Design, synthesis and SAR of antitubercular benzylpiperazine ureas

Article abstract of DOI:10.1007/s11030-020-10158-3

Abstract: N-furfuryl piperazine ureas disclosed by scientists at GSK Tres Cantos were chosen as antimycobacterial hits from a phenotypic whole-cell screen. Bioisosteric replacement of the furan ring in the GSK Tres Cantos molecules with a phenyl ring led to molecule (I) with an MIC of 1?μM against Mtb H37Rv, low cellular toxicity (HepG2 IC₅₀ ~ 80?μM), good DMPK properties and specificity for Mtb. With the aim of delineating the SAR associated with (I), fifty-five analogs were synthesized and screened against Mtb. The SAR suggests that the piperazine ring, benzyl urea and piperonyl moieties are essential signatures of this series. Active compounds in this series are metabolically stable, have low cellular toxicity and are valuable leads for optimization. Molecular docking suggests these molecules occupy the Q0 site of QcrB like Q203. Graphic Abstract: Bioisosteric replacement of N-furfuryl piperazine-1-carboxamides yielded molecule (I) a novel lead with satisfactory PD, metabolism, and toxicity profiles.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-020-10158-3

Molecular Diversity
https://doi.org/10.1007/s11030-020-10158-3
ORIGINAL ARTICLE
Design, synthesis and SAR of antitubercular benzylpiperazine ureas
Sohal Satish1 · Rohan Chitral1 · Amitkumar Kori1 · Basantkumar Sharma1 · Jayashree Puttur1 · Afreen A. Khan2 ·
Deepali Desle2 · Kavita Raikuvar2 · Aaron Korkegian3 · Elvis A. F. Martis2 · Krishna R. Iyer2 · Evans C. Coutinho2 ·
Tanya Parish3 · Santosh Nandan1
Received: 10 September 2020 / Accepted: 17 November 2020
© Springer Nature Switzerland AG 2021
Abstract
N-furfuryl piperazine ureas disclosed by scientists at GSK Tres Cantos were chosen as antimycobacterial hits from a pheno-
typic whole-cell screen. Bioisosteric replacement of the furan ring in the GSK Tres Cantos molecules with a phenyl ring led
to molecule (I) with an MIC of 1 μM against Mtb H37Rv, low cellular toxicity (HepG2 IC50 ~80 μM), good DMPK proper-
ties and specifcity for Mtb. With the aim of delineating the SAR associated with (I), ffty-fve analogs were synthesized and
screened against Mtb. The SAR suggests that the piperazine ring, benzyl urea and piperonyl moieties are essential signatures
of this series. Active compounds in this series are metabolically stable, have low cellular toxicity and are valuable leads for
optimization. Molecular docking suggests these molecules occupy the Q0 site of QcrB like Q203.
Graphic Abstract
Bioisosteric replacement of N-furfuryl piperazine-1-carboxamides yielded molecule (I) a novel lead with satisfactory PD,
metabolism, and toxicity profles.
Keywords Antitubercular · Lead compounds · Benzylpiperazine ureas · QcrB inhibitors
Introduction
Electronic supplementary material The online version of this
Tuberculosis (TB) claims more than 10 million lives
article (https://doi.org/10.1007/s11030-020-10158-3) contains
annually and is further compounded by the emergence of
supplementary material, which is available to authorized users.
MDR-TB and XDR-TB [1] which pose challenges for the
prevention, treatment, and control of TB. Discovery of
new chemotypes is the need of the hour. New chemotypes
revealed by scientists at GSK [2] have provided hits for
researchers both in academia and industry to develop as
antitubercular agents. We describe here our eforts at identi-
fying a hit and optimizing it to a lead which has the potential
of being developed as an antitubercular agent.
* Santosh Nandan
santosh.nandan@gmail.com
1
Ambernath Organics Pvt. Ltd., 222, The Summit Business
Bay, Andheri (E), Mumbai 400 093, India
2
Department of Pharmaceutical Chemistry, Bombay College
of Pharmacy, Kalina, Santacruz (E), Mumbai 400 098, India
3
TB Discovery Research, Infectious Disease Research
Our eforts began by searching for a hit in the database pub-
lished by scientists at GSK Tres Cantos [2, 3]. We recognized a
Institute, 1616 Eastlake Avenue E, Suite 400, Seattle,
WA 98102, USA
Vol.:(0123456789)
1 3

Molecular Diversity
Table 1 Aralkylpiperazine ureas disclosed by scientists at GSK Tres Cantos
GSK number
Structure
H37Rv
TOX50
MIC (μM)
HepG2 (μM)
TCMDC-143657
2.50
2.00
1.25
2.00
>100
>100
>100
>100
TCMDC-143653
TCMDC-143650
TCMDC-143652
Fig. 2 Molecule (I)—a piperonyl piperazine urea
antimycobacterial activity. We were gratifed to see that mol-
ecule (I) when tested against Mtb H37Rv returned an MIC of
1 μM (vide infra). This result coupled with the lack of cytotox-
icity (HepG2~80 μM, vide infra) led us to launch a program
of synthesis of analogs of (I) aimed at delineating the SAR.
The synthesis of benzylpiperazine ureas (3) is described in
Scheme 1. The monoprotected piperazine benzyl carbamate
(1) was reductively alkylated with aromatic aldehydes using
sodium triacetoxyborohydride [NaBH(OAc)3]. In the case of
the 2,6-dimethylpiperazine monoprotected carbamate, the reduc-
tive alkylation was not successful; however, alkylation with the
piperonyl bromide gave the required compound in satisfactory
yield. The benzyl carbamate moiety in molecules (2) in Scheme 1
was removed by catalytic hydrogenation, and the ureas (3) were
prepared using isocyanates or by base-catalyzed reaction of the
alkylated piperazine with benzyl carbamates of the respective
amines. In the second leg of Scheme 1, the monoprotected pip-
erazine benzyl carbamate (1) was protected with the tert-butyl-
oxycarbonyl (BOC) group to give (1a). The benzyl carbamate
was removed by catalytic hydrogenation, followed by reductive
amination to give (2a). The BOC group was removed using trif-
luoroacetic acid, and the ureas were prepared using isocyanates.
The synthesis of molecules with the piperidine replac-
ing the piperazine is depicted in Scheme 2. The piperidine
amides (7) were prepared by reductive amination of ethyl
nipecotate (5) with piperonal, followed by hydrolysis and
coupling with benzylamine.
Fig. 1 Piperazine pyrazole and arylvinyl piperazine amides reported
by Boshof et al
set of furfurylpiperazinyl ureas (Table 1) with good antimyco-
bacterial activity (MIC’s 1 to 3 μM, MABA assay on H37Rv)
which is non-cytotoxic (IC50 >100 μM, HepG2) and with
attractive physicochemical properties (molecular weight~350,
solubility~250 μM, 5<Chrom LogD>3). Interestingly, one
molecule (TCMDC-143653) inhibited the growth of Mtb
(pX50 =5.6) in an intracellular assay [2, 3] beside inhibiting
isoniazid and rifampicin-resistant Mtb strains, suggesting that
InhA and RNA polymerase may not be the targets of these
molecules [2, 3]. Boshof et al. have reported that piperazine
pyrazolyl amides (Fig. 1) inhibit the growth of Mtb via inhibi-
tion of QcrB [4], a novel antimycobacterial target. A closely
related set of molecules, arylvinylpiperazine amides (Fig. 1),
has also been reported by Cole et al. to inhibit QcrB of Mtb [5].
The furan ring in the GSK Tres Cantos compounds
(Table 1) is known to raise stability and metabolic issues [6].
The arylvinylpiperazine amides (GW86072X, Fig. 1) have
a conjugated double bond that could lead to toxicity [7]. To
address these structural imperfections in an otherwise good
set of molecules, we decided to replace the furan ring with
the bioisosteric phenyl [Molecule (I), Fig. 2] and test it for
1 3

Molecular Diversity
Scheme 1 Synthesis of benzyl
piperazine ureas
Reagents and conditions: (i) (a) AraCHO, NaBH(OAc)3,
dichloroethane, N2, refux yield 60–87% or (b) AraCH2Br,
sodium carbonate (Na2CO3), toluene, N2. Refux yield
40–50%; (ii) H2,10% Pd–C, methanol, rt yield 80–90%;
(iii) (a) ArbCH2NCO, dichloroethane, rt yield 40–75% or
(b) ArbCH2NHCO2CH2Ph (Cbz-phenyl amine), 1,8-diaz-
abicyclo[5.4.0]undec-7-ene (DBU), acetonitrile, N2, refux
yield 40–72%; (iv) BOC-anhydride, triethylamine, dichlo-
romethane, N2, 0 °C-rt yield 85%; (v) trifuoroacetic acid,
dichloromethane, N2, 0 °C to rt.
Scheme 2 Synthesis of piperidine amides (7)
Reagents and conditions: (i) Piperonal, NaBH(OAc)3,
dichloroethane, refux yield 65%; (ii) KOH, MeOH/H2O,
refux;(iii) ArCH2NH2, hexafuorophosphate azabenzotriazole
tetramethyl uronium (HATU), N,N-diisopropylethylamine
(DIPEA), N,N-dimethylformamide (DMF), rt yield 60.23%.
Scheme 3 Synthesis of 4-aryl-
methyl piperidines
1 3

Molecular Diversity
Reagents and conditions: (i) ArCH2NCO, triethylamine,
dichloroethane, rt yield 55%; (ii) sodium hydride (NaH),
tetrahydrofuran (THF), 0 °C to rt yield 52%; (iii) 10%
Pd/C, H2, methanol, rt yield 61.27%.
The reverse substitution pattern on the piperidine
(Scheme 3) was obtained by reacting piperidine-4-one (8)
with benzyl isocyanate to give (9) which was followed by
a Horner–Wadsworth–Emmons reaction to yield (10) and a
fnal reduction of the double bond to give (11). The pyrro-
lidine and homopiperazine replacements for the piperazine
ring used benzyloxycarbonyl protected 3-aminopyrrolidine
and homopiperazine as starting materials and followed the
same sequence as in Scheme 1.
Fig. 3 Division of molecule (I) into four regions A, B, C, D to aid
analysis of the SAR
Changing the size of the piperazine ring or opening the
ring to make acyclic ethylene diamine derivatives leads to
inactive molecules (17, 20, 21 and 12 in Table 2). Thus,
we conclude that the piperazine ring is essential for anti-
mycobacterial activity.
Design and structure–activity relationships
Since we planned to screen our molecules in a whole-
cell assay (vide infra), it was necessary to avoid making
large structural perturbations, as the SAR would then be
difcult to interpret. Therefore, only small incremental
changes were made to (I) to understand the importance
and contribution of each structural feature of molecule (I).
Our design was based on dissecting the molecule into four
diferent regions as shown in Fig. 3. Tanimoto coefcients
were calculated using KNIME platform to ensure that the
molecules were not dissimilar. The MIC90 was determined
against Mtb H37Rv (ATCC 25618), under aerobic condi-
tions [8].
Our second set of molecules (Table 3) was designed
and synthesized to probe the structural requirement of the
urea moiety (C in Fig. 3). Replacing the urea moiety with
a carbamate led to an equipotent molecule (22 in Table 3),
whereas changing the urea moiety to an amide (23) led to
loss of activity. In (22) and (23), isosteric replacement of
the CH2 with heteroatoms O or N, gives the ether amide
(24) or amino amide (25), respectively; the latter is inactive.
Alkylation of the urea to an N-methyl urea (26) leads to a
small loss in activity.
Next, we decided to target the substitution pattern on
the two aryl rings to understand the scope for modifcation
around these two rings (Table 4). The piperonyl ring (A in
Fig. 3) could be replaced with a 3,4-dimethoxybenzyl (27 in
Table 4), or a 3-methoxybenzyl (28) group with little or no
loss of activity. The 4-methoxybenzyl (29) and the 3-meth-
oxy-4-methylbenzyl (35) derivatives, as well as isomers of
dimethoxybenzyl (32, 33), are inactive. Surprisingly, the
2-methyl-3-methoxybenzyl (36) and 2-chloro-3-methoxy-
benzyl (48) derivatives are not completely inactive suggest-
ing that the 3,4-disubstitution pattern is optimum to main-
tain activity. Since the 3-methoxybenzyl group (28) shows
activity like (I), we positioned other substituents at the meta
position without much success. Replacing the piperonyl ring
with a benzo-1,4-dioxolanyl (30) or a 2,2-difuorobenzo-
1,3-dioxolanyl ring (31) leads to complete loss of activity.
Interestingly, the 3-ethoxybenzyl (40) and the 3-trifuo-
romethoxybenzyl (43) substituents give molecules with a
signifcant loss of activity. Introduction of a methyl group
as in (44), also abolishes activity. Replacing the piperonyl
ring with a thienyl ring (46, 47) also gives compounds with
reduced activity. These results suggest that the piperonyl
ring is a key motif of activity.
Each region—A, B, C, D in molecule (I) (Fig. 3)—was
sequentially altered, assuming that the original molecule
and its derivatives would be inhibiting the same target
within the mycobacterial cell and thus follow a logical
SAR. Thus, while modifcations were being made in a par-
ticular region for, e.g., A—probing the conformational,
electronic and steric properties—the remainder of the
molecule (i.e., B, C, D) was held constant. This lead to
four sets of molecules that are very similar (as judged by
the Tanimoto coefcient) to the starting molecule (I). The
initial focus was to determine which part of the piperazine
ring (B in Fig. 3) is an important contributor to the anti-
mycobacterial activity of (I). Systematic changes to the N
atoms, ring size, and introduction of methyl groups were
made (Table 2), to probe substitutions tolerated on the
piperazine ring. The results show that both N atoms are
important for activity, as changing to piperidine (18 and
19 in Table 2) abolishes activity. We believe that the pip-
erazine ring (being strongly basic) is protonated at physi-
ological pH, making an important contribution to receptor
interaction; hence, alteration abolishes activity. Introduc-
tion of a single methyl group adjacent to the piperonyl
nitrogen (13 in Table 2) or adjacent to the urea nitrogen
(14 in Table 2) is tolerated with small loss of activity.
Replacement of the benzyl moiety in (I) with a
cyclohexyl (49 in Table 5) or cyclohexylmethyl (50) leads
1 3

