitsunobu reaction. For the amino substitution although
we were able to prepare the monomer using high dilution method but the resulting diarylurea
was found to yield benzimidazole (as confirmed by the NMR) during the HBr mediated
demethylation step. Again, BBr3 mediated demethylation for this system was found sluggish in
nature (data not shown). Therefore, we were only able to synthesize compounds having methyl,
trifluoromethyl, m-nitro and halides groups at three different positions of the first aromatic ring.
The diaryl ureas 1 - 20 were synthesized by the reaction of their corresponding amines
(un-substituted and substituted) with 3- and 4-Methoxyphenyl isocyanate in dichloromethane
(DCM) (Scheme 1)23. Aforesaid method was quite successfully utilized for the m-Nitroaniline (-I
& -M), however, for o-Nitroaniline and p-Nitroaniline we opted for the other alternative. The
strong –I and –M effect of the nitro group is presumed to be responsible for diminishing the
nucleophilicity of the amine. The demethylation steps were accomplished with HBr (50% in
acetic acid) at 70 °C. Thereafter, the three different aliphatic alcohols were coupled with their
respective phenol counterpart via Mitsunobu coupling reaction24 to yield final compounds 41 -
72.
6

ACCEPTED MANUSCRIPT
O
(i)
R1
R2
R1
OCH3
+
N
N
NH2
OCN
H
H
R1
H; m-NO2
Comp
Comp
R1
R1
R2
R2
11
12
13
14
15
16
17
18
19
20
p-F
o-Cl
m-Cl
p-Cl
o-Br
m-Br
p-Br
o-CF3
m-CF3
p-CF3
m-OCH3
m-OCH3
m-OCH3
m-OCH3
m-OCH3
m-OCH3
m-OCH3
m-OCH3
m-OCH3
m-OCH3
1
2
3
4
5
6
7
8
9
10
H
H
m-OCH3
p-OCH3
m-OCH3
p-OCH3
m-OCH3
p-OCH3
m-OCH3
m-OCH3
m-OCH3
m-OCH3
o-CH3; m-CH3; p-CH3
o-F; m-F; p-F
o-Cl; m-Cl; p-Cl
o-Br; m-Br; p-Br
o-CF3; m-CF3; p-CF3
m-NO2
m-NO2
o-CH3
o-CH3
m-CH3
p-CH3
o-F
m-F
Allyl
alcohol
O
O
R1
OH
(iii)
O
N
N
H
R1
H
N
N
H
41: R1 = H; 42: R1 = NO2(m); 43: R1 = CH3(o)
H
21: R1 = H; 22: R1 = NO2(m)
23: R1 = CH3(o)
O
1-Butanol
O
3
R1
(ii)
N
N
H
H
44: R1 = H; 45: R1 = NO2(m); 46: R1 = CH3(o)
(iii)
O
O
O
R1
Prenol
R1
N
N
OH
N
N
H
H
H
H
24: R1 = H; 25: R1 = NO2(m); 26: R1 = CH3(o)
47: R1 = H; 48: R1 = NO2(m); 49: R1 = CH3(o)
27: R1 = CH3(m); 28: R1 = CH3(p); 29: R1 = F(o)
30: R1 = F(m); 31: R1 = F(p); 32: R1 = Cl(o)
33: R1 = Cl(m); 34: R1 = Cl(p); 35: R1 = Br(o)
36: R1 = Br(m); 37: R1 = Br(p); 38: R1 = CF3(o)
39: R1 = CF3(m); 40: R1 = CF3(p)
Allyl
alcohol
O
R1
N
N
O
H
H
50: R1 = H; 51: R1 = NO2(m); 52: R1 = CH3(o)
1-Butanol
O
R1
N
N
O
3
H
H
53: R1 = H; 54: R1 = NO2(m); 55: R1 = CH3(o)
56: R1 = H; 57: R1 = NO2(m); 58: R1 = CH3(o)
59: R1 = CH3(m); 60: R1 = CH3(p); 61: R1 = F(o);
62: R1 = F(m); 63: R1 = F(p); 64: R1 = Cl(o)
65: R1 = Cl(m); 66: R1 = Cl(p); 67: R1 = Br(o)
68: R1 = Br(m); 69: R1 = Br(p); 70: R1 = CF3(o)
71: R1 = CF3(m); 72: R1 = CF3(p);
O
Prenol
R1
N
N
O
H
H
Scheme 1: Reagents and conditions: (i) DCM (dry), 3-5 h, rt; (ii) HBr/AcOH, 10 h, 70 °C; (iii) DIAD/TPP in THF,
6-8 h, rt.
The previous scheme did not work well (yield<5%) because of the poor reactivity of o
and p-Nitroaniline and m-Cyanoaniline towards the isocyanates. To overcome the reactivity issue
for the groups with -I & -M effect, we used o and p-Nitrophenyl isocyanate and m-Cyanophenyl
isocyanate and reacted them with 3- and 4-Anisidine to obtain 73 – 76. The use of anisidine
increased the nucleophilicity of the amine, whereas, presence of nitro and cyano groups
enhanced the electrophilicity of the isocyante. This combination worked well (yield >75%) and
made it possible to prepare compounds 78 – 80 and 84 – 86 (Scheme 2).
7

ACCEPTED MANUSCRIPT
(i)
O
R1
R1
R2
OCH3
+
H2N
NCO
N
N
H
H
R1
Comp
R1
R2
o-NO2
p-NO2
m-CN
73
74
75
76
o-NO2
p-NO2
m-CN
m-CN
m-OCH3
m-OCH3
m-OCH3
p-OCH3
CN
CN
OH
OR2
(iv)
O
O
R2 OH
N
N
H
N
N
H
H
H
77
78: R2 = Allyl
79: R2 = n-Butyl
80: R2 = prenyl
(ii) or (iii)
(iv)
O
O
R1
R1
Prenol
N
H
N
H
OH
N
N
O
H
H
81: R1 = o-NO2
82: R1 = p-NO2
83: R1 = m-CN
84: R1 = o-NO2
85: R1 = p-NO2
86: R1 = m-CN
Scheme 2: Reagents and conditions: (i) DCM (dry), 3-5 h, rt; (ii) HBr/AcOH, 10 h, 70 °C; (iii) BBr3 in
DCM, 10 h, rt; (iii) DIAD/TPP in THF, 6-8 h, rt.
For the carboxylic acid derivative we started with ethyl 3-aminobenzoate and 3-
Methoxyphenyl isocyanate. The demethylation step was carried out in presence of HBr at
o
70 C followed by the esterification of the carboxylic group (89). The carboxylic group
was protected again to resist its participation in Mitsunobu reaction. Thereafter, the
phenolic OH group was coupled with prenyl alcohol under Mitsunobu reaction condition
followed the de-esterification of the carboxylic group with LiOH (90) (Scheme 3).
8