Molecular Diversity
Table 2 Modifcations made on piperazine element of I (B in Fig. 3)
Compound
Y
MIC90
(µM)
Tanimoto simi- Compound
larity
Y
MIC90
(µM)
Tanimoto
similarity
H37Rv
H37Rv
I
1.0
1.00
17
>20
1.00
12
13
>20
5.6
0.89
0.80
18
19
>20
>20
0.89
0.89
14
15
4.5
0.80
0.80
20
21
>20
>20
0.89
0.89
>20
16
15.0
0.80
Table 3 Modifcations on the urea element of I (C in Fig. 3).
shift of these NH protons with activity. Replacing the
benzyl group with acyclic aliphatic substituents (61, 62)
leads to inactive molecules. Replacing the benzyl with a
cyclohexyl methyl leads to a molecule (50) which is equi-
potent to (I), suggesting that the hydrophobicity might be
important for activity in this portion (D) of the molecule.
We selected 5 molecules from the above sets and
profled them for their antibacterial activity (S. aureus,
E. coli and S. typhi), cytotoxicity (HepG2 cells IC50) and
metabolic stability (Table 6). The metabolic stability [9]
(rat liver microsomes) studies show that (I) is degraded
slowly with a clearance of 2.90 mL/min/Kg. Compound
27 was found to be least susceptible to oxidative biotrans-
formation, while 28 is metabolically more labile. The low
hepatic clearance observed in the case of compound (I)
suggests that it would have high oral bioavailability and a
long half-life when administered orally. The cytotoxicity
results indicate these molecules in general to be less cyto-
toxic and have an attractive therapeutic index. All mol-
ecules tested for antibacterial activity were inactive at the
highest concentration tested (32 mg/L), suggesting that the
molecules exert their antimycobacterial efect by inhibit-
ing a mycobacteria specifc pathway or biomolecule.
The cytotoxicity and antibacterial activity data parallel
the observations of Cole et al. [5] and Boshof et al. [4].
The structural similarity with arylvinylpiperazine amides
Compound
Y
MIC90 (µM)
H37Rv
Tanimoto
similarity
22
23
24
25
26
C(O)OCH2
C(O)CH2CH2
C(O)CH2O
C(O)CH2NH
C(O)N(Me)CH2
1.9
16.0
6.7
>20
5.9
0.88
0.88
0.88
1.00
0.78
to molecules with activities like (I). Changing the size and
type of the ring and decreasing or extending the carbon
chain lead to loss of activity (59, 63, 64). We also made
eforts to place substituents on the aromatic ring. In most
cases, it results in inactive molecules (53, 55, 57, 65). The
4-chloro (54) and 4-fuoro (56) derivatives are the only
compounds to show some activity. The only exchange-
able proton in these structures is the carbamoyl NH which
1
is attached to the benzyl moiety. Examination of the H-
NMR spectra reveals no clear correlation of the chemical
1 3

Molecular Diversity
Table 4 Modifcations on the piperonyl element of I (A in Fig. 3).
Compound
Y
MIC90 (µM)
H37Rv
Tanimoto
similarity
Compound
Y
MIC90 (µM)
Tanimoto
similarity
H37Rv
27
28
29
30
31
32
1.0
0.89
0.89
0.89
1.00
0.80
0.89
38
39
40
41
42
43
>20
0.70
0.78
0.89
0.78
0.78
0.80
2.3
>20
12.0
>20
>20
15.0
16.0
>20
>20
>20
33
34
>20
>20
0.89
0.89
44
45
>20
>20
0.78
0.80
35
36
>20
9.4
0.89
0.89
46
47
11.0
11.0
0.78
0.78
37
6.5
0.78
48
11.0
0.80
coupled with the antibacterial and cytotoxicity results sug-
gests that these compounds may be acting via inhibition of
QcrB. Molecular docking of Q203 and (I) into the QcrB
homology model shows that molecule I occupies the Q0 site
(the stigmatellin binding site) in a manner like Q203 [10]
(Fig. 4). The benzodioxole moiety of (I) occupies the same
pocket as the trifuromethoxyphenyl group of Q203.
Conclusions
In conclusion, modifcation of the GSK Tres Cantos mol-
ecules by replacement of the furan moiety with a phenyl
ring led to molecule (I). In an attempt to delineate the
SAR around (I), ffty-fve analogs were synthesized and
screened against Mtb. From this series, four molecules
1 3

Molecular Diversity
Table 5 Modifcations on the benzyl element of I (D in Fig. 3).
Compound
Y
MIC90 (µM)
H37Rv
Tanimoto
similarity
Compound
Y
MIC90 (µM)
H37Rv
Tanimoto
similarity
49
50
51
6.2
1.3
13
0.89
0.89
1.00
58
59
60
>20
0.80
0.89
0.80
20
>20
52
>20
0.80
61
>20
0.89
53
54
>20
15.0
0.89
0.89
62
63
>20
>20
0.89
0.89
55
56
57
>20
8.0
0.89
0.89
0.89
64
65
>20
>20
1.00
0.89
>20
Table 6 Profle of selected hits
Compound
MIC
Clearance
HepG2 cytotox- cLogP
Antibacterial MIC (mg/L)
(S. aureus, E. coli and S.
typhi)
H37Rv(µM)
(mL/min/Kg) icity IC50 (μM)
I
1.0
1.0
2.3
6.2
1.3
2.90
0.36
4.47
3.54
3.85
81.00
50.80
38.63
52.43
47.43
2.67
2.42
2.49
2.81
3.00
>32
>32
>32
>32
27
28
49
50
Not tested
(27, 28, 49 and 50) were found to be like (I) in terms of
antimycobacterial activity, toxicity and metabolic profle.
It is likely that the piperonyl piperazine ureas also inhibit
QcrB as they are structurally very similar to the arylvinyl-
piperazine amides disclosed by Cole et al.; however, this
series of molecules does not possess the Michael acceptor
that is present in the molecules disclosed by Cole et al.
The compounds have low cellular toxicity and do not
inhibit the growth of other bacteria, suggesting that they
might target a specifc mycobacterial pathway or biomol-
ecule. Salient features of the SAR are the requirement of
a benzylpiperazinyl urea and a piperonyl moiety. In addi-
tion, metabolism studies with microsomes show that (I)
is not metabolized and cleared rapidly. The low molecular
weight and the good solubility of this compound make it
an ideal candidate for a lead optimization program.
1 3

Molecular Diversity
Fig. 4 Docking of molecule
(I) (cyan sticks) and Q203
(magenta sticks) into the homol-
ogy model of M tuberculosis
QcrB
terminated after 48 h and then worked up. The mixture was
extracted with DCM; the organic layer was washed consecu-
tively with sat. NaHCO3, water and brine. The organic layer
was dried over anhydrous sodium sulfate (Na2SO4) and then
concentrated under reduced pressure to obtain Cbz-pipera-
zine; 900 mg (51%).
Experimental section
Chemistry
Materials and methods
Method B (For Cbz-protection of 3-aminopyrrolidine)
[12]: 3-Aminopyrrolidine (4.0 g, 25.14 mmol) was dissolved
in 55 mL of DCM; triethylamine (10.5 mL, 75.44 mmol)
was then added to the solution. The solution was cooled to
0 °C; benzyl chloroformate (0.35 mL, 2.51 mmol) was added
dropwise over a period of 20 min. The reaction mixture was
slowly warmed to room temperature and stirred overnight.
The mixture was washed consecutively with water and brine
and then dried over anhydrous Na2SO4. The organic layer
was concentrated under reduced pressure to obtain 3-amino
Cbz-pyrrolidine; 410 mg (44.5%).
Commercially available reagents and solvents were used
without any purifcation. Solvents were commercially pur-
chased and dried before use wherever necessary. Experi-
ments were carried out under nitrogen atmosphere except
where water was used as solvent. The progress of the reac-
tions was monitored by TLC using Merck precoated silica
plates (60 F254). Flash column chromatography was carried
out with silica gel (230–400 mesh) column; visualization
of TLC spots was done in a UV chamber at 254 nm and
220 nm and by staining with ninhydrin or PMA. 1H and 13
C
spectra were recorded in DMSO-d6 using TMS as an internal
standard on a Bruker 400 MHz spectrometer at room tem-
perature. Chemical shift values quoted are in parts per mil-
lion (ppm), and coupling constants are in Hertz (Hz). Mass
spectra and HPLC were recorded on a Agilent Quadrupole
LCMS (6120), and HRMS was recorded on a Waters Q-TOF
micro-mass spectrometer (ESI–MS).
Method C (For Carbamates in the DBU coupling reac-
tion): Benzylamine (0.5 g, 3.99 mmol) was taken in 20 mL
of water and stirred at room temperature. K2CO3 (1.1 g,
7.99 mmol) was added to this solution, and this was followed
by addition of benzyl chloroformate (0.8 mL, 4.79 mmol).
The reaction was complete in 5–6 h. The mixture was
extracted with chloroform; the organic fraction was sepa-
rated, washed with brine, dried over anhydrous Na2SO4 and
then concentrated under reduced pressure to obtain the crude
product. The crude was purifed by silica gel column chro-
matography, eluting with 1% MeOH/chloroform to provide
pure Cbz-benzylamine (850 mg, 82.2%). Note: All carba-
mates needed for the synthesis of compounds 49–65 were
prepared using Method C.
Experimental procedure
(I) General procedure for synthesis of carbamates
[benzyloxycarbonyl (Cbz) ‑protection of amine].
Method A [11]: Piperazine (1.5 g, 17.44 mmol) was dis-
solved in water (60 mL); 3 N HCl (25 mL) was added to
adjust the pH to 2–3. To this stirred solution, benzyl chloro-
formate (2.97 g, 17.44 mmol) was added in 3 portions over
a period of 15–18 h; pH 2–3 was maintained at regular
intervals using 3 N NaOH. The progress of the reaction was
monitored by TLC (7% MeOH in DCM). The reaction was
(II) General procedure for reductive amination
Cbz-piperazine (0.15 g, 0.68 mmol) and piperonal (122 mg,
0.81 mmol) were dissolved in dry DCE. To this, ace-
tic acid (11 μL, 0.2 mmol) was added and the reaction
1 3

Molecular Diversity
mixture stirred. Then, sodium triacetoxyborohydride (3 eq,
2.04 mmol, 431 mg) was added and the mixture refuxed
under N2 atmosphere. The progress of the reaction was
monitored by TLC. The reaction was complete in 4 h. The
reaction mixture was quenched with sat. NaHCO3, diluted
with water and then extracted with dichloromethane. The
organic layer was washed with water then brine, dried over
anhydrous Na2SO4, and concentrated under reduced pressure
to get the crude product. The crude was purifed by silica gel
chromatography eluting with 40% EtOAc in Pet. ether which
aforded the pure product 22; 210 mg (87%).
reaction mixture was warmed to room temperature and stir-
ring continued. The reaction was found to be complete in
4 h. The reaction mixture was quenched with sat. NaHCO3
and then extracted with DCM. The organic layer was washed
with brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure to obtain the product. This product
was directly used in the next step without purifcation.
(VI) General procedure for the synthesis of urea
derivatives using benzyl isocyanate
The above procedure was followed for reductive amina-
tion of monoprotected diamines and aldehydes. Time taken
for completion of the reaction with different aldehydes
ranged from 3 to 18 h, yield of the reaction ranged from 60
to 87%.
Piperazine intermediate (obtained from Cbz deprotection of
compound 2; Scheme 1) (0.28 g, 1.2 mmol) was dissolved
in 5.0 mL dry DCE and stirred at room temperature under
N2. Benzyl isocyanate (0.18 mL, 1.2 mmol) was added and
stirring continued. On completion of the reaction, the reac-
tion mixture was quenched with water and then extracted
with DCM. The organic fraction was washed with brine,
dried over anhydrous Na2SO4 and concentrated to obtain the
desired crude product. The crude was purifed by silica gel
column chromatography, eluting with (1% MeOH/CHCl3)
to obtain compound I (75% yield). The compounds 14, 15,
17 and 21 were synthesized following the sequence (1→3)
in Scheme 1.
(III) General procedure for removal of the Cbz group
[13–15]
Cbz-piperazine (0.35 g, 0.94 mmol) was dissolved in meth-
anol (0.2 M) and stirred at room temperature. 10% Pd–C
(5 mol %) was added to the reaction mixture, and H2 gas
was bubbled into it. The reaction was monitored by TLC
and was completed in 2 h. The reaction mixture was diluted
with methanol and fltered through a Celite bed and the resi-
due washed with methanol. The fltrate was concentrated
to yield the product. This was directly used in the next step
without purifcation. The yield of this reaction ranged from
80 to 90%.
(Time taken for completion of reaction with diferent car-
bamates ranged from 16 to 28 h, yield of the reaction ranged
from 40 to 75%.).
*The compounds 13, 16, 20 were synthesized following
the sequence (1→3) in Scheme 1.
(VII) Procedure for synthesis of compound (18)
(IV) Procedure for Boc protection of amine
A. Procedure for synthesis of aryl phosphonate [16].
Cbz-2,6-dimethylpiperazine (1.0 gm, 4.27 mmol, 1 eq) was
dissolved in 50 mL dry DCM and stirred at 0 °C under N2.
Triethylamine (2.97 mL, 21.36 mmol, 5.0 eq) was added to
the reaction mixture, followed by Boc-anhydride (5.8 mL,
25.63 mmol, 6.0 eq), and the mixture warmed to room
temperature. The reaction was monitored by TLC and was
completed in 2 h. The reaction mixture was quenched with
water and then extracted with DCM. The organic layer was
separated, washed with brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure to obtain the crude
product. The crude product was purifed by silica gel column
chromatography to provide the Boc protected compound
(isolated yield 85%).
Trimethyl phosphite (2 eq) was added to a stirred solution
of the aryl bromide (1 eq) in xylene (0.3 M). The solution
was heated to 100 °C and the reaction continued overnight
(16 h). On completion of the reaction, the solvent was
removed under reduced pressure. The residue was washed
with water and then extracted thrice with EtOAc. The com-
bined organic fraction was then washed with brine and dried
over anhydrous Na2SO4. The organic layer was concentrated
under reduced pressure to obtain a crude residue which was
purifed by column chromatography to a colorless oil.
B. Procedure for Horner–Wadsworth–Emmons reaction
[16].
(V) Procedure for removal of Boc group
A solution of the aryl phosphonate (0.257 g, 0.94 mmol,
1 eq) in dry THF (1 mL) was added to a stirred solution of
NaH (0.027 g, 1.1 mmol, 1.2 eq) in dry THF (1 mL) at room
temperature under N2. The solution was stirred at room tem-
perature for 15 min. A solution of the 4-piperidone (0.22 g,
Benzyl-4-(t-butoxy)-3,5-dimethylpiperazine-1-carboxylate
(400 mg, 1.19 mmol, 1.0 eq) was dissolved in (6 mL) dry
DCM. TFA (0.92 mL, 11.97 mmol, 10 eq) was added to the
reaction mixture slowly keeping the reaction at 0 °C. The
1 3

Molecular Diversity
0.94 mmol, 1 eq) in dry THF (2.5 mL) was added slowly to
the above solution and stirred overnight at room tempera-
ture. On completion of the reaction, the reaction mixture
was quenched with dil. HCl and then diluted with water and
fnally extracted thrice with CHCl3. The combined organic
layer was washed with brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure to obtain the crude
product. The crude was purifed by column chromatogra-
phy to obtain the pure product (52% yield). This was then
reduced by catalytic hydrogenation using 10% Pd/C in meth-
anol to obtain compound 18.
Procedure B
(a) The intermediate (110 mg, 0.374 mmol, 1 eq) obtained
from procedure A was taken in dry DCM (3 mL); tri-
ethylamine (0.05 ml, 0.556 mmol, 1.5 eq) was added to
this solution, followed by addition of aniline (0.07 ml,
0.74 mmol, 2 eq) at room temperature under N2 atmos-
phere, and the mixture stirred for 17 h. The reaction
mixture was evaporated under vacuum, and the residue
was taken up in DCM. The organic layer was washed
with water, then brine, then dried over anhydrous
Na2SO4 and finally concentrated to obtain a crude
residue which was purifed by silica gel chromatogra-
phy, eluting with 70% EtOAc/Pet. ether, compound 25
(74 mg, 57%).
(VIII) Procedure for synthesis of compound (23)
(Intermediate obtained after deprotection of compound 3
(Scheme 1) and cinnamic acid was coupled using HATU
followed by reduction with 10% Pd/C, with bubbling of H2
gas to obtain compound 23)
(b) The intermediate (150 mg, 0.50 mmol, 1 eq) obtained
from procedure A was dissolved in acetone (3 mL).
Anhydrous K2CO3 (139 mg, 1.01 mmol, 2 eq) was
added, which was followed by the addition of phenol
(94 mg, 1.01 mol, 2 eq), and the mixture refuxed over-
night. The reaction mixture was diluted with EtOAc
and washed with water, then brine, then dried over
anhydrous Na2SO4 and fnally concentrated to obtain
a crude product which was purifed by silica gel chro-
matography, eluting with 55% EtOAc/Pet. ether, com-
pound 24 (111 mg, 62.3%).
Cinnamic acid (269 mg, 1.81 mmol) was dissolved in
5 mL of dry DMF; HATU (1.3 g, 3.6 mmol) was added,
followed by addition of DIPEA (0.55 mL, 3.18 mmol), and
the reaction mixture was stirred at room temperature under
N2 for 30 min. Piperazine intermediate (obtained from Cbz
deprotection of compound 2 (Scheme 1) (200 mg, 0.9 mmol)
was added, and the reaction mixture was stirred for 16 h.
DMF was evaporated under reduced pressure; the residue
was mixed with DCM, washed frst with water then with
brine. The DCM layer was then dried over anhydrous
Na2SO4 and concentrated under reduced pressure to obtain
the crude product. The residue was purifed by silica gel
chromatography, eluting with (50% EtOAc/Pet. ether) to
obtain compound 23 (210 mg, 67%).
(X) Procedure for synthesis of compound (26)
NaH (0.025 g, 0.42 mmol) was taken in 1 mL of DMF and
stirred at 0 °C. A solution of compound I (0.1 g, 0.28 mmol)
in 2 mL dry DMF was added to this slowly and stirred for
10–15 min. Methyl iodide (0.022 mL, 0.33 mmol) was added
to the solution and stirred at room temperature overnight.
The reaction was completed in 16 h. DMF was evaporated
under reduced pressure, and the residue was diluted with
chloroform and washed frst with water then with brine; the
chloroform layer was separated and dried over anhydrous
Na2SO4 and fnally concentrated under reduced pressure to
obtain the crude product. The crude was purifed by silica
gel chromatography, eluting with (3% MeOH/CHCl3) to
obtain compound 26 (79 mg, 75%).
(IX) Procedure for synthesis of compounds 24 and 25
The intermediate obtained after deprotection of compound 2
(Scheme 1) was subjected to Procedure A, followed by Pro-
cedure B (see below) to synthesize compounds 24 and 25.
Procedure A
To a solution of piperazine intermediate (obtained by Cbz
deprotection of compound 2) (1.05 g) in dry DCM (25 mL),
DIPEA (1.24 mL) was added and stirred under N2 atmos-
phere. To this, 0.45 mL of chloroacetyl chloride was added
gradually over a period of 5 min and left stirring overnight.
Reaction was completed in 18 h. The reaction was quenched
with sat. NaHCO3 and extracted with DCM. The organic
fraction was washed with brine, dried over Na2SO4, and
concentrated to obtain the crude product. This was purifed
by silica gel chromatography eluting with 60% EtOAc/Pet.
ether to obtain the pure product (0.98 gm, 75%). This was
used in procedure B.
(XI) Procedure for synthesis of compound (30)
(Intermediate* obtained after reductive amination of
3,4-dihydroxy benzaldehyde and Cbz-piperazine was used
in this reaction)
K2CO3 (0.08 g, 0.58 mmol, 2 eq) was added to a solution
of piperazine derivative* (0.1 g, 0.29 mmol, 1 eq) in 3 mL of
ethylene glycol. The reaction mixture was heated to 120 °C
1 3

Molecular Diversity
and stirred under N2. Dibromoethane (54 µL, 0.58 mmol,
2 eq) was added to the reaction and heating continued at
120 °C. The reaction was complete in 3 h. At the end of the
reaction, the mixture was diluted with water and extracted
into EtOAc. The organic fraction was washed with brine,
then dried over Na2SO4, and fnally concentrated under
reduced pressure to obtain the crude product. The crude
product was purifed by silica gel chromatography, elut-
ing with 30% EtOAc/Pet. ether, to obtain the pure product.
(56 mg, 52.3%).
(XIV) General procedure for the synthesis of com‑
pounds 49–65 [19].
Piperazine intermediate (obtained from Cbz deprotection
of compound 2) (250 mg, 1.13 mmol, 1.0 eq) and Z-pro-
tected amine (317 mg, 1.36 mmol, 1.2 eq) were dissolved
in 6 mL of CAN; this was followed by the addition of DBU
(0.2 mL, 1.36 mmol, 1.2 eq). The reaction mixture was kept
stirring at 80 °C under N2 atmosphere. The reaction was
complete in 26 h. The reaction mixture was quenched with
water and then extracted with chloroform. The organic layer
was washed with brine, dried over anhydrous Na2SO4 and
concentrated under reduced pressure to obtain the crude
product. The crude was purifed by silica gel chromatogra-
phy eluting with (2% MeOH/CHCl3) to provide 49 (278 mg,
71.2%).
This product was subjected to catalytic hydrogenation
using 10% Pd/C and H2 gas. The reduced product was used
for synthesis of compound 30, following the general proce-
dure for synthesis of urea derivative.
(XII) Procedure for bromination of aromatic alcohol
[17]
(Time taken for completion of the reaction with diferent
carbamates ranged from 16 to 28 h, yield of the reaction
ranged from 40 to 72%.)
The alcohol (17) (1 eq) was dissolved in DCM and stirred at
0 °C under nitrogen atmosphere. A solution of PBr3 (1.3 eq)
in DCM was added dropwise to the reaction mixture over a
period of 10–15 min, and the reaction was stirred at room
temperature under N2. The reaction was completed after
16 h. The reaction mixture was quenched with sat. NaHCO3
solution and then extracted into DCM. The DCM layer was
washed with brine and dried over anhydrous Na2SO4. This
was concentrated under reduced pressure to obtain the crude
product as an oil. This was directly used for the next step
without any purifcation.
Note: All carbamates (Cbz-protected amines) used for the
synthesis of compounds listed below were prepared using
Method C (Procedure for Cbz-protection).
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑benzylpip‑
erazine‑1‑carboxamide (I)
The title compound was synthesized by following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 74.56%); it was found by HPLC to be 95.78% pure; IR
(KBr): v/cm−1 =3428.81 (NH stretch), 2927.41 and 2852.2
(CH stretch of CH2), 1617.02 (C=O stretch), 1540.85 (C=C
stretch), 1243.86 (C–N stretch), 1044.26 (C–O stretch);
1H NMR (400 MHz, DMSO-d6) δ 7.28 (t, J=7.3 Hz, 2H,
Ar–H), 7.26–7.18 (m, 3H, Ar–H), 7.01 (t, J=5.76 Hz, 1H;
NH), 6.82–6.84 (m, 2H; Ar–H), 6.73 (d, J= 7.88 Hz, 1H;
Ar–H), 5.97 (s, 2H; O–CH2–O), 4.22 (d, J =5.72 Hz, 2H;
Bn CH2), 3.37 (s, 2H; piperonyl CH2), 3.30 (br.s, 4H; pip-
(XIII) Procedure for alkylation of piperazine [18].
This procedure was used in the synthesis of compounds
15 and 45. Compound 45 was synthesized by alkylation of
Cbz-piperazine with 2-(benzo[d] [1, 3] dioxol-5-yl)propanal,
followed by Cbz deprotection and reaction with isocyanate.
A mixture of Cbz-piperazine (1 eq) and sodium carbon-
ate (2 eq) was taken in toluene (0.3 M) and stirred at 80 °C
for 10–15 min. A solution of the aryl bromide (1.2 eq) was
added to the stirred solution slowly and stirring contin-
ued. The solution was refuxed for 24 h. The reaction was
stopped, quenched with water and the product extracted with
thrice with EtOAc. The combined organic layer was washed
with brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure to obtain the crude product. This
was purifed by column chromatography to obtain the pure
product (40–50% yield).
erazine CH2), 2.28 (t, J=4.60 Hz, 4H; piperazine CH2); 13
C
NMR (400 MHz, DMSO-d6) δ 157.37 (C=O), 147.18 (Ar
CH), 146.16 (Ar CH), 141.01 (Ar CH), 131.71 (Ar CH),
128.04 (Ar CH), 126.94 (Ar CH), 126.34 (Ar CH), 121.98
(Ar CH), 109.07 (Ar CH), 107.81 (Ar CH), 100.75 (Ar CH),
61.63 (CH2), 52.26 (CH2), 43.39 (CH2); positive ion HRMS
(C20H23N3O3) calculated 354.1818 found 354.1848.
1‑{(2‑[[((Benzo[d] [1, 3] dioxol‑5‑yl)methyl)methyl]
amino]ethyl}‑3‑benzyl‑1‑methylurea (12)
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivative. (Isolated
yield 69.68%); IR (KBr): v/cm−1 = 3389.28 (NH stretch),
1 3