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CO2C2H5
NH2
CO2C2H5
O
OCH3
(i)
+
OCN
N
N
OCH3
H
H
87
CO2CH3
CO2H
(ii)
(iii)
O
O
N
N
H
OH
N
N
OH
H
H
H
88
O
89
CO2H
(iv)
(v)
N
N
H
O
H
90
Scheme 3: Reagents and conditions; (i) DCM (dry), 4 h, rt; (ii) HBr/AcOH, 12 h, 70 °C; (iii) SOCl2/MeOH, reflux 5
h; (iv) DIAD/TPP in THF, 8 h, rt; (v) LiOH/H2O, 4 h, rt.
For all the synthesized molecules, only one aromatic ring of diaryl urea is substituted
with different groups having altered I and M effect, whereas, the second one bears only an
aliphatic chain at para and meta positions. To compare the effect of different substituents in first
aromatic ring, compound 41, 44, 47, 50, 53 and 56 were prepared (Scheme 1), where the first
aromatic ring is un-substituted but the second aromatic ring is substituted by all the three
aliphatic chains at the meta and para positions.
2.2 Antimycobacterial activity:
The antimycobacterial activities and cytoxicities all 39 compounds are given in Tables 1-
3. The bactericidal activity was determined against M. Smegmatis (mc2155) and two other Mtb
strains mc26030 (non pathogenic strain) and H37Rv (pathogenic). The Mtb strain mc26030 is
derived from H37Rv, which is lacking of region of difference 1 (RD1) as well as is an auxotroph
for pantothenic acid.25 MIC values were determined by three different methods, namely, serial
dilution method26 (for mc2155), agar plate method27 (for Mtb mc26030) and Microplate Alamer
Blue assay (MABA)28,29 (for Mtb H37Rv). Mammalian HepG2 cell line was used to determine
their cytoxicity and the corresponding IC50 values were calculated from their dose response
curve. Thereafter, the best compounds were taken forward to ascertain their direct effect on
mycolic and fatty acid biosynthesis by 14C-radiolebelled assay. Finally, their potential
immunomodulatory activity was tested by measuring different cytokines levels in infected
peritoneal macrophage to probe an alternative pathway for the clearance of mycobacterium
species by this series of molecules.
9

ACCEPTED MANUSCRIPT
Correlation between +I vs -I effect and activity:
The first set of compounds 41 44 47, 50, 53 and 56 were prepared as a reference
,
,
to correlate the activity of different substituents on the first aromatic ring. Therefore, for
this set of molecules the first aromatic ring was kept un-substituted and the second
aromatic ring was substituted with three different aliphatic chains at meta and para
positions. This set of molecules showed moderate activity against mc2155, mc26030 and
H37Rv strains except for compound 47. The MIC values for 47 were found 25
µg/ml, 30
µ
g/ml and 43.1
µ
g/ml respectively against mc2155, mc26030 and H37Rv strains.
However, for this set of molecules allyl and prenyl substitutions (second aromatic ring)
showed better effect against H37Rv strain (>75%) compared to n-butyl one (<60%) at 50
µ
g/ml concentration. The small discrepancies of the MIC values against mc26030 and
H37Rv strains can be attributed for the difference in their susceptibility profile and for the
methods being used to determine the drug efficacy (solid agar plates versus broth
medium). The cytotoxicity for these molecules was found to vary between 182.4 to 124.1
µ
g/ml. Replacement of the thiourea moiety with its bioisostere (urea) showed a promising
effect in order to decrease their cytotoxicity level compared with isoxyl.13
To compare only the role of +I and –I effect, we have prepared another two sets of
molecules 43, 46, 49, 52, 55, 58, 59 and 60 (with +I effect) and 70 - 72 (with –I effect). Initial
screening of 43, 46, 49, 52, 55, 58, 59 and 60 on different mycobacterium species showed the
preference of +I group at ortho and para positions for their better activity, whereas, out of the
six variants of this set the optimum activity was found for compound 58 with a prenyl group at
the meta position on the second aromatic ring (Table 1). The MIC values were found 12.5 µg/ml,
1 µg/ml and 5.2 µg/ml against mc2155, mc26030 and H37Rv strains respectively. This finding
confirmed us about the preferred position of the prenyl chain for second aromatic ring. After
optimization of the position and nature of aliphatic chain another two variants 60 and 59 were
prepared to find out the effect of methyl group at meta and para positions. The MIC values were
found 32.2 µg/ml and 10.5 µg/ml for 60and 59 respectively against H37Rv strain. The collective
antimycobacterial activity of 58, 59 and 60 confirmed the perpetuation of the ortho position for
an +I group. Relative activity of 58 and 60 also provided an indication about the effect of steric
congestion near the urea bond. Now to validate the above mentioned inference another set of
compounds 70 - 72 were synthesized, where the methyl group was substituted by CF3 having a
10

ACCEPTED MANUSCRIPT
strong –I effect and the MIC values were found to increased over >50 µg/ml against H37Rv
strain but surprisingly improved against mc2155 strain. Comparing the activity of these sets of
molecules it can be concluded that the +I groups dominate over the –I groups for their improved
bactericidal activity against H37Rv strain but a reverse effect was encountered against mc2155
strain (Table 1).
Table 1: Role of +I and –I effect on antimycobacterial activity.
R3
O
O
R4
R2
N
N
H
H
R1
MIC (µg/ml) MIC (µg/ml)
(H37Rv)
(% Inhibition)
MIC (µg/ml)
(mc2155)
Cytotoxicity
(µg/ml)
(mc26030)
Comp.
44
53
41
50
47
56
46
55
43
52
49
58
59
60
70
71
72
R1
H
R4
R2
H
H
H
H
H
H
H
H
H
H
H
H
R3
H
nBu(p)
nBu(m)
>50 (41%)
>50 (57%)
>50 (84%)
>50 (73%)
43.1
>100
50-100
>100
20
>200
>200
>200
100
25
132.5
124.1
182..4
150.6
124.7
168.2
130.5
175.1
99.2
H
H
H
H
allyl(p)
H
H
allyl(m)
H
H
prenyl(p)
prenyl(m)
nBu(p)
30
H
H
>50 (79%)
7.0
30
>200
100
>200
50
CH3
CH3
CH3
CH3
CH3
CH3
H
H
100
>100
100
>100
100
1
H
nBu(m)
>50 (70%)
24.5
H
allyl(p)
H
allyl(m)
>50 (68%)
>50 (61%)
5.2
>200
50
102.4
109.6
163.4
175.3
140.6
71.4
H
prenyl(p)
prenyl(m)
prenyl(m)
prenyl(m)
prenyl(m)
prenyl(m)
prenyl(p)
H
12.5
50
CH3
H
H
32.2
nd
H
CH3
H
10.5
nd
12.5
12.5
3.12
6.25
CF3
H
H
>50 (45%)
40.2
nd
CF3
H
H
nd
135.6
142.2
H
CF3
>50 (69%)
nd
nd = not determined
Correlation between –I & -M vs –I & +M effect and activity:
To understand the outcome of different substitutions having –I
& –M and –I & +M effect,
we synthesized four sets of molecules. For the first three sets the first aromatic ring is
substituted with nitro, cyano and carboxylic groups having –I & –M effect, whereas, for
the fourth set of molecules it is substituted by three different halides (F, Cl and Br) having
–I
&
+M effect. Among the others, nitro has the strongest –I
&
–M effect. Therefore, we
11