Molecular Diversity
2926.45 and 2795.31 (CH stretch of CH2), 1633.41 (C=O
stretch), 1536.02 (C=C stretch), 1241.93 (C–N stretch),
DMSO-d6) δ 7.27–2.22 (m, 2H; Ar–H); 7.20–7.16 (m, 3H,
Ar–H); 7.02 (t, J=5.54 Hz, 1H; NH), 6.83 (d, J=10.36 Hz,
2H; Ar–H), 6.73 (d, J=7.915 Hz, 1H; Ar–H), 5.97 (s, 2H;
O–CH2–O), 4.20 (d, J = 5.52 Hz, 2H; Bn CH2), 3.80 (d,
J=13.36 Hz, 1H; piperazine CH), 3.60 (dd, 2H, J=26.34,
12.63 Hz; CH2), 3.10 (d, J=13.24 Hz, 1H; piperazine CH),
2.90 (t, J = 10.10 Hz, 1H; piperazine CH), 2.73 (dd, 1H,
J = 12.59,8.28 Hz, piperazine CH), 2.55(s, overlapping
with DMSO, 1H), 2.49–2.39(m, 1H, piperazine CH), 1.96
(t, J = 9.42 Hz, 1H; piperazine CH), 1.03 (d, J= 6.00 Hz,
3H); positive ion HRMS (C21H25N3O3) calculated 368.1974
found 368.1977.
1
1036.55 (C–O stretch); H NMR (400 MHz, DMSO-d6) δ
7.29–7.15 (m, 5H; Ar–H), 6.88 (app. t, 1H; NH), 6.84–6.78
(m, 2H; Ar–H), 6.70 (d, J = 7.80 Hz, 1H; Ar–H), 5.96 (s,
2H; O–CH2–O), 4.20 (d, J=5.80 Hz, 2H; Bn CH2), 3.37 (s,
2H; piperonyl CH2), 3.34 (br.s, 2H; CH2), 2.8 (s, 3H; CH3),
2.42 (br.s, 2H; CH2), 2.09 (s, 3H; CH3); positive ion HRMS
(C20H25N3O3) calculated 356.1974 found 356.1941.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑ben‑
zyl‑2‑methylpiperazine‑1‑carboxamide (13)
The title compound was synthesized by following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 45.2%); it was found by HPLC to be 96.39% pure; IR
(KBr): v/cm−1 =3469.31 (NH stretch), 2924.52 and 2811.7
(CH stretch of CH2), 1628.59 (C=O stretch), 1545.67 (C=C
stretch), 1449.24 (CH for CH3), 1252.54 (C–N stretch),
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑ben‑
zyl‑2,6‑dimethylpiperazine‑1‑carboxamide (15)
The title compound was synthesized by following the gen-
eral procedure for synthesis of urea derivative. (Isolated
yield 74.31%); IR (KBr): v/cm−1 = 3225.36 (NH stretch),
2918.73 and 2879.2 (CH stretch of CH2), 1617.98 (C=O
stretch), 1536.02 (C=C stretch), 1400.07 (CH for CH3),
1
1035.59 (C–O stretch); H NMR (400 MHz, DMSO-d6) δ
7.28(t, 2H, J = 7.389 Hz, Ar–H), 7.16–7.24(m, 3H); 6.98
(t, J=5.70 Hz, 1H; NH), 6.84 (t, 2H, J=6.53 Hz; Ar–H),
6.74 (d, J=7.80 Hz, 1H; Ar–H), 5.97 (d, 2H, J =3.32 Hz,
O–CH2–O), 4.25 (overlapping dd, J=15.41, 5.78 Hz, 1H,
piperazine H), 4.18 (overlapping dd, J= 15.52, 5.75 Hz,
1H piperazine H); 4.11(br.s, 1H, piperazine CH); 3.70 (d,
J=12.72 Hz, 1H; piperazine H), 3.39(d, J=13.19 Hz, 1H
piperazine H); 3.28 (d, J = 13.216 Hz, 1H, piperazine H);
2.92 (td, J = 2.50, 12.35 Hz, 1H; piperazine H), 2.72 (d,
J=10.17 Hz, 1H; piperazine H), 2.56 (d, J=11.04 Hz, 1H;
piperazine H), 1.99 (dd, J=3.35, 10.98 Hz, 1H; piperazine
H), 1.86 (td, J=3.03, 11.82 Hz, 1H; piperazine H), 1.12 (d,
J=6.548 Hz, 3H; CH3); 13C NMR (400 MHz, DMSO-d6)
δ 158.05 (C=O), 157.01 (Ar CH), 147.23 (Ar CH), 146.11
(Ar CH), 141.16 (Ar CH), 140.88 (Ar CH) (Ar CH), 132.05
(Ar CH), 128.18 (Ar CH), 128.04 (Ar CH), 126.96 (Ar CH),
126.89 (Ar CH), 126.52 (Ar CH), 126.32 (Ar CH), 121.060
(Ar CH), 108.70 (Ar CH), 107.86 (Ar CH), 100.74 (O-CH2),
61.54 (CH2), 56.99 (CH & CH2), 52.75 (CH2), 46.09 (CH2),
43.38 (CH2), 42.95 (CH2), 15.37 (CH3); positive ion HRMS
(C21H25N3O3) calculated 368.1974 found 368.1996.
1
1242.9 (C–N stretch), 1042.34 (C–O stretch); H NMR
(400 MHz, DMSO-d6) δ 7.28(t, J = 7.42 Hz, 2H; Ar–H),
7.22–7.16 (m, 3H; Ar–H), 6.9–6.83 (m, 3H; Ar–H & NH),
6.78 (d, J =7.88 Hz, 1H; Ar–H), 5.98 (s, 2H; O–CH2–O),
4.24 (d, J = 5.56 Hz, 2H; Bn CH2), 4.01 (app.t, 2H; pip-
erazine CH2), 3.37 (s, 2H; piperonyl CH2), 2.60–2.57 (m,
2H; piperazine CH2), 2.02–1.98 (m, 2H; piperazine CH2),
1.19 (d, J = 6.59 Hz, 6H; CH3*2); positive ion HRMS
(C22H27N3O3) calculated 382.2131 found 382.2129.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑ben‑
zyl‑3,5‑dimethylpiperazine‑1‑carboxamide (16)
The title compound was synthesized by following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 64.21%); it was found by HPLC to be 96.49% pure;
IR (KBr): v/cm−1 = 3433.64 (NH stretch), 2923.56 and
2852.2 (CH stretch of CH2), 1630.52 (C=O stretch), 1540.85
(C=C stretch), 1401.03 (CH for CH3), 1242.9 (C–N stretch),
1
1037.52 (C–O stretch); H NMR (400 MHz, DMSO-d6) δ
7.27 (t, J=7.4 Hz, 2H; Ar–H), 7.19–7.16 (m, 3H; Ar–H),
7.04 (t, J=5.28 Hz, 1H, NH), 6.89 (s, 1H; Ar–H), 6.83–6.76
(m, 2H; Ar–H), 5.95 (s, 2H; O–CH2–O), 4.21 (d, J=5.56 Hz,
2H; Bn CH2), 3.81–3.78 (m, 2H; piperazine CH*2), 3.63 (s,
2H; piperonyl CH2), 2.54 (br.s merged with DMSO, 2H;
piperazine CH2), 2.48 (br.s, 2H; piperazine CH2), 0.90 (d,
J=5.85 Hz, 6H; CH3*2); positive ion HRMS (C22H27N3O3)
calculated 382.2131 found 382.2143.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑ben‑
zyl‑3‑methylpiperazine‑1‑carboxamide (14)
The title compound was synthesized by following the
general procedure for synthesis of urea derivative (iso-
lated yield 57.71%); it was found by HPLC to be 98.62%
pure; IR (KBr): v/cm−1 = 3428.81 (NH stretch), 2919.7
and 2857.02 (CH stretch of CH2), 1622.8 (C=O stretch),
1559.17 (C=C stretch), 1498.42 (CH for CH3), 1267 (C–N
1
stretch), 1037.52 (C–O stretch); H NMR (400 MHz,
1 3

Molecular Diversity
4‑(Benzo[d] [1, 3] dioxol‑5‑ylmethyl)‑N‑ben‑
zyl‑1,4‑diazepane‑1‑carboxamide (17)
3‑[(Benzo[d] [1, 3] dioxol‑5‑ylmethyl)amino]‑N‑ben‑
zylpyrrolidine‑1‑carboxamide (20)
The title compound was synthesized by following the gen-
eral procedure for synthesis of urea derivative. (Isolated
yield 73.27%); IR (KBr): v/cm−1 = 3343.96 (NH stretch),
2922.59 and 2855.1 (CH stretch of CH2), 1625.7 (C=O
stretch), 1536.99 (C=C stretch), 1249.65 (C–N stretch),
The title compound was synthesized following the general
procedure for synthesis of urea derivatives (isolated yield
45%); it was found by HPLC to be 98.75% pure; IR (KBr):
v/cm−1 =3286.11 (NH stretch), 2923.56 and 2847.38 (CH
stretch of CH2), 1620.88 (C=O stretch), 1545.67 (C=C
stretch), 1246.75 (C–N stretch), 1041.37 (C–O stretch);
1H NMR (400 MHz, DMSO-d6) δ 7.29–7.23 (m, 4H;
Ar–H); 7.18(t, J=6.81 Hz, 1H); 6.9 (s, 1H; Ar–H), 6.81 (d,
J = 7.85 Hz, 1H; Ar–H), 6.76 (d, J= 7.92 Hz, 1H; Ar–H),
6.62 (t, J=5.88 Hz, 1H; NH), 5.95 (s, 2H; O–CH2–O), 4.2
(d, J=5.92 Hz, 2H; Bn CH2), 3.56 (s, 2H; piperonyl CH2),
3.39–3.32 (m, 2H; pyrrolidine CH2), 3.22–3.12 (m, 2H; pyr-
rolidine CH2), 3.03 (dd, 1H, J=4.98, 10.072 Hz, pyrrolidine
CH), 1.93–1.86 (m, 1H; pyrrolidine CH), 1.70–1.62 (m, 1H;
pyrrolidine CH);); positive ion HRMS (C20H23N3O3) calcu-
lated 354.1818 found 354.1575.
1
1036. (C–O stretch); H NMR (400 MHz, DMSO-d6) δ
7.30–7.18 (m, 5H; Ar–H), 6.85–6.72 (m, 3H; Ar–H & NH),
6.73 (d, J =7.98 Hz, 1H; Ar–H), 5.97 (s, 2H; O–CH2–O),
4.22 (d, J = 5.76 Hz, 2H; Bn CH2), 3.48 (m, 2H; pipero-
nyl CH2), 3.41–3.39 (m, 4H; homopiperazine CH2), 2.54
(m, 4H; homopiperazine CH2), 1.71 (m, 2H; homopipera-
zine CH2); positive ion HRMS (C21H25N3O3) calculated
368.1974 found 368.1894.
4‑[(2H‑1,3‑Benzodioxol‑5‑yl)methyl]‑N‑benzylpiper‑
idine‑1‑carboxamide (18)
The title compound was synthesized by following the
general procedure for synthesis of urea derivative (iso-
lated yield 61.27%); it was found by HPLC to be 99.09%
pure; IR (KBr): v/cm−1 = 3338.18 (NH stretch), 2910.06
and 2886.92 (CH stretch of CH2), 1623.77 (C=O stretch),
1544.7 (C=C stretch), 1246.75 (C–N stretch), 1098.26 (C–O
stretch); 1H NMR (400 MHz, DMSO-d6) δ 7.29–7.16 (m,
5H; Ar–H), 6.96 (br.s, 1H; NH), 6.78 (d, J = 7.8 Hz, 1H,
overlapping; Ar–H), 6.74 (s, 1H, overlapping, Ar–H), 6.60
(d, J=5.80 Hz, 1H; Ar–H), 5.94 (s, 2H; O–CH2–O), 4.20
(d, J=5.24 Hz, 2H; Bn CH2), 3.93 (m, 2H; piperonyl CH2),
2.62–2.56 (m, 2H; piperidine CH2), 2.48 (m, 2H; piperidine
CH2), 1.60 (br.s, 1H; piperidine CH), 1.51–1.47 (m, 2H;
piperidine CH2), 1.03–0.94 (m, 2H; piperidine CH2); posi-
tive ion HRMS (C21H24N2O3) calculated 353.1865 found
353.1882.
1‑[(1‑(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]pyrroli‑
din‑3‑yl)‑3‑benzylurea (21)
The title compound was synthesized following the general
procedure for synthesis of urea derivatives (isolated yield
1
42.11%); it was found by HPLC to be 99.86% pure; H
NMR (400 MHz, DMSO-d6) δ 7.30–7.26 (m, 2H; Ar–H),
7.21–7.19 (m, 3H; Ar–H), 6.82 (apparent t, J=8.47 Hz, 2H;
Ar–H), 6.73 (d, J=7.88 Hz, 1H; Ar–H), 6.25 (t, J=5.74 Hz,
1H; NH), 6.08 (d, J = 7.60 Hz, 1H; NH), 5.96 (s, 2H;
O–CH2–O), 4.16 (d, J=5.92 Hz, 2H; Bn CH2), 4.03–4.02
(m, 1H; pyrrolidine CH), 3.43 (dd, J=24.24 Hz, 12.86 Hz,
2H; piperonyl CH2), 2.65–2.54 (m, 2H; pyrrolidine CH2),
2.31–2.24 (m, 2H; pyrrolidine CH2), 2.09–2.05 (m, 1H; pyr-
rolidine CH), 1.45–1.42(m, 1H); 1.27 (d, J=6.46 Hz, 3H);
positive ion ESI MS m/z (relative intensity) 354.0 [(M+H)+,
100].
1‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑benzylpi‑
peridine‑4‑carboxamide (19)
Benzyl 4‑(benzo[d] [1, 3] dioxol‑5‑ylmethyl)pipera‑
zine‑1‑carboxylate (22)
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivatives. (Isolated
It was found by HPLC to be 98.76% pure; IR (KBr): v/
1
yield 60.23%); H NMR (400 MHz, DMSO-d6) δ 8.24 (t,
cm−1 = 3493.42 (NH stretch), 1633.41 (C=O stretch),
1
J=5.8 Hz, 1H; NH), 7.31 (t, J=7.76 Hz, 2H; Ar–H), 7.21
(m, 3H; Ar–H), 6.84 (m, 2H; Ar–H), 6.74 (d, J=7.96 Hz,
1H; Ar–H), 5.97 (s, 2H; O–CH2–O), 4.24 (d, J =5.88 Hz,
2H; Bn CH2), 3.38 (s, 2H; piperonyl CH2), 2.82 (d,
J = 11.01 Hz,, 2H; piperidine CH2), 2.18 (m, 1H; piperi-
dine CH), 1.96–1.89 (app.t, 2H; piperidine CH2), 1.68–1.54
(m, 4H; piperidine CH2); positive ion ESI MS m/z (relative
intensity) 353.2 [(M+H)+, 100].
1241.93 (C–N stretch), 1036.55 (C–O stretch): H NMR
(400 MHz, DMSO-d6) δ 7.37–7.29 (m, 5H; Ar–H), 6.84 (s,
1H, overlapping; Ar–H), 6.81 (s, 1H, overlapping; Ar–H),
6.73 (d, J =7.84 Hz, 1H; Ar–H), 5.97 (s, 2H; O–CH2–O),
5.05 (s, 2H; Bn CH2), 3.57–3.37 (m, 6H; piperonyl CH2
& Piperazine CH2), 2.33–2.29 (m, 4H; piperazine CH2);
13C NMR (400 MHz, DMSO-d6) δ 154.33(C=O), 147.20
(Ar CH), 146.18(Ar CH), 131.62(Ar CH) 136.88(Ar CH),
128.38(Ar CH), 127.79(Ar CH), 127.50(Ar CH), 121.98(Ar
CH), 109.05(Ar CH), 107.82(Ar CH), 100.76(O–CH2–O),
1 3

Molecular Diversity
66.12 (CH2), 61.52(CH2), 52.05(CH2), 43.46(CH2); posi-
tive ion HRMS (C20H22N2O4) calculated 355.1658 found
355. 1570.
J=5.00 Hz, 2H; O=C-CH2), 3.47 (br.s, 4H), 3.39 (s, 2H;
piperonyl CH2), 2.31(br.s, 2H; piperazine CH2), 2.29 (br.s,
2H; piperazine CH2); positive ion HRMS (C20H23N2O3) cal-
culated 354.1818 found 354.1787.
1‑(4‑(Benzo[d] [1, 3] dioxol‑5‑ylmethyl)pipera‑
zin‑1‑yl)‑3‑phenylpropan‑1‑one (23)
4‑(Benzo[d] [1, 3] dioxol‑5‑ylmethyl)‑N‑ben‑
zyl‑N‑methylpiperazine‑1‑carboxamide (26)
It was found by HPLC to be 95.42% pure; IR (KBr): v/
cm−1 = 3444.24 (NH stretch), 2920.66 and 2853.17 (CH
stretch of CH2), 1639.2 (C=O stretch), 1539.88 (C=C
stretch), 1244.83 (C–N stretch), 1037.52 (C–O stretch),
752.0 and 700.99 (CH stretch for aromatic with fve adjacent
IR (KBr): v/cm−1 = 3433.64 (NH stretch), 2924.52 and
2807.85 (CH stretch of CH2), 1629.55 (C=O stretch),
1481.06 (C=C stretch), 1435.74 (CH for N-CH3), 1243.86
(C–N stretch), 1036.55 (C–O stretch); it was found by
1
1
hydrogens); H NMR (400 MHz, DMSO-d6) δ 7.26–7.20
HPLC to be 95.64% pure; H NMR (400 MHz, DMSO-
(m, 4H; Ar–H), 7.17–7.13 (m, 1H; Ar–H), 6.83 (s, 1H, over-
lapping; Ar–H), 6.81 (s, 1H, overlapping; Ar–H), 6.73 (d,
J=7.92 Hz, 1H; Ar–H), 5.97 (s, 2H; O–CH2–O), 3.41–3.32
(m, 6H; piperazine CH2 & piperonyl CH2), 2.77 (t,
J=8.04 Hz, 2H; CH2), 2.57 (t, J=8.12 Hz, 2H; CH2), 2.24
(br.s, 4H; piperazine CH2); positive ion HRMS (C21H24N2O)
calculated 353.1865 found 353.1844.
d6) δ 7.34–7.32 (m, 2H; Ar–H), 7.25–7.20 (m, 3H; Ar–H),
6.84 (overlapped. s, 1H; Ar–H), 6.83(overlapped. d,
J=10.13 Hz, 1H); 6.73 (d, J=7.85 Hz, 1H; Ar–H), 4.3 (s,
2H; O–CH2–O), 3.38 (s, 2H; N-CH2), 3.10 (br.s, 4H; pipera-
zine CH2), 2.64 (s, 3H; N-CH3), 2.33 (br.s, 4H; piperazine
CH2); 13C NMR (400 MHz, DMSO-d6) δ 163.40 (C=O),
160.98 (Ar CH), 156.38 (Ar H), 142.83 (Ar H), 142.76 (Ar
H), 137.08 (Ar H), 130.25(Ar CH), 130.17(Ar CH), 128.34
(Ar CH), 127.74(Ar CH), 122.91(Ar CH), 113.68(Ar CH),
113.61(Ar CH), 113.47(Ar CH), 113.40 (Ar CH), 65.45
(CH2), 43.30 (CH3); positive ion HRMS (C21H25N3O3) cal-
culated 368.1974 found 368.1925.
1‑{4‑[(2H‑1,3‑Benzodioxol‑5‑yl)methyl]pipera‑
zin‑1‑yl}‑2‑phenoxyethan‑1‑one (24)
The title compound was synthesized following proce-
dure B using phenol. (Isolated yield 62%); IR (KBr): v/
cm−1 = 3479.92 (NH stretch), 2921.63 and 2854.13 (CH
stretch of CH2), 1658.48 (C=O stretch), 1593.88 (C=C
stretch), 1245.79 (C–N stretch), 1080.91 (C–O–C stretch),
N‑Benzyl‑4‑(3,4‑dimethoxybenzyl)piperazine‑1‑car‑
boxamide (27)
1
1034.62 (C–O stretch); H NMR (400 MHz, DMSO-d6) δ
The title compound was synthesized following the general
procedure for synthesis of urea derivative (isolated yield
62.36%); it was found by HPLC to be 96.60% pure; IR
(KBr): v/cm−1 =3334.32 (NH stretch), 2929.34 and 2833.88
(CH stretch of CH2), 1626.66 (C=O stretch), 1514.81 (C=C
stretch), 1261.22 (C–N stretch), 1025.94 (C–O stretch); 1H
NMR (400 MHz, DMSO-d6) δ 7.28 (t, J = 7.30 Hz, 2H;
Ar–H), 7.22–7.16 (m, 3H; Ar–H), 7.04 (t, J=5.68 Hz, 1H;
NH), 6.88 (s, 1H; Ar–H, overlapping), 6.86 (s, 1H; Ar–H,
overlapping), 6.79 (d, J = 8.12 Hz, 1H; Ar–H), 4.21 (d,
J=5.72 Hz, 2H; Bn CH2), 3.71 (d, J=2.00 Hz, 6H; OCH3),
3.38 (s, 2H; CH2), 3.30 (br.s, 4H; piperazine CH2), 2.28 (br.s,
4H; piperazine CH2); 13C NMR (400 MHz, DMSO-d6) δ
157.40 (C=O), 148.55 (Ar C–OCH3), 147.82 (Ar C–OCH3),
141.02 (Ar CH), 130.21 (Ar CH), 128.14 (Ar CH), 128.04
(Ar CH), 126.99 (Ar CH), 126.45 (Ar CH), 126.33 (Ar CH),
120.98 (Ar CH), 112.51 (Ar CH), 111.49 (Ar CH), 61.75
(CH3), 55.45 (CH2), 55.40 (CH3), 52.35 (CH2), 43.44 (CH2),
43.40 (CH2); positive ion HRMS (C21H27N3O3) calculated
370.2131 found 370.2138.
7.26 (t, J=7.72 Hz, 2H; Ar–H), 6.94–6.82 (m, 5H; Ar–H),
6.75 (d, J =7.84 Hz, 1H; Ar–H), 5.97 (s, 2H; O–CH2–O),
4.75 (s, 2H; Bn CH2), 3.44–3.39 (m, 6H; piperazine & piper-
onyl CH2), 2.37 (br.s, 2H; piperazine CH2), 2.29 (br.s, 2H;
piperazine CH2); positive ion HRMS (C20H22N2O4) calcu-
lated 355.1658 found 355.1604.
1‑[((4‑(Benzo[d] [1, 3] dioxol‑5‑yl)methyl)pipera‑
zin‑1‑yl]‑2‑(phenylamino)ethanone (25)
The title compound was synthesized following procedure B
using aniline (isolated yield 57%); it was found by HPLC to
be 97.03% pure; IR (KBr): v/cm−1 =3398.92 (NH stretch),
2918.73 and 2886.92 (CH stretch of CH2), 1605.45 (NH
bending for secondary amine), 1652.7 (C=O stretch),
1585.2 (C=C stretch), 1441.53 (CH2 bending), 1239.04
(C–N stretch), 1031.73 (C–O stretch); 1H NMR (400 MHz,
DMSO-d6) δ 7.05 (t, J = 7.68 Hz, 2H; Ar–H), 6.86–6.82
(m, 2H; Ar–H), 6.72 (d, J = 7.84 Hz, 1H; Ar–H), 6.62 (d,
J=8.4 Hz, 2H; Ar–H), 6.53 (t, J=7.4 Hz, 1H; Ar–H), 5.97
(s, 2H; O–CH2–O), 5.48 (t, J=4.84 Hz, 1H; NH), 3.84 (d,
1 3