ACCEPTED MANUSCRIPT
selected this one to optimize the aliphatic side chain and its preferred position in the
second aromatic ring. For this reason we had prepared compounds 42
84 and 85 with the nitro at meta ortho and para positions respectively. The relative
activity of the compounds 42 45 48 51 54 57 showed a clear preference for the prenyl
, 45, 48, 51, 54, 57,
,
,
,
,
,
,
chain at meta position as observed previously for compound 58. Only compond 57 of this
set showed significant effect on three mycobacterium species, the respective MIC value
were 6
However, a small improvement in activity was observed for para nitro substitution over
the ortho one. The similar trend was observed for compounds 78 79 80 and 86, where
the first aromatic ring was substituted with a nitrile group (-I -M) at meta position.
µg/ml, 2 µg/ml and 3.21 µ
g/ml against H37Rv, mc26030 and mc2155 strains.
,
,
&
Their relative activity also probed the preference of prenyl group at meta position, as
observed for methyl and nitro substitutions. The best activity was found for compound 86
and the respective MIC values were 9.2 µg/ml and 25 µ
g/ml against H37Rv and mc2155.
Similar to the previous sets of molecules same trend was observed for the molecule (90
)
having a carboxy substitution at meta position (Table 2).
Table 2: Role of -I and –M effect on antimycobacterial activity.
R1
O
R4
R2
N
N
H
H
R3
R1
MIC (µg/ml) MIC (µg/ml)
(H37Rv)
MIC (µg/ml) Cytotoxicity
Comp
(mc26030)
(µg/ml)
R2
R3
R4
(mc2150)
49
54
51
42
57
48
84
85
79
78
80
86
90
H
H
NO2
NO2
NO2
NO2
NO2
NO2
H
H
H
H
H
H
H
O-nBu(m)
O-nBu(p)
>50 (75%)
>50 (79%)
50-100
50
>200
>200
92.2
97.3
H
O-allyl(m)
>50 (76%)
45.5
5
50
105.6
111.4
10-100
100
H
O-allyl(p)
H
O-prenyl(m)
O-prenyl(p)
O-prenyl(m)
O-prenyl(m)
O-nBu(p)
.
6.0
2
3.21
113.7
142.6
>50 (69%)
>100
>200
H
H
NO2
H
>50 (43%)
>50 (94%)
nd
nd
>200
100
61.4
91.6
NO2
H
H
CN
H
>50 (22%)
>50 (66%)
>100
>100
>200
>200
206.7
103.5
H
CN
H
O-allyl(p)
H
CN
H
O-prenyl(p)
O-prenyl(m)
O-prenyl(m)
.>500 (82%)
9.2
5
nd
nd
50
25
186.4
172.6
132.6
H
CN
H
H
CO2H
H
.
16.5
12.5
nd = not determined
12

ACCEPTED MANUSCRIPT
To find out the amendment of activity with –I & +M effect we had prepared another three
sets of molecules with three different halides 61 – 69. For these three halids (F, Cl and Br) –I
effect decreases with the increase of atomic number, whereas, the +M effect increases with the
increase of atomic number. Compounds 61, 62 and 63 with respective fluoro substitution at
ortho, meta and para positions were found not to be effective against mycobacterium species
rather than it increases the cytotoxicity level. However, a trivial improvement was observes for
the meta substitution (62) compared to 61 and 63. For the chloro series (64 - 66) the activity
trend was found similar to that of fluoro series (Table 3). Nevertheless, the activity profile was
improved for the chloro series along with an improvement in their cytotoxicity level. For the
bromo (67 - 69) series the activity was improved to a significant amount compared to their fluoro
and chloro analogues. Compound 68 showed the best antimycobaterial activity among all other
halide derivatives and its MIC values were found was found to improved by two folds against
H37Rv and mc2155 strains.
Table 3: Role of -I and +M effect on antimycobacterial activity.
R1
O
R2
N
N
O
H
H
R3
R1
MIC (µg/ml)
(H37Rv)
MIC (µg/ml) Cytotoxicity
(µg/ml)
Entry
R2
R3
(mc2150)
>50 (68%)
>50 (89%)
100
25
102.8
94.0
F
H
F
H
H
63
62
61
66
65
64
H
H
Cl
H
H
Br
H
H
H
F
>50 (45%)
>50 (75%)
50
82.4
50-100
122.3
H
H
Cl
H
H
48.6
25
50
114.3
128.2
135.6
Cl
H
>50 (64%)
>50 (86%)
H
50-100
69
68
67
Br
H
H
26.5
38.7
12.5
25
148.1
131.9
Br
.
The comparative activity of these three different halide derivatives clearly showed
that the inductive effect ( ) played a decisive role in enhancing the antimycobaterial
I
activity and decreasing of the cytotoxicity level. Similar to the methyl derivatives a small
favourable steric effect near the urea linkage was also observed for these halide series.
13

ACCEPTED MANUSCRIPT
2.3 Effect on fatty acid and mycolic acid biosynthesis:
To probe the effect of synthesized molecules on mycobacterium fatty acid (FA)
and mycolic acid (MA) biosynthesis, we have selected eight different compounds from
each different set. The radio-labeled [1-14C]-acetate was used to determine
radiometrically their inhibitory effect on different fatty acids and mycolic acid
biosynthesis. Among the eight molecules compound 47 did not showed any inhibitory
activity on the biosynthesis of FA and MA as evidenced from the TLC profile (Fig 2),
whereas, the other compounds 46, 57, 58, 86 and 90 showed their selective inhibitory
activity on MA biosynthesis. For all these five compounds only the epoxy mycolate
biosynthesis of M. smegmatis was found to get inhibited but the other two α and α’
mycolate biosynthesis were remained almost unaffected. This finding clearly showed the
importance of the aromatic substitution (first aromatic ring) for inhibition of mycolic acid
biosynthesis. However, for compound 62 and 71 weak inhibitory activity was observed
for epoxy mycolate biosynthesis, but interestingly an additional band was observe below
the epoxy mycolate (Fig 2).
FAME
α
α′
Epoxy
57 46 58 90 86 47
71 62 DM Control
FAME = Fatty acid methyl ester; α = α-mycolic acid
α’ = α’-mycolic acid; Epoxy = Epoxy-mycolic acid
Fig 2: Inhibition of Fatty acid and Mycolic acid biosynthesis by the synthesized compounds. Compound
concentrations are 5 times to their MIC value.
There are two fatty acid synthetases (FAS I and FAS II) present in M. smegmatis
(mc2155).30 Among these two, FAS I is known to be responsible for the synthesis of C16 to C18
long fatty acid, whereas, further elongation and functionalization takes place in the FAS II
module to yield meromycolic acid.31 However, the synthesized molecules did not show any
inhibitory activity upon fatty acid biosynthesis.
14

ACCEPTED MANUSCRIPT
2.4 Immunomodulatory activity:
Mtb is an obligatory aerobic, intracellular pathogen, which has a predilection for
the lung tissue rich in oxygen supply. Phagocytosis of Mtb by alveolar macrophages is the
first event in the host-pathogen relationship that decides the outcome of infection. In
general, the event of phagocytic killing takes place either by toxic reactive oxygen
intermediates (ROI) or reactive nitrogen intermediates (RNI) or by both. However, RNI is
known to play a pivotal role during phagocytosis against tubercle bacilli.32
In order to determine the production of RNI, we determined the inducible nitric oxide
synthase (iNOS2) expression in BALB/c derived peritoneal macrophage (representative of all
tissue macrophages) infected by mc2155 strain, which is primarily responsible for the synthesis
of RNI from arginine via NADPH mediated pathway.32 A significant increase of iNOS2
expression in the infected macrophage treated with compound 58 and 86 compared to infected
macrophage alone evidently indicated their immunoprotective role against infection (Fig 3).
However, compound 57 showed a little effect on macrophage mediated RNI generation as
evidenced from the relative iNOS2 expression level for infected and infected but drug treated
macrophage (Fig 4A).
15

ACCEPTED MANUSCRIPT
Fig 3: Effect of 58 on iNOS2 expression (A), proinflammatory (IL-12 and IFN-γ) (B-C) and anti-inflammatory (IL-
10) (D) cytokines expression by RT-PCR. Data were represents here from one of three independent experiments
yielded similar kind of results. Corresponding bars are the mean of the respective densitometry data (mean ± SD).
*p<0.05; **p<0.01 indicates significant differences compared with indicated respective control. UIM = Uninfected
macrophage; IM = Infected macrophage; SUT-58 = compound ID for molecule 58.
Proinflammatory cytokines play a decisive role against mycobacterium infection.33 The
IFN-γ is known to trigger the macrophage activation for production of RNI via iNOS2, whereas,
IL-12 get induced during phagocytosis of tubercle bacilli as a natural immunoprotection against
Mtb infection.34 However, down-regulation of these cytokines is responsible for the disease
progression. Therefore, we examined the expression level of interferon-γ (IFN-γ) and
interleukin-12 (IL-12) to validate the effect of the synthesized compounds on proinflammatory
cytokine oozing during infection. In case of 58 and 86 an increase of IL-12 and IFN-γ expression
level between infected and infected with treated macrophage clearly demonstrated their potential
to up-regulate the expression of proinflammatory cytokines in infected macrophage (Fig 3B, 3C
and Fig 5B, 5C). Unlike the other two compounds, 57 did not show any positive activity on their
expression level against infection.
16

ACCEPTED MANUSCRIPT
Fig 4: Effect of 57 on iNOS2 expression (A), proinflammatory (IL-12 and IFN-γ) (B-C) and anti-inflammatory (IL-
10) (D) cytokines expression by rt-PCR. Data were represents here from one of three independent experiments
yielded similar kind of results. Corresponding bars are the mean of the respective densitometry data (mean ± SD).
*p<0.05; **p<0.01 indicates significant differences compared with indicated respective control. UIM = Uninfected
macrophage; IM = Infected macrophage; SUT-57 = compound ID for molecule 57.
Interleukin-10 (IL-10) is an anti-inflammatory cytokine and its up-regulation is known to
be responsible for the tuberculosis progression.35 Therefore, the expression of IL-10 was
determined in order to find out immunomodulatory activity of the synthesized compounds. In
case of compound 57, 58, and 86 treated infected macrophage a down-regulation of the IL-10
was observed compared to the up-regulation of IL-10 level in infected macrophage.
17

ACCEPTED MANUSCRIPT
Fig 5: Effect of 86 on iNOS2 expression (A), proinflammatory (IL-12 and IFN-γ) (B-C) and anti-
inflammatory (IL-10) (D) cytokines expression by RT-PCR. Data were represents here from one of three
independent experiments yielded similar kind of results. Corresponding bars are the mean of the respective
densitometry data (mean ± SD). *p<0.05; **p<0.01 indicates significant differences compared with
indicated respective control. UIM = Uninfected macrophage; IM = Infected macrophage; SUT-86 = compound ID
for molecule 86.
The differential expression level of iNOS2, IFN-γ, IL-12 and IL-10 for the infected and
treated macrophages compared to uninfected and infected macrophages unambiguously confirms
the immunomodulatory activity of these synthesized molecules.
The cytotoxicity (IC50) of the synthesized compounds against peritoneal macrophages
was found in the range of 180.9 - 58.1 µg/ml (Table 4) which is almost 16 – 5 higher than the
isoxyl (Table 5).
18

ACCEPTED MANUSCRIPT
Table 4: Cytotoxicity (IC50) against mammalian cell (Peritoneal Macrophage).
Comp Cytotoxicity Comp Cytotoxicity
Comp
Cytotoxicity
(µg/ml)
68.2
Comp
Cytotoxicity
(µg/ml)
116.7
115.3
96.7
(µg/ml)
152.6
87.5
(µg/ml)
78.4
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
78
79
80
84
85
86
90
90.3
83.7
122.1
108.9
75.2
121.7
74.3
87.1
105.6
96.7
180.9
169.1
58.1
152.9
141.8
94.1
125.7
109.5
131.2
81.3
115.3
119.4
131.8
140.2
69.4
75.2
140.5
160.3
127.6
159.4
120.4
142.5
Table 5: Antimycobacterial activity and cytotoxicity (IC50) values for Isoniazide and Isoxyl.
Comp
Cytotoxicity
(µg/ml) HepG2
>200
MIC (µg/ml)
H37Rv
0.11
MIC (µg/ml)
mc26030
0.05
MIC (µg/ml)
mc2155
6.25
Cytotoxicity (µg/ml)
macrophage
>200
Isoniazid
Isoxyl
2.3
nd
25
31.3
12.5
3. Conclusion:
Unsymmetrical diaryl ureas are shown to have a significant effect against Mtb. The
SAR study of the synthesized compounds demonstrated the positional preference of
different functional groups in both of the aromatic rings. For the first aromatic ring ortho
and meta positions were found to be the preferred one for the groups having +I and –I
& -
M
effect respectively. The best activity was found for 58 which has the strongest +I effect
and for 57 with the strongest –I
& –M effect than the others. However, compounds having
–I and –I
& +M showed moderate antitubercular activity. On the other hand, for the
second aromatic ring, best activity was found for the molecules having prenyl group at the
meta position, whereas, except compound 46, other aliphatic substitutions showed
moderate activity. In this series it was found that the substitution of thiourea moiety with
its bioisostere not only reduces the cytotoxicity by 40 folds (comp. 58 and isoxyl) but also
can disengage the participation of FMO (Flavin monooxygenase). Moreover, these series
19

ACCEPTED MANUSCRIPT
of molecules are quite distinctive in the sense of their selective inhibitory activity towards
mycolic acid biosynthesis.
The Mtb is known to suppress the immune response of the host cell during
infection. Therefore, rejuvenation of the immune response of macrophage has an
important role to clear the bacterium. Interestingly, molecules 58 and 86 showed a
significant impact to re-educate the immune response in favour to the host. Therefore,
these molecules are not only capable to kill the mycobacterium in a direct killing method
by perturbation of the mycolic acid biosynthesis but also able to clear the mycobacterium
in a macrophage mediated pathway by upregulation of the proinflammatory and
downregulation of the anti-inflammatory cytokines.
Altogether, these findings have a deep impact on the development of potent
antimycobacterial molecules with dual killing effect and high selectivity.
4. Experimental:
4.1 Reagents and instrumentation:
All the chemicals were purchased from Sigma–Aldrich, Alfa Aesar and Merck
Chemicals. Column chromatographic separations were performed using silica gel (100–200
mesh). Solvents were dried and distilled following standard procedures. TLC was carried out on
pre-coated plates (Merck silica gel 60, f254), and the spots were visualized with UV light or by
1
charring the plates dipped in 10% PMA solution in methanol or 5% H2SO4/vanillin/EtOH. H
13
NMR (300, 400 MHz) and C NMR (75, 100 MHz) spectra were recorded on a Bruker NMR
spectrometer (δ scale). UV–vis measurements were made using a Perkin Elmer UV–vis
spectrophotometer (Model Lambda 25). Mass spectra had been recorded using Waters Mass
Spectrometer (model XevoG2QTof).
4.2 Synthesis of urea derivatives: General method:
Method A: 3- and 4-Methoxyphenyl isocyanate (individually as per the scheme 1) was
added drop wise to a stirred solution of the corresponding amine in dichloromethane
(DCM) and was allowed to stir at room temperature until the completion of reaction
(checked by TLC). The organic solvent was then evaporated under reduced pressure.
20