Molecular Diversity
N‑Benzyl‑4‑(3‑methoxybenzyl) piperazine‑1‑carbox‑
amide (28)
N‑Benzyl‑4‑[(2,2‑difuorobenzo[d] [1, 3] dioxol‑5‑yl)
methyl]piperazine‑1‑carboxamide (31)
The title compound was synthesized following the general
procedure for synthesis of urea derivative (isolated yield
74.33%); it was found by HPLC to be 96.39% pure; IR
(KBr): v/cm−1 =3301.54 (NH stretch), 2910.06 and 2858.95
(CH stretch of CH2), 1620.88 (C=O stretch), 1549.52 (C=C
stretch), 1266.04 (C–N stretch), 1045.23 (C–O stretch); 1H
NMR (400 MHz, DMSO-d6): δ 7.29–7.16 (m, 5H; Ar–H),
7.03 (t, J = 5.72 Hz, NH, 1H), 6.87–6.85 (m, 2H; Ar–H),
6.82–6.79 (dd, J = 2.08 Hz, J = 7.16 Hz, 2H; Ar–H) 4.21
(d, J=5.76 Hz, 2H; Bn CH2), 3.72 (s, 3H; OCH3), 3.43 (s,
2H; CH2), 3.32–3.30 (m, 4H; piperazine CH2), 2.39–2.31
(m, 4H; piperazine CH2); 13C NMR (400 MHz, DMSO-
d6) δ 159.22 (C=O), 157.37 (Ar C–OCH3), 141 (Ar CH),
139.58 (Ar CH), 129.18 (Ar CH), 128.04 (Ar CH), 126.94
(Ar CH), 126.35 (Ar CH), 120.99 (Ar CH), 114.27 (Ar CH),
112.31 (Ar CH), 61.9 (CH3), 54.91 (CH2), 52.43 (CH2),
43.40 (CH2); positive ion HRMS (C20H25N3O2) calculated
340.2025 found 340.2071.
The title compound was synthesized following the general
procedure for synthesis of urea derivative (isolated yield
65%); it was found by HPLC to be 99.88% pure; IR (KBr):
v/cm−1 = 3340.1 (NH stretch), 2922.59 and 2800.13 (CH
stretch of CH2), 1625.7 (C=O stretch), 1544.7 (C=C stretch),
1250.61 (C–N stretch), 1144.55 (C-F stretch), 1037.52 (C–O
stretch); 1H NMR (400 MHz, DMSO-d6) δ 7.34–7.32 (m,
2H; Ar–H), 7.29–7.24 (m, 2H; Ar–H), 7.20–7.16 (m, 3H,
Ar–H), 7.13 (d, J=8.24 Hz, 1H; Ar–H), 7.05 (t, J=5.76 Hz,
NH, 1H), 4.20 (d, J= 5.68 Hz, 2H; Bn CH2), 3.48 (s, 2H;
piperonyl CH2), 3.32 (br.s merged with DMSO water, 4H;
piperazine CH2), 2.30 (app.t, 4H; piperazine CH2); posi-
tive ion HRMS (C20H21N3O2F2) calculated 390.1629 found
390.1663.
N‑Benzyl‑4‑(2,3‑dimethoxybenzyl)piperazine‑1‑car‑
boxamide (32)
The title compound was synthesized by following the gen-
eral procedure for synthesis of urea derivatives (isolated
yield 55.3%); it was found by HPLC to be 98.38% pure; IR
(KBr): v/cm−1 =3318.89 (NH stretch), 2931.27 and 2803.99
(CH stretch of CH2), 1621.84 (C=O stretch), 1549.52 (C=C
stretch), 1261.22 (C–N stretch), 1003.77 (C–O stretch); 1H
NMR (400 MHz, DMSO- d6) δ 7.29–7.26 (m, 2H; Ar–H),
7.16–7.22 (m, 3H; Ar–H), 7.05–6.99 (m, Ar–H & NH, 2H);
6.94–6.6.89 (m, 2H; Ar–H), 4.21 (d, J=5.64 Hz, 2H; Bn
CH2), 3.78 (s, 3H; OCH3), 3.7 (s, 3H; OCH3) 3.45 (s, 2H;
CH2), 3.69 (overlapping with DMSO water, br.s, 4H; pipera-
zine CH2), 2.31 (br.s, 4H; piperazine CH2). positive ion HRMS
(C21H27N3O3) calculated 370.2131 found 370.2194.
N‑Benzyl‑4‑(4‑methoxybenzyl) piperazine‑1‑carbox‑
amide (29)
The title compound was synthesized following the general
procedure for synthesis of urea derivative. (Isolated yield
64.53%); IR (KBr): v/cm−1 =3339.14 (NH stretch), 2921.63
and 2853.17 (CH stretch of CH2), 1625.7 (C=O stretch),
1544.7 (C=C stretch), 1248.68 (C–N stretch), 1032.69
1
(C–O stretch); H NMR (400 MHz, DMSO-d6) δ 7.27 (t,
J = 7.73 Hz, 2H; Ar–H), 7.22–7.16 (m, 5H; Ar–H), 7.02
(t, J=5.60 Hz, NH, 1H), 6.86 (d, J=8.41 Hz, 2H; Ar–H),
4.21 (d, J=5.71 Hz, 2H; Bn CH2), 3.38 (overlapping with
DMSO, s, 2H; CH2), 3.30–3.25 (m, 4H; piperazine CH2),
2.31–2.16 (m, 4H; piperazine CH2); positive ion HRMS
(C20H25N3O2) calculated 340.2025 found 340.1774.
N‑Benzyl‑4‑[(2,5‑dimethoxyphenyl)methyl]pipera‑
zine‑1‑carboxamide (33)
N‑Benzyl‑4‑((2,3‑dihydrobenzo[b] [1, 4] dioxin‑6‑yl)
The title compound was synthesized by following the gen-
eral procedure for synthesis of urea derivatives (isolated
yield 63.58%); it was found by HPLC to be 97.77% pure; IR
(KBr): v/cm−1 =3312.14 (NH stretch), 2921.63 and 2851.24
(CH stretch of CH2), 1623.77 (C=O stretch), 1548.56 (C=C
stretch), 1267.97 (C–N stretch), 1051.01 (C–O stretch); 1H
NMR (400 MHz, DMSO-d6) δ 7.30–7.25 (m, 2H; Ar–H),
7.22–7.16 (m, 3H; Ar–H), 7.04 (t, J=5.72 Hz, NH, 1H), 6.89
(overlapping s, 1H; Ar–H), 6.87(overlapping d, J=8.68, 1H),
6.77 (dd, J=8.69, 2.95 Hz, 1H; Ar–H), 4.20 (d, J=5.68 Hz,
2H; Bn CH2), 3.69 (d, J=8.48 Hz, 6H; 2*OCH3), 3.43 (s, 2H;
CH2), 3.32 (s, 4H; overlapping with DMSO water, piperazine
CH2), 2.30 (br.s, 4H; piperazine CH2); positive ion HRMS
(C21H27N3O3) calculated 370.2131 found 370.2135.
methyl)piperazine‑1‑carboxamide (30)
The title compound was synthesized by the above-
1
mentioned procedure (Isolated yield 42.62%); H NMR
(400 MHz, DMSO-d6): δ 7.29–7.18 (m, 5H; Ar–H), 7.03(t,
J=5.60 Hz, 1H; NH), 6.78–6.71 (m, 3H; Ar–H), 4.21 (m,
6H; O-CH2-CH2-O & Bn CH2), 3.33–3.32 (m, 6H; pipera-
zine CH2 & N-CH2), 2.27 (m, 4H; piperazine CH2); positive
ion ESI MS m/z (relative intensity) 368.0 [(M+H)+, 100].
1 3

Molecular Diversity
(Ar–CH), 126.99 (Ar–CH), 126.34 (Ar–CH), 125.64
(Ar–CH), 125.09 (Ar–CH), 122.21 (Ar–CH), 109.35
(Ar–CH), 60.31 (OCH3), 55.34 (CH2), 52.60 (CH2), 43.49
(CH2), 43.39 (CH2), 11.07 (CH3); positive ion HRMS
(C21H27N3O2) calculated 354.2182 found 354.2168.
N‑Benzyl‑4‑[(3,4,5‑trimethoxyphenyl)methyl]
piperazine‑1‑carboxamide (34)
The title compound was synthesized by following the gen-
eral procedure for synthesis of urea derivatives (isolated
yield 58.32%); it was found by HPLC to be 98.46% pure; IR
(KBr): v/cm−1 =3359.39 (NH stretch), 2927.41 and 2854.13
(CH stretch of CH2), 1622.8 (C=O stretch), 1544.7 (C=C
stretch), 1261.22 (C–N stretch), 1095.37 (C–O stretch); 1H
NMR (400 MHz, DMSO-d6) δ 7.29–7.26 (m, 2H; Ar–H),
7.22–7.16(overlap. m, 3H, Ar–H); 7.03 (t, J=5.47 Hz, NH,
1H), 6.96 (d, J=8.52 Hz, 1H; Ar–H), 6.75 (d, J=8.52 Hz,
1H; Ar–H), 4.20 (d, J=5.56 Hz, 2H; Bn CH2), 3.76 (s, 6H;
OCH3), 3.72(s, 3H, OCH3); 3.43, (s, 2H; CH2), 3.32 (br.s,
4H; piperazine CH2), 2.31 (br.s, 4H; piperazine CH2); posi-
tive ion HRMS (C22H29N3O4) calculated 400.2236 found
400.2236.
N‑Benzyl‑4‑(3‑methylbenzyl) piperazine‑1‑carboxa‑
mide (37)
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 64.2%); it was found by HPLC to be 98.44% pure; IR
(KBr): v/cm−1 =3318.89 (NH stretch), 2912.95 and 2848.35
(CH stretch of CH2), 1622.8 (C=O stretch), 1540.85 (C=C
stretch), 1267.97 (C–N stretch); 1H NMR (400 MHz, DMSO-
d6) δ 7.28 (overlapping t, 2H; J=7.38 Hz, Ar–H), 7.21 (t,
2H, J=6.917 Hz, Ar–H), 7.18(d, J=4.43 Hz, 2H, Ar–H),
7.09 (d, J=5.68 Hz, 2H, Ar–H); 7.04(t, J=7.76 Hz, Ar–H &
NH, overlapping, 2H), 4.20 (d, J=5.68 Hz, 2H; NH–CH2),
3.42 (s, 2H; Bn CH2), 3.30 (t, J=5.16 Hz, 4H; piperazine
CH2), 2.28 (d, J = 8.12 Hz, 7H; piperazine CH2 & CH3,
overlapping); 13C NMR (400 MHz, DMSO-d6) δ 157.33
(C=O), 141 (Ar–CH), 137.79 (Ar–CH), 137.18 (Ar–CH),
129.48 (Ar–CH), 128.03 (Ar–CH), 127.58 (Ar–CH), 126.93
(Ar–CH), 126.34 (Ar–CH), 125.96 (Ar–CH), 62.04 (CH2),
52.45 (CH2), 43.42 (CH2), 20.97 (CH3); positive ion HRMS
(C20H25N3O) calculated 324.2076 found 324.2100
N‑Benzyl‑4‑[(3‑methoxy‑4‑methylphenyl)methyl]
piperazine‑1‑carboxamide (35)
The title compound was synthesized following the general
procedure for synthesis of urea derivative (isolated yield
55.23%); it was found by HPLC to be 97.71% pure; IR
(KBr): v/cm−1 =3346.85 (NH stretch), 2919.7 and 2851.24
(CH stretch of CH2), 1617.98 (C=O stretch), 1586.16 (C=C
stretch), 1262.18 (C–N stretch), 1040.41 (C–O stretch); 1H
NMR (400 MHz, DMSO-d6) δ 7.29–7.26 (m, 2H; Ar–H,
overlapping), 7.22–7.18 (m, 3H; Ar–H), 7.00 (m, Ar–H
& NH, 2H), 6.84 (s, 1H; Ar–H), 6.75 (d, J=7.44 Hz, 1H;
NH), 4.20 (d, J=5.52 Hz, 2H; Bn CH2), 3.75 (s, 3H; OCH3),
3.42 (s, 2H; CH2), 3.31 (s, 4H; piperazine CH2, overlap-
ping with DMSO water), 2.29 (m, 4H; piperazine CH2), 2.10
(m, 3H; CH3); positive ion HRMS (C21H27N3O2) calculated
354.2182 found 354.2180.
N‑Benzyl‑4‑[(3‑cyanophenyl)methyl]pipera‑
zine‑1‑carboxamide (38)
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 61.78%); it was found by HPLC to be 98.73% pure;
1H NMR (400 MHz, DMSO-d6) δ 7.74 (overlapping, s,
1H); 7.72 (overlapping, d, J=7.88 Hz, 2H; Ar–H), 7.65 (d,
J = 7.56 Hz, 1H; Ar–H), 7.53 (t, J = 7.46 Hz, 1H; Ar–H),
7.30–7.26 (m, 2H; Ar–H), 7.23–7.17(m, 3H, Ar–H); 7.05
(br.s, NH, 1H), 4.20 (d, J=5.20 Hz, 2H; Bn CH2), 3.53 (s,
2H; CH2), 3.32 (overlapping with DMSO water, m, 4H;
piperazine CH2), 2.30 (s, 4H; piperazine CH2); positive ion
HRMS (C20H22N4O) calculated 335.1872 found 335.1847.
N‑Benzyl‑4‑(3‑methoxy‑2‑methylbenzyl)pipera‑
zine‑1‑carboxamide (36)
The title compound was synthesized following the general
procedure for synthesis of urea derivative (isolated yield
65.13%); it was found by HPLC to be 99.22% pure; IR
(KBr): v/cm−1 =3333.36 (NH stretch), 2928.38 and 2802.06
(CH stretch of CH2), 1627.63 (C=O stretch), 1539.88 (C=C
stretch), 1257.36 (C–N stretch), 1048.12 (C–O stretch); 1H
NMR (400 MHz, DMSO-d6): δ 7.28 (t, J = 7.46 Hz, 2H;
Ar–H), 7.23–7.16 (m, 3H; Ar–H), 7.08 (t, J=7.88 Hz, Ar
H, 1H); 7.04 (t, J=5.72 Hz, NH, 1H), 6.84 (t, J=8.40 Hz,
2H; Ar–H), 4.21 (d, J = 5.68 Hz, 2H; NH–CH2), 3.75 (s,
3H; OCH3), 3.39 (s, 2H; CH2), 3.32 (br.s, 4H; piperazine
CH2), 2.29 (br.s, 4H; piperazine CH2), 2.14 (s, 3H; CH3);
13C NMR (400 MHz, DMSO-d6) δ 157.35 (C=O), 157.27
(Ar C–OCH3), 141.02 (Ar–CH), 137.07 (Ar–CH), 128.04
N‑Benzyl‑4‑[(3‑chlorophenyl)methyl]pipera‑
zine‑1‑carboxamide (39)
The title compound was synthesized following the general
procedure for synthesis of urea derivative. (Isolated yield
52.3%); IR (KBr): v/cm−1 =3322.75 (NH stretch), 2918.73
and 2851.24 (CH stretch of CH2), 1624.73 (C=O stretch),
1540.85 (C=C stretch), 1264.11 (C–N stretch) 725.10 (C–Cl
stretch); 1H NMR (400 MHz, DMSO-d6) δ 7.38–7.31 (m,
1 3