ACCEPTED MANUSCRIPT
Crude residues thus obtained were purified over silica gel column chromatography using
hexanes and ethyl acetate as eluent.
Method B: To a stirred solution of p-anisidine in DCM (dry) three different phenyl
isocyanates were added (individually as per the scheme 2) and the stirring was continued
at room temperature until the completion of reaction (checked by TLC). The organic
solvent was then evaporated under reduced pressure. Crude residues thus obtained were
purified over silica gel column chromatography using hexanes and ethyl acetate.
Demethylation: General method
Method A: 50% Hydrobromic acid in acetic acid was added to a suspension of the
methyl ether of urea derivatives dissolved in acetic acid. The clear solution was
maintained at 70 °C for 12 hrs. After that the reaction mixture was cooled and quenched
with 10% sodium bicarbonate. Then the organic portion was extracted with ethyl acetate.
The combined organic layers were washed with water followed by brine solution and then
dried over sodium sulphate and evaporated to afford the crude product. The crude thus
obtained was purified by silica gel column chromatography using hexanes and ethyl
acetate.
Method B: A solution of the methyl ether of urea derivatives in DCM (dry) was
maintained at 0-5 °C followed by the addition of 1M solution of Borontribromide (BBr3)
in DCM. The stirring was continued at room temperature for 4-6 h. After completion of
the reaction (checked by TLC) it was quenched with 10% sodium bicarbonate and
extracted with DCM (3X100 ml). Combined organic layers were then washed with water
followed by brine solution and then dried over sodium sulphate and evaporated to afford
the crude product. The crude thus obtained was purified by silica gel column
chromatography using hexanes and ethyl acetate.
General procedure for the Mitsunobu reaction:
To a solution of alcohol in dry tetrahydrofuran (THF) triphenyl phosphine (PPh3) and
DIAD were added consecutively and stirred at RT for 15 minutes. Thereafter, the
corresponding phenolic compound was added slowly to the reaction mixture under
nitrogen atmosphere and the stirring was continued for 6-8 hours at room temperature.
21

ACCEPTED MANUSCRIPT
After completion of reaction (by TLC), solvent was evaporated and the crude was
partitioned between water and ethyl acetate (3X100 ml). The combined organic layers
were then washed with water followed by brine solution and dried over sodium sulphate.
The crude thus obtained was purified by column chromatography using a mixture of
hexanes /ethyl acetate. The purified compounds were dissolved in THF and treated with
5% aqueous Lithium hydroxide solution (THF/H2O 4:1) for 1 h at room temperature to
remove the DIAD by-products (as observed from their 1H-NMR). After that the THF was
removed under reduced pressure and the solid was filtered. The solid thus obtained was
dried completely and subjected for spectral analysis and bioassay. However, lithium
hydroxide treatment was not carried out for the Cyano derivatives 78, 79, 80 and 86.
1-(4-(allyloxy)phenyl)-3-phenylurea (41):
To a solution of Allyl alcohol (0.028 g; 0.48 mmol) in dry THF, TPP (0.149 g; 0.57 mmol) and
DIAD (0.115 g; 0.57 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 21 (0.1 g; 0.44 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
o
1
59.8%; white solid; m.p. 158-160 C. H NMR (acetone-d6) (300 MHz) – δ 4.38-4.40 (m, 2H),
5.09 (dd, J = 1.5, 10.3 Hz, 1H), 5.26 (dt, J = 1.8, 17.4 Hz, 1H), 5.88-5.94 (m, 1H), 6.73-6.85 (m,
3Ar-H), 7.09-7.14 (m, 2Ar-H), 7.29 (dd, J = 1.8, 6.6 Hz, 2Ar-H), 7.37-7.40 (m, 2Ar-H), 7.84 (bs,
13
1N-H), 7.94 (brs, 1N-H). C NMR (acetone-d6) (75 MHz) - δ 68.7, 114.8, 116.3, 118.4, 184.4,
120.3, 120.4, 121.8, 128.6, 133.2, 134.1, 140.2, 153.0, 154.2. HRMS (ESI+): m/z calcd for
C16H17N2O2 [M+H]+: 269.1290; found: 269.1293.
1-(4-(allyloxy)phenyl)-3-(3-nitrophenyl)urea (42):
To a solution of Allyl alcohol (0.024 g; 0.40 mmol) in dry THF, TPP (0.125 g; 0.48 mmol) and
DIAD (0.096 g; 0.46 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 22 (0.1 g; 0.366 mmol) was added slowly to the reaction mixture under
nitrogen atmosphere and the stirring was continued for 8 hours at room temperature. After
aqueous workup the crude was purified by column chromatography (16% EtOAc in Hexane).
1
Yield: 52.8%; pale yellow solid; m.p. 182-184 oC. H NMR (acetone-d6) (300 MHz) – δ 4.40-
22

ACCEPTED MANUSCRIPT
4.43 (m, 2H), 5.11 (dd, J = 1.5, 10.5 Hz, 1H), 5.28 (dd, J = 1.8, 17.1 Hz, 1H), 5.88-6.01 (m, 1H),
6.75-6.79 (m, 2Ar-H), 7.31 (dd, J = 2.1, 9.0 Hz, 2Ar-H), 7.41 (t, J = 8.4 Hz, 1Ar-H), 7.67-7.70
(m, 2Ar-H), 8.00 (brs, 1N-H), 8.50 (brs, 1N-H), 8.51 (t, J = 2.1 Hz, 1Ar-H). 13C NMR (acetone-
d6) (75 MHz) – δ 68.7, 112.6, 114.9, 116.2, 116.4, 120.8, 120.9, 124.1, 124.2, 129.8, 132.6,
134.1, 141.7, 148.8, 152.9, 154.6. HRMS (ESI+): m/z calcd for C16H16N3O4 [M+H]+: 314.1141 ;
found: 314.1145.
1-(4-(allyloxy)phenyl)-3-(o-tolyl)urea (43):
To a solution of Allyl alcohol (0.026 g; 0.45 mmol) in dry THF, TPP (0.141 g; 0.54 mmol) and
DIAD (0.108 g; 0.54 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 23 (0.1 g; 0.41 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
1
61.2%; white solid; m.p. 176-178 oC. H NMR (acetone-d6) (300 MHz) – δ 2.13 (s, 3H), 4.39-
4.41 (m, 2H), 5.10 (dd, J = 1.8, 10.5 Hz, 1H), 5.27 (dd, J = 1.8, 17.4 Hz, 1H), 5.89-5.98 (m, 1H),
6.73-6.83 (m, 3Ar-H), 7.01 (m, 2Ar-H), 7.31 (dd, J = 2.4, 6.9 Hz, 3Ar-H), 7.80-7.83 (m, 1N-H),
8.14 (brs, 1N-H). 13C NMR (acetone-d6 + CDCl3) (75 MHz) – δ 22.8, 74.9, 120.6, 122.7, 126.6,
128.0, 129.9, 132.2, 134.1, 135.9, 137.8, 139.2, 142.7, 159.2, 160.6. HRMS (ESI+): m/z calcd
for C17H19N2O2 [M+H]+: 283.1447; found: 283.1447.
1-(4-butoxyphenyl)-3-phenylurea (44):
To a solution of n-Butyl alcohol (0.036 g; 0.48 mmol) in dry THF, TPP (0.149 g; 0.57 mmol)
and DIAD (0.115 g; 0.57 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 21 (0.1 g; 0.44 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
1
76.6%; white solid; m.p. 168-170 oC. H NMR (acetone-d6) (300 MHz) – δ 0.83 (t, J = 7.5 Hz,
3H), 1.32-1.39 (m, 2H), 1.57-1.62 (m, 2H), 3.82 (t, J = 6.3, 2H), 6.72 (dd, J = 2.1, 6.6 Hz, 2Ar-
H), 6.83 (t, J = 7.2 Hz, 1Ar-H), 7.13 (t, J = 8.1 Hz, 2Ar-H), 7.28 (dd, J = 2.1, 6.9 Hz, 2Ar-H),
13
7.38-7.41 (m, 2Ar-H), 7.80 (brs, 1N-H), 7.91 (brs, 1N-H). C NMR (acetone-d6) (75 MHz) - δ
23

ACCEPTED MANUSCRIPT
13.4, 19.2, 31.5, 67.8, 114.7, 118.6, 120.7, 122.0, 128.8, 133.1, 140.5, 153.0, 155.0. HRMS
(ESI+): m/z calcd for C17H21N2O2 [M+H]+: 285.1603; found: 285.1601.
1-(4-butoxyphenyl)-3-(3-nitrophenyl)urea (45):
To a solution of n-Butyl alcohol (0.030 g; 0.40 mmol) in dry THF, TPP (0.125 g; 0.48 mmol)
and DIAD (0.096 g; 0.48 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 22 (0.1 g; 0.37 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (16% EtOAc in Hexane). Yield:
48.2%; pale yellow solid; m.p. 196-198 oC. 1H NMR (acetone-d6) (300 MHz) – δ 0.83 (t, J = 7.5
Hz, 3H), 1.32-1.40 (m, 2H), 1.58-1.63 (m, 2H), 3.83 (t, J = 6.6 Hz, 2H), 6.75 (dd, J = 2.1, 6.9
Hz, 2Ar-H), 7.30 (d, J = 9.3 Hz, 2Ar-H), 7.41 (t, J = 8.4 Hz, 1Ar-H), 7.67-7.69 (m, 2Ar-H), 7.99
(brs, 1N-H), 8.45 (brs, 1N-H), 8.51(t, J = 2.4 Hz, 1Ar-H). 13C NMR (d6-DMSO) (100 MHz) – δ
13.7, 18.7, 30.8, 67.3, 112.0, 114.6, 116.0, 120.5, 124.2, 130.0, 132.1, 141.3, 148.1, 152.6, 154.2.
HRMS (ESI+): m/z calcd for C17H20N3O4 [M+H]+: 330.1454; found: 330.1459.
1-(4-butoxyphenyl)-3-(o-tolyl)urea (46):
To a solution of n-Butyl alcohol (0.0336 g; 0.45 mmol) in dry THF, TPP (0.141 g; 0.54 mmol)
and DIAD (0.108 g; 0.54 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 23 (0.1 g; 0.41 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
1
54.6%; off white solid; m.p. 180-182 oC. H NMR (acetone-d6) (300 MHz) – δ 0.97 (t, J = 7.2
Hz, 3H), 1.46-1.51 (m, 2H), 1.72-1.74 ( m, 2H), 2.27 (s, 3H), 3.96 (t, J = 6.0 Hz, 2H), 6.85-6.97
(m, 3Ar-H), 7.15 (brs, 2Ar-H), 7.44 (d, J = 7.2 Hz, 2Ar-H), 7.96 (d, J = 8.1 Hz, 1N-H), 8.26 (brs,
13
1N-H). C NMR (d6-DMSO) (100 MHz) – δ 13.7, 17.9, 18.7, 30.8, 67.3, 114.6, 119.7, 120.9,
122.4, 126.1, 127.2, 130.1, 132.8, 137.6, 152.9, 153.8. HRMS (ESI+): m/z calcd for C18H23N2O2
[M+H]+: 299.1760; found: 299.1764.
24

ACCEPTED MANUSCRIPT
1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-phenylurea (47):
To a solution of Prenyl alcohol (0.0415 g; 0.48 mmol) in dry THF, TPP (0.149 g; 0.57 mmol)
and DIAD (0.115 g; 0.57mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 21 (0.1 g; 0.44 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
1
42.3%; off white solid; m.p. 112-114 oC. H NMR (acetone-d6) (300 MHz) – δ 1.61 (d, J = 9.0
Hz, 6H), 4.37 (d, J = 6.3 Hz, 2H), 5.31 ( t, J = 5.1 Hz, 1H), 6.69-6.74 (m, 2Ar-H), 6.82 (t, J = 7.2
Hz, 1Ar-H), 7.12 (t, J = 8.1 Hz , 2Ar-H), 7.25-7.29 (m, 2Ar-H) 7.38 (d, J = 7.8 Hz, 2Ar-H), 7.82
(brs, 1N-H), 7.94 (brs, 1N-H). 13C NMR (acetone-d6) (75 MHz) – δ 17.4, 25.0, 64.8, 114.8,
118.4, 118.5, 120.4, 120.5, 121.8, 121.9, 128.7, 132.9, 136.6, 140.2, 152.9, 154.6. HRMS
(ESI+): m/z calcd for C18H21N2O2 [M+H]+: 297.1603; found: 297.1606.
1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(3-nitrophenyl)urea (48):
To a solution of Prenyl alcohol (0.035 g; 0.40 mmol) in dry THF, TPP (0.125 g; 0.48 mmol) and
DIAD (0.096 g; 0.48 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 22 (0.1 g; 0.37 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (16% EtOAc in Hexane). Yield:
53.7%; pale brown solid; m.p. 164-166 oC. 1H NMR (acetone-d6) (300 MHz) – δ 1.62 (d, J = 7.8
Hz, 6H), 4.39 (d, J = 6.6 Hz, 2H), 5.32 (t, J = 1.5 Hz, 1H), 6.75 (dd, J = 1.2, 9 Hz, 2Ar-H), 7.31
(m, 2Ar-H), 7.41-7.45(m, 1Ar-H), 7.67-7.72 (m, 2Ar-H), 8.01 (brs, 1N-H), 8.46 (brs, 1N-H),
8.46-8.53 (m, 1Ar-H). 13C NMR (acetone-d6) (75 MHz) – δ 17.3, 24.9, 64.8, 112.6, 114.8, 116.2,
120.4, 120.9, 124.1, 129.7, 132.3, 136.6, 141.6, 148.8, 152.7, 154.9. HRMS (ESI+): m/z calcd
for C18H20N3O4 [M+H]+: 342.1454; found: 342.1456.
1-(4-((3-methylbut-2-ene-1-yl)oxy)phenyl)-3-(o-tolyl)urea (49):
To a solution of Prenyl alcohol (0.039 g; 0.45 mmol) in dry THF, TPP (0.141 g; 0.54 mmol) and
DIAD (0.108 g; 0.54 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 23 (0.1 g; 0.41 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
25