Molecular Diversity
3H; Ar–H), 7.29–7.28 (m, 3H; Ar–H), 7.24–7.18 (m, 3H;
Ar–H), 7.06 (t, J=5.6 Hz, NH, 1H), 4.22 (d, J=5.64 Hz,
2H; Bn CH2), 3.57 (s, 2H; CH2), 3.30 (s, overlapping with
DMSO water, 4H; piperazine CH2), 2.32 (app. t, J=4.47 Hz,
4H; piperazine CH2); positive ion HRMS (C19H22N3OCl)
calculated 344.1530 found 344.1551.
stretch), 1283.39 (C–N stretch) 1257.36 (C-F stretch); 1H
NMR (400 MHz, DMSO-d6) δ 7.73–7.71 (m, 2H; Ar–H),
7.64 (d, J=7.40 Hz, 1H; Ar–H), 7.53 (t, J=7.72 Hz, 1H;
Ar–H), 7.37–7.29 (m, Ar–H & NH, 5H), 5.05 (s, 2H; Bn
CH2), 3.53 (s, 2H; CH2), 3.39–3.32 (br.s, 4H; piperazine
CH2), 2.32 (t, J=4.78 Hz, 4H; piperazine CH2); positive ion
HRMS (C19H22N3OF) calculated 328.1825 found 328.1780.
N‑Benzyl‑4‑[(3‑ethoxyphenyl)methyl]pipera‑
zine‑1‑carboxamide (40)
N‑Benzyl‑4‑[3‑(trifuoromethoxy)benzyl]pipera‑
zine‑1‑carboxamide (43)
The title compound was synthesized following the general
procedure for synthesis of urea derivative. (Isolated yield
64.23%);Was found by HPLC to be 99.00% pure; IR (KBr):
v/cm−1 = 3328.53 (NH stretch), 2924.52 and 2880.17 (CH
stretch of CH2), 1626.66 (C=O stretch), 1546.63 (C=C
stretch), 1260.25 (C–N stretch) 1051.01 (C-F stretch); 1H
NMR (400 MHz, DMSO-d6) δ 7.29–7.26 (m, 2H; Ar–H),
7.22–7.18 (m, 4H; Ar–H), 7.04 (t, J = 5.60 Hz, NH, 1H),
6.85 (overlap. s, 1H; Ar–H); 6.84(overlap. d, J = 7.35 Hz,
1H); 6.72 (d, J = 9.00 Hz, 1H), 4.22 (d, J = 5.64 Hz, 2H;
Bn CH2), 3.98 (dd, J = 13.86 & 6.90 Hz, 2H; OCH2), 3.42
(s, 2H; CH2), 3.33 (overlap with DMSO water, br.s, 4H;
piperazine CH2), 2.22 (br.s, 4H; piperazine CH2), 1.30 (t,
J = 7.00 Hz, 3H); positive ion HRMS (C21H27N3O2) cal-
culated 354.2182 found 354.2223.
The title compound was synthesized following the general
procedure for synthesis of urea derivative (isolated yield
67.23%); it was found by HPLC to be 98.05% pure; IR
(KBr): v/cm−1 =3329.5 (NH stretch), 2923.56 and 2853.17
(CH stretch of CH2), 1626.66 (C=O stretch), 1542.77 (C=C
stretch), 1286.29 (C–N stretch) 1254.47 (C-F stretch); 1H
NMR (400 MHz, DMSO-d6) δ 7.45 (t, J = 5.68 Hz, 1H;
Ar–H), 7.34–7.16 (m, 8H; Ar–H), 7.05 (t, J=5.72 Hz, NH,
1H), 4.22 (d, J=5.68 Hz, 2H; Bn CH2), 3.53 (s, 2H; CH2),
3.32 (br.s, 4H; piperazine CH2), 2.32 br.s, 4H; piperazine
CH2); positive ion HRMS (C20H22N3O2F3) calculated
394.1742 found 394.1719.
N‑Benzyl‑4‑[(2‑methylphenyl)methyl]pipera‑
zine‑1‑carboxamide (44)
N‑Benzyl‑4‑[(3‑nitrophenyl)methyl]pipera‑
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 61.6%); it was found by HPLC to be 98.86% pure;
IR (KBr): v/cm−1 = 3314.07 (NH stretch), 2922.59 and
2851.24 (CH stretch of CH2), 1629.55 (C=O stretch),
1547.59 (C=C stretch), 1455.99 (C–C stretch for aro-
matics),1367.28 (CH bend for CH3), 1294.97 (C–N
zine‑1‑carboxamide (41)
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 55.89%); it was found by HPLC to be 98.73% pure;
1H NMR (400 MHz, DMSO-d6) δ 8.15 (overlapping. s,
1H; Ar–H), 8.12(overlapping. d, J = 81.7 Hz, 2H);7.77 (d,
J = 7.36 Hz, 1H; Ar–H), 7.61 (t, J = 7.84 Hz, 1H; Ar–H),
7.30–7.26(m, 2H; Ar–H), 7.23–7.17 (m, 3H; Ar–H). 7.06
(t, J=5.6 Hz, NH, 1H), 4.22 (d, J=5.56 Hz, 2H; Bn CH2),
3.60 (s, 2H; CH2), 3.32 (overlapping with DMSO water.
br.s, 4H; piperazine CH2), 2.42 (br.s, 4H; piperazine CH2);
positive ion HRMS (C19H22N4O3) calculated 355.1770
found 355.1782.
1
stretch); H NMR (400 MHz, DMSO-d6) δ 7.31–7.27(m,
2H; Ar–H), 7.24–7.18(m, 4H; Ar–H), 7.14–7.13(m, 3H;
Ar–H), 7.05 (br.s, NH, 1H), 4.20 (d, J = 4.74 Hz, 2H;
Bn CH2), 3.40 (s, 2H; CH2), 3.32 (br.s, 4H; piperazine
CH2), 2.32 (br.s, 7H; piperazine CH2 & CH3); positive ion
HRMS (C20H25N3O) calculated 324.2076 found 324.2040.
4‑[1‑(Benzo[d] [1, 3] dioxol‑5‑yl)ethyl]‑N‑benzylpip‑
erazine‑1‑carboxamide (45)
N‑Benzyl‑4‑[(3‑fuorophenyl)methyl]pipera‑
zine‑1‑carboxamide (42)
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivative. (Isolated
yield 64.56%); 1H NMR (400 MHz, DMSO-d6) δ 7.26 (t,
J = 7.40 Hz, 2H; Ar–H), 7.20–7.18 (m, 3H, Ar–H), 7.00
(br.s NH, 1H), 6.85–6.83 (m, 2H; Ar–H), 6.81–6.73 (m,
1H; Ar–H), 5.97 (s, 2H; O–CH2–O), 4.21 (d, J = 5.6 Hz,
2H; Bn CH2) 3.32 (br.s, 4H; piperazine CH2), 2.48–2.24
The title compound was synthesized following the general
procedure for synthesis of urea derivative (isolated yield
65.23%); it was found by HPLC to be 98.86% pure; IR
(KBr): v/cm−1 =3339.14 (NH stretch), 2922.59 and 2852.2
(CH stretch of CH2), 1625.7 (C=O stretch), 1543.74 (C=C
1 3

Molecular Diversity
(m, 5H; piperazine CH2 & CH), 1.25 (m, 3H; CH3); posi-
tive ion ESI MS m/z (relative intensity) 368.2 [(M + H)+,
100].
4H; piperazine CH2); positive ion HRMS (C20H24N3O2Cl)
calculated 374.1635 found 374.1627.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑cyclohexyl‑
piperazine‑1‑carboxamide (49)
N‑Benzyl‑4‑(thiophen‑2‑ylmethyl) piperazine‑1‑car‑
boxamide (46)
Was found by HPLC to be 98.74% pure; 1H NMR (400 MHz,
DMSO-d6) δ 6.83 (d, J=8.0 Hz, 2H, Ar–H, overlapping),
6.72 (d, J=7.88 Hz, 1H; Ar–H), 6.08 (d, J=7.64 Hz, 1H;
NH), 5.9 (s, 2H; O–CH2–O), 3.35 (s, 2H, CH2), 3.23 (br.s,
4H; piperazine CH2), 2.25 (br.s, 4H; piperazine CH2),
1.71–1.63 (m, 4H; Cy H), 1.52–1.55 (m, 1H; Cy CH),
1.01–1.22 (m, 6H; CyH); 13C NMR (400 MHz, DMSO-d6)
δ 156.69(C=O), 147.18(Ar CH), 146.13(Ar CH), 131.77(Ar
CH), 121.95(Ar CH), 109.05(Ar CH), 107.80(Ar CH),
100.74 (O–CH2–O), 61.68 (CH2), 52.29(CH2), 49.06(CH),
43.38(CH2), 33.11(CH2), 25.36(CH2), 25.08(CH2); posi-
tive ion HRMS (C19H27N3O3) calculated 346.2131 found
346.2152.
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 71.91%); it was found by HPLC to be 99.76% pure;
IR (KBr): v/cm−1 = 3309.25 (NH stretch), 1627.63 (C=O
stretch), 1548.56 (C=C stretch), 1295.93 (C–N stretch),
694.24 (C-S stretch); 1H NMR (400 MHz, DMSO-d6) δ 7.42
(t, J=3.04 Hz, 1H; thienyl H), 7.29–7.26 (m, 2H; Ar–H),
7.22–7.16 (m, 3H; Ar–H), 7.03(t, J = 5.56 Hz, 1H; NH),
6.959–6.951 (m, 2H; thienyl H), 4.21 (d, J=5.64 Hz, 2H;
NH–CH2), 3.68 (s, 2H, thienyl CH2), 3.30–3.32 (m, 4H; pip-
erazine CH2), 2.35–2.33 (m, 4H; piperazine CH2); 13C NMR
(400 MHz, DMSO-d6) δ 157.35 (C=O), 141.28 (ArCH),
140.98 (ArCH), 128.04 (ArCH), 126.93 (ArCH), 126.53
(ArCH), 126.35 (thienyl CH), 126.19 (thienyl CH), 125.50
(thienyl CH), 56.21 (CH2), 52.21 (CH2), 43.42 (CH2); posi-
tive ion HRMS (C17H21N3OS) calculated 316.1484 found
316.1487.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)
methyl]‑N‑(cyclohexylmethyl)piperazine‑1‑carbox‑
amide (50)
The title compound was synthesized using cyclohexylmeth-
ylamine carbamate following the general procedure for syn-
thesis of 49 (isolated yield 42.90%); it was found by HPLC
to be 97.93% pure; IR (KBr): v/cm−1 =3393.14 (NH stretch),
2924.52 and 2849.31 (CH stretch of CH2), 1616.06 (C=O
stretch), 1548.56 (C=C stretch), 1260.25 (C–N stretch),
N‑Benzyl‑4‑[(thiophen‑3‑yl)methyl]pipera‑
zine‑1‑carboxamide (47)
The title compound was synthesized following the gen-
eral procedure for synthesis of urea derivative (isolated
yield 71.23%); it was found by HPLC to be 99.73% pure;
IR (KBr): v/cm−1 = 3308.29 (NH stretch), 1628.59 (C=O
1
1040.41 (C–O stretch); H NMR (400 MHz, DMSO-d6) δ
6.83 (overlap.s, 1H; Ar–H), 6.82 (overlap. d, J = 8.10 Hz,
1H, Ar–H); 6.72 (d, J = 7.92 Hz, 1H; Ar–H), 6.36 (t,
J=5.59 Hz, 1H; NH), 5.97 (s, 2H; O–CH2–O), 3.36 (s, 2H;
piperonyl CH2), 3.30–3.22 (br.s, 4H; piperazine CH2), 2.82
(t, J=6.24 Hz, 2H; NH–CH2-Cy), 2.31–2.25 (br.s, 4H; pip-
erazine CH2), 1.62–1.60 (m, 5H; Cy H), 1.35–1.32 (m, 1H;
Cy H), 1.15–1.01 (m, 3H; Cy H); 0.84–0.77(m, 2H); 13C
NMR (400 MHz, DMSO-d6) δ157.44(C=O), 147.17(Ar
CH), 146.15(Ar CH), 131.72(Ar CH), 121.97(Ar CH),
109.06(Ar CH), 107.80(Ar CH), 100.74(O–CH2–O), 61.65
(CH2), 52.26(Ar CH), 46.34 (CH2), 43.41(CH2), 37.73(CH),
30.48 (CH2), 26.16 (CH2), 25.45 (CH2); positive ion HRMS
(C20H29N3O3) calculated 360.2287 found 360.2253.
1
stretch), 1547.59 (C=C stretch), 694.24 (C-S stretch); H
NMR (400 MHz, DMSO-d6): δ 7.4(dd, J=3.00 Hz, 4.72 Hz,
1H; thienyl H), 7.26–7.16 (m, 3H; Ar–H), 7.29–7.26 (m,
3H; Ar–H & NH), 7.02 (m, thienyl H & NH, 2H), 4.20 (d,
J=5.68 Hz, 2H; Bn CH2), 3.47 (s, 2H, thienyl CH2), 3.37
(overlap with DMSO water, br.s, 4H; piperazine CH2),
2.32–2.25 (br.s, 4H; piperazine CH2); positive ion HRMS
(C17H21N3OS) calculated 316.1484 found 316.1460.
N‑Benzyl‑4‑(2‑chloro‑3‑methoxybenzyl)pipera‑
zine‑1‑carboxamide (48)
The title compound was synthesized following the general
procedure for synthesis of urea derivative (isolated yield
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑phenylpip‑
erazine‑1‑carboxamide (51)
1
58.45%); it was found by HPLC to be 99.08% pure; H
NMR (400 MHz, DMSO-d6) δ 7.28–7.25 (m, 3H; Ar–H),
7.25–7.19 (m, 3H; Ar–H), 7.09–7.03 (m, Ar–H & NH, 3H),
4.20 (d, J=5.52 Hz, 2H; Bn CH2), 3.85 (s, 3H; OCH3), 3.55
(s, 2H; CH2), 3.31 (br.s, 4H; piperazine CH2), 2.36 (br.s,
The title compound was synthesized using aniline carba-
mate following the general procedure for synthesis of 49
(isolated yield 68.6%); it was found by HPLC to be 95.01%
pure; IR (KBr): v/cm−1 = 3325.64 (NH stretch), 1644.02
1 3