ACCEPTED MANUSCRIPT
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
25.5%; yellow solid; m.p. 52-53 oC; 1H NMR (300 MHz) (CDCl3) – δ 1.74 (s, 3H), 1.80 (s, 3H),
2.20 (s, 3H), 4.49 (d, J = 6.9 Hz, 2H), 5.46-5.50 (m, 1H), 6.28 (s, 1N-H), 6.40 (s, 1N-H), 6.85-
6.90 (m, 2Ar-H), 7.11 (t, J = 7.5 Hz, 1Ar-H), 7.20-7.26 (m, 4Ar-H), 7.59 (d, J = 7.8 Hz, 1Ar-H).
13C NMR (100 MHz) (d6-DMSO) – δ 17.9, 18.0, 25.4, 64.4, 114.8, 119.7, 120.2, 120.8, 122.4,
126.1, 127.2, 130.1, 132.8, 136.7, 137.6, 152.8, 153.5. HRMS (ESI+): m/z calcd for C19H23N2O2
[M+H]+: 311.1760; found: 311.1757.
1-(3-(allyloxy)phenyl)-3-phenylurea (50):
To a solution of Allyl alcohol (0.028 g; 0.48 mmol) in dry THF, TPP (0.149 g; 0.57 mmol) and
DIAD (0.115 g; 0.57 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 24 (0.1 g; 0.4381 mmol) was added slowly to the reaction mixture under
nitrogen atmosphere and the stirring was continued for 8 hours at room temperature. After
aqueous workup the crude was purified by column chromatography (20% EtOAc in Hexane).
1
Yield: 45.2%; off white solid; m.p. 142-144 oC. H NMR (acetone-d6) (300 MHz) – δ 4.40-4.43
(m, 2H), 5.11 (dd, J = 1.5, 10.5 Hz, 1H), 5.29 (dd, J = 1.5, 17.1 Hz, 1H), 5.90-5.93 (m, 1H), 6.41
(dd, J = 2.4, 8.1 Hz, 1Ar-H), 6.85-6.87 (m, 2Ar-H), 7.00-7.03 (m, 1Ar-H), 7.14 (t, J = 8.4 Hz,
13
2Ar-H), 7.22 (t, J = 2.1 Hz, 1Ar-H), 7.40 (d, J = 7.8 Hz, 2Ar-H), 8.01 (brs, 2N-H). C NMR
(acetone-d6) (75 MHz) – δ 68.3, 105.2, 108.2, 111.0, 116.5, 118.5, 122.1, 128.7, 129.4, 133.9,
140.0, 141.3, 152.7, 159.2. HRMS (ESI+): m/z calcd for C16H17N2O2 [M+H]+: 269.1290; found:
269.1291.
1-(3-(Allyloxy)phenyl)-3-(3-nitrophenyl)urea (51):
To a solution of Allyl alcohol (0.024 g; 0.40 mmol) in dry THF, TPP (0.125 g; 0.48 mmol) and
DIAD (0.096 g; 0.48 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 25 (0.1 g; 0.366 mmol) was added slowly to the reaction mixture under
nitrogen atmosphere and the stirring was continued for 8 hours at room temperature. After
aqueous workup the crude was purified by column chromatography (16% EtOAc in Hexane).
o
1
Yield: 59.2%; pale yellow solid; m.p. 126-128 C; H NMR (300 MHz) (acetone-d6) – δ 4.56-
4.59 (m, 2H), 5.26 (dd, J = 1.5, 10.8 Hz, 1H), 5.40-5.48 (m, 1H), 6.03-6.16 (m, 1H), 6.64 (dd, J
= 2.4, 8.4 Hz, 1Ar-H), 7.02-7.05 (m, 1Ar-H), 7.20 (t, J = 8.1 Hz, 1Ar-H), 7.37 (t, J = 2.1 Hz,
26

ACCEPTED MANUSCRIPT
1Ar-H), 7.57 (t, J = 8.1 Hz, 1Ar-H), 7.81-7.88 (m, 2Ar-H), 8.35 (brs, 1N-H), 8.67 (t, J = 2.1 Hz,
1N-H, 1Ar-H). 13C NMR (75 MHz) (acetone-d6 + CDCl3) – δ 73.9, 111.1, 114.8, 116.6, 118.2,
121.3, 122.4, 129.4, 134.9, 138.4, 153.0, 157.4, 162.1, 164.1. HRMS (ESI+): m/z calcd for
C16H16N3O4 [M+H]+: 314.1141 ; found: 314.1138.
1-(3-(allyloxy)phenyl)-3-(o-tolyl)urea (52):
To a solution of Allyl alcohol (0.026 g; 0.45 mmol) in dry THF, TPP (0.141 g; 0.54 mmol) and
DIAD (0.108 g; 0.54 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 26 (0.1 g; 0.41 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield: 69.4;
1
white solid; m.p. 144-146 oC. H NMR (acetone-d6) (300 MHz) – δ 2.27 (s, 3H), 4.54-4.56 (m,
2H), 5.23-5.27 (m, 1H), 5.43 (dd, J = 1.5, 17.1 Hz, 1H), 6.13-6.16 (m, 1H), 6.59 (dd, J = 2.4, 8.4
Hz, 1Ar-H), 6.97-7.01(m, 2Ar-H), 7.14-7.19 (m, 3Ar-H), 7.37 (t, J = 2.1 Hz, 1Ar-H), 7.53 (brs,
13
1N-H), 7.94 (d, J = 8.4 Hz, 1Ar-H), 8.47 (brs, 1N-H). C NMR (d6-DMSO) (100 MHz) – δ
17.9, 68.0, 104.5, 107.8, 110.5, 117.2, 121.1, 122.7, 126.1, 127.5, 129.5, 130.2, 133.7, 137.3,
141.1, 152.6, 158.6. HRMS (ESI+): m/z calcd for C17H19N2O2 [M+H]+: 283.1446; found:
283.1451.
1-(3-butoxyphenyl)-3-phenylurea (53):
To a solution of n-Butyl alcohol (0.036 g; 0.48 mmol) in dry THF, TPP (0.149 g; 0.57 mmol)
and DIAD (0.115 g; 0.57 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 24 (0.1 g; 0.44 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
o
1
56.8%; off white solid; m.p. 138-140 C. H NMR (acetone-d6) (300 MHz) – δ 0.98 (t, J = 7.2
Hz, 3H), 1.47-1.54 (m, 2H), 1.73-1.76 (m, 2H), 3.98 (t, J = 6.3 Hz, 2H), 6.57 (dt, J = 0.6, 8.1 Hz,
1Ar-H), 7.01-0.96 (m, 2Ar-H), 7.16 (t, J = 8.1 Hz, 1Ar-H), 7.25 (t, J = 8.4 Hz, 2Ar-H), 7.35 (t, J
13
= 2.1 Hz, 1Ar-H), 7.55 (d, J = 8.4 Hz, 2Ar-H), 8.14 (brs, 2N-H). C NMR (acetone-d6) (75
MHz) – δ 13.2, 19.1, 31.3, 67.3, 105.1, 108.2, 110.7, 118.6, 122.1, 128.7, 129.4, 140.0, 141.3,
152.6, 159.9. HRMS (ESI+): m/z calcd for C17H21N2O2 [M+H]+: 285.1603; found: 285.1608.
27

ACCEPTED MANUSCRIPT
1-(3-butoxyphenyl)-3-(3-nitrophenyl)urea (54):
To a solution of n-Butyl alcohol (0.030 g; 0.40 mmol) in dry THF, TPP (0.125 g; 0.48 mmol)
and DIAD (0.096 g; 0.48 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 25 (0.1 g; 0.37 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (16% EtOAc in Hexane). Yield:
68.5%; Pale yellow solid; m.p. 108-110 oC. 1H NMR (CDCl3 + acetone-d6) (300 MHz) – δ 0.95
(t, J = 7.2 Hz, 3H), 1.41-1.48 (m, 2H), 1.69-1.74 (m, 2H), 3.88 (t, J = 6.6 Hz, 2H), 6.62 (d, J =
7.5 Hz, 1Ar-H), 6.80 (d, J = 7.8 Hz, 1Ar-H), 6.99 (s, 1Ar-H), 7.15 (t, J = 8.1 Hz, 1Ar-H), 7.36 (t,
J = 8.4 Hz, 1Ar-H), 7.50 (brs, 1N-H), 7.72 (d, J = 8.1 Hz, 1Ar-H), 7.80-7.85 (m, 1Ar-H), 7.85
(brs, 1N-H), 8.1 (s, 1Ar-H). 13C NMR (CDCl3) (75 MHz) – δ 13.8, 19.2, 31.3, 67.8, 107.8, 110.6,
113.2, 114.3, 117.8, 125.5, 129.7, 130.0, 138.5, 139.6, 148.5, 153.4, 160.0 HRMS (ESI+): m/z
calcd for C17H20N3O4 [M+H]+: 330.1454; found: 330.1457.
1-(3-butoxyphenyl)-3-(o-tolyl)urea (55):
To a solution of n-Butyl alcohol (0.0336 g; 0.45 mmol) in dry THF, TPP (0.141 g; 0.54 mmol)
and DIAD (0.108 g; 0.54 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 26 (0.1 g; 0.41 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
1
65.8%; white solid; m.p. 120-122 oC. H NMR (acetone-d6) (300 MHz) – δ 0.97 (t, J = 7.5 Hz,
3H), 1.46-1.54 (m, 2H), 1.72-1.77 (m, 2H), 2.27 (s, 3H), 3.97 (t, J = 6.3 Hz, 2H), 6.56 (dd, J =
2.4, 8.1 Hz, 1Ar-H), 6.95-6.99 (m, 2Ar-H), 7.12-7.18 (m, 3Ar-H), 7.36 (t, J = 1.8 Hz, 1Ar-H),
7.53 (brs, 1N-H), 7.94 (d, J = 8.1 Hz, 1Ar-H), 8.45 (brs, 1N-H). 13C NMR (acetone-d6) (75 MHz)
– δ 13.3, 17.3, 19.1, 31.3, 67.3, 104.9, 108.1, 110.6, 121.7, 123.1, 126.2, 128.1, 129.4, 130.2,
137.7, 141.5, 152.9, 159.9. HRMS (ESI+): m/z calcd for C18H23N2O2 [M+H]+: 299.1759; found:
299.1766.
1-(3-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-phenylurea (56):
To a solution of Prenyl alcohol (0.0415 g; 0.48 mmol) in dry THF, TPP (0.149 g; 0.57 mmol)
and DIAD (0.115 g; 0.57 mmol) were added consecutively and stirred at RT for 15 minutes.
28

ACCEPTED MANUSCRIPT
Thereafter, comp. 24 (0.1 g; 0.44 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
1
48.3%; off white solid; m.p. 124-126 oC. H NMR (acetone-d6) (300 MHz) – δ 1.60 (d, J = 9.0
Hz, 6H), 4.40 (d, J = 6.6 Hz, 2H), 5.31-5.34 (m, 1H), 6.43 (dd, J = 1.8, 7.5 Hz, 1Ar-H), 6.82-
6.88 (m, 2Ar-H), 7.02 (t, J = 8.1 Hz, 1Ar-H), 7.11-7.19 (m, 3Ar-H), 7.41 (d, J = 8.7 Hz, 2Ar-H),
8.0 (brs, 2N-H). 13C NMR (acetone-d6) (75 MHz) – δ 17.4, 25.0, 64.5, 67.5, 105.2, 108.3, 110.8,
118.6, 120.3, 122.1, 128.7, 129.4, 131.3, 136.8, 140.1, 141.3, 152.7, 159.6. HRMS (ESI+): m/z
calcd for C18H21N2O2 [M+H]+: 297.1603; found: 297.1606.
1-(3-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(3-nitrophenyl)urea (57):
To a solution of Prenyl alcohol (0.035 g; 0.40 mmol) in dry THF, TPP (0.125 g; 0.48 mmol) and
DIAD (0.096 g; 0.48 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 25 (0.1 g; 0.37 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (16% EtOAc in Hexane). Yield:
1
64.1%; yellow solid; m.p. 82-84 oC. H NMR (acetone-d6) (300 MHz) – δ 1.63 (d, J = 5.8 Hz,
6H), 4.41 (d, J = 6.6 Hz, 2H), 5.33 (t, J = 1.2 Hz, 1H), 6.46-6.48 (m, 1Ar-H), 6.88-6.91 (m, 1Ar-
H), 7.05 (t, J = 8.1 Hz, 1Ar-H), 7.18 (t, J = 2.1 Hz, 1Ar-H), 7.43 (t, J = 8.4 Hz, 1Ar-H), 7.68-
7.74 (m, 2Ar-H), 8.19 (brs, 1N-H), 8.52 (s, 1N-H) 8.53 (s, 1Ar-H). 13C NMR (CDCl3) (75 MHz)
– δ 18.2, 25.8, 64.8, 106.6, 110.0, 112.3, 113.8, 117.2, 119.6, 125.0, 129.6, 138.2, 139.5, 140.4,
148.6, 152.9, 156.6, 159.6. HRMS (ESI+): m/z calcd for C18H20N3O4 [M+H]+: 342.1454; found:
342.1455.
1-(3-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(o-tolyl)urea (58):
To a solution of Prenyl alcohol (0.039 g; 0.45 mmol) in dry THF, TPP (0.141 g; 0.54 mmol) and
DIAD (0.108 g; 0.54 mmol) were added consecutively and stirred at RT for 15 minutes.
Thereafter, comp. 26 (0.1 g; 0.41 mmol) was added slowly to the reaction mixture under nitrogen
atmosphere and the stirring was continued for 8 hours at room temperature. After aqueous
workup the crude was purified by column chromatography (20% EtOAc in Hexane). Yield:
1
51.3%; off white solid; m.p. 130-132 oC. H NMR (acetone-d6) (300 MHz) – δ 1.62 (d, J = 7.8
29