Molecular Diversity
1
(C=O stretch), 1598.7 (C=C stretch), 1244.83 (C–N stretch),
789.70 (C–Cl stretch); H NMR (400 MHz, DMSO-d6)
1
1037.52 (C–O stretch); H NMR (400 MHz, DMSO-d6) δ
δ 7.33 (d, J = 8.32 Hz, 2H; Ar–H), 7.20 (d, J = 8.24 Hz,
2H; Ar–H), 7.08 (t, J = 5.72 Hz, 1H; NH), 6.84 (overlap,
s, 1H); 6.83 (overlap, d, J= 8.44 Hz, 2H; Ar–H), 6.73 (d,
J=7.96 Hz, 1H; Ar–H), 5.97 (s, 2H; O–CH2–O), 4.17 (d,
2H, J=5.66 Hz, Bn CH2), 3.36 (s, 2H; piperonyl CH2), 3.3
(br.s, 4H; piperazine CH2), 2.25 (br.s, 4H; piperazine CH2);
positive ion HRMS (C20H22N3O3Cl) calculated 388.1428
found 388.1437.
8.40 (s, 1H; NH), 7.4 (d, J =7.84 Hz, 2H; Ar–H), 7.20 (t,
J = 7.60 Hz, 2H; Ar–H), 6.90 (t, J = 7.32 Hz, 1H; Ar–H),
6.88–6.83 (m, 2H; Ar–H), 6.70 (d, J=7.84 Hz, 1H; Ar–H),
5.90 (s, 2H; O–CH2–O), 3.41–3.40 (m, 6H; piperazine CH2
& piperonyl CH2), 2.34–2.33(m, 4H; piperazine CH2). posi-
tive ion HRMS (C19H21N3O3) calculated 340.1661 found
340.1674.
4‑[(2H‑1,3‑Benzodioxol‑5‑yl)methyl]‑N‑[(2‑fuoro‑
phenyl)methyl]piperazine‑1‑carboxamide (55)
(R)‑4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑(1‑phe‑
nylethyl)piperazine‑1‑carboxamide (52)
The title compound was synthesized using 2-fuoroben-
zylamine carbamate following the general procedure for
synthesis of 49. (isolated yield 53.33%); it was found by
HPLC to be 97.94% pure; IR (KBr): v/cm−1 =3348.78 (NH
stretch), 1626.66 (C=O stretch), 1539.88 (C=C stretch),
1255.43 (C–N stretch), 1037.42 (C–O stretch) 755 (C-H
stretch for Aromatic with four hydrogen atoms); 1H NMR
(400 MHz, DMSO-d6): δ 7.26 (dd, J = 16.22 & 8.41 Hz,
2H; Ar–H), 7.10 (dd, J=16.50 & 8.55 Hz, 2H; Ar–H), 7.01
(app.t, 1H; NH), 6.84 (overlap. s, 1H; Ar–H), 6.83(overlap.
d, J=9.76 Hz, 1H); 6.74 (d, J=8.0 Hz, 1H; Ar–H), 5.97 (s,
2H; O–CH2–O), 4.25 (d, J=5.23 Hz, 2H; Bn CH2), 3.37 (s,
2H; piperonyl CH2), 3.31 (overlap with DMSO water, br.s,
4H; piperazine CH2), 2.30 (br.s, 4H; piperazine CH2); posi-
tive ion HRMS (C20H22N3O3F) calculated 372.1723 found
372.1665.
The title compound was synthesized using (R)-α-
methylbenzyl amine carbamate following the general proce-
dure for synthesis of 49 (isolated yield 46.5%); it was found
by HPLC to be 98.06% pure; IR (KBr): v/cm−1 = 3325.64
(NH stretch), 1626.66 (C=O stretch), 1539.88 (C=C stretch),
1375.96 (CH bend for CH3), 1253.5 (C–N stretch), 1035.59
(C–O stretch); 1H NMR (400 MHz, DMSO-d6) δ 7.27–7.24
(m, 4H; Ar–H), 7.18–7.16 (m, 1H; NH), 6.84 (overlap.
s, 1H; Ar–H), 6.83(overlap. d, J = 8.20 Hz, 1H, Ar–H);
6.74–6.72 (m, 2H), 5.97 (s, 2H; O–CH2–O), 4.80–4.77 (m,
1H; CH), 3.36 (overlap with DMSO water. s, 2H; piperonyl
CH2), 3.28 (br.s, 4H; piperazine CH2), 2.26 (br.s, 4H; pip-
erazine CH2), 1.30 (d, J=7.04 Hz, 3H; CH3); positive ion
HRMS (C21H25N3O3) calculated 368.1974 found 368.1978.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑(2‑methylb‑
enzyl)piperazine‑1‑carboxamide (53)
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)
methyl]‑N‑(4‑fuorobenzyl)piperazine‑1‑carboxam‑
ide (56)
The title compound was synthesized using 2-methylben-
zyl amine carbamate following the general procedure for
the synthesis of 49 (isolated yield 46.28%); it was found
by HPLC to be 99.81% pure; 1H NMR (400 MHz, DMSO-
d6) δ 7.15–7.10 (m, 2H; Ar–H), 6.88 (t, J = 5.46 Hz,
1H; NH), 6.85 (overlap. s, 1H; Ar–H), 6.84(overlap. d,
J=2.18 Hz, 1H); 6.73 (d, J=7.72 Hz, 1H; Ar–H), 5.97 (s,
2H; O–CH2–O), 4.17 (d, J=5.44 Hz, 2H; Bn CH2), 3.35 (s,
2H; piperonyl CH2), 3.31 (br.s, 4H; piperazine CH2), 2.30
(br.s, 4H; piperazine CH2), 2.24 (s, 3H; CH3); positive ion
HRMS (C21H25N3O3) calculated 368.1974 found 368.1996.
The title compound was synthesized using 4-fuoroben-
zylamine carbamate following the general procedure for
synthesis of 49 (isolated yield 63.33%); it was found by
HPLC to be 96.27% pure; IR (KBr): v/cm−1 =3339.14 (NH
stretch), 1615.09 (C=O stretch), 1541.81 (C=C stretch),
1245.79 (C–N stretch), 1042.34 (C–O stretch) 824 (C-H
stretch for aromatics with two adjacent hydrogen atoms);
1H NMR (400 MHz, DMSO-d6) δ 7.26–7.23 (m, 2H;
Ar–H), 7.12–7.07 (m, 2H; Ar–H), 7.05 (t, J=5.99 Hz, 1H,
NH); 6.83 (overlapped. d, J=8.64 Hz, 2H; Ar–H), 6.74 (d,
J=7.92 Hz, Ar–H, 1H), 5.97 (s, 2H; O–CH2–O), 4.18 (d,
J=5.60 Hz, 2H; Bn CH2), 3.36 (s, 2H; piperonyl CH2), 3.28
(br.s, 4H; Piperazine CH2), 2.27 (app.t, J = 4.23 Hz, 4H;
Piperazine CH2); 13C NMR (400 MHz, DMSO-d6) δ162.35
(C-F), 159.94 (C=O), 156.32 (Ar CH), 137.10 (Ar CH),
135.92 (Ar CH), 129.05 (Ar CH), 128.92 (Ar CH), 128.33
(Ar CH), 127.77 (Ar CH), 127.73 (Ar CH), 115.06 (Ar CH),
114.84 (Ar CH), 65.38 (CH2), 43.09 (CH2); positive ion
HRMS (C20H22N3O3F) calculated 372.1723 found 372.1739.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑(4‑chlo‑
robenzyl)piperazine‑1‑carboxamide (54)
The title compound was synthesized using 4-chloroben-
zylamine carbamate following the general procedure for
synthesis of 49. (Isolated yield 65.87%); IR (KBr): v/
cm−1 = 3354.57 (NH stretch), 2928.38 and 2858.95 (CH
stretch of CH2), 1617.02 (C=O stretch), 1535.06 (C=C
stretch), 1244.83 (C–N stretch), 1040.41 (C–O stretch),
1 3

Molecular Diversity
positive ion HRMS (C18H25N3O3) calculated 332.1974
found 332.1912.
4‑[(2H‑1,3‑Benzodioxol‑5‑yl)methyl]‑N‑[(4‑methyl‑
phenyl)methyl]piperazine‑1‑carboxamide (57)
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑isobutyl‑
The title compound was synthesized using 4-methylbenzyl
amine carbamate following the general procedure for the
synthesis of 49 (isolated yield 61.33%); it was found by
HPLC to be 98.77% pure; IR (KBr): v/cm−1 =3361.32 (NH
stretch), 1619.91 (C=O stretch), 1543.74 (C=C stretch),
piperazine‑1‑carboxamide (60)
The title compound was synthesized using isobutylamine
carbamate following the general procedure for synthesis of
49 (isolated yield 45.50%); it was found by HPLC to be
98.08% pure; 1H NMR (400 MHz, DMSO-d6) δ 6.82 (over-
lap. s, 1H; Ar–H), 6.82(overlap. d, J=8.17 Hz, 1H, Ar–H);
6.72 (d, J=7.84 Hz, 1H; Ar–H), 6.45 (t, J=5.36 Hz, 1H;
NH), 5.99 (s, 2H; O–CH2–O), 3.35 (s, 2H; piperonyl CH2),
3.25 (br.s, 4H; Piperazine CH2), 2.80 (t, J = 6.24 Hz, 2H;
CH2), 2.25 (br.s, 4H; Piperazine CH2), 1.68–1.61 (m, 1H;
CH), 0.78 (d, J = 6.68 Hz, 6H; CH3); positive ion HRMS
(C17H25N3O3) calculated 320.1974 found 320.1930.
1
1246.75 (C–N stretch), 1037.52 (C–O stretch); H NMR
(400 M Hz, DMSO-d6) δ 7.15 (t, J = 5.80 Hz, 1H; NH),
7.01–6.84 (m, 4H; Ar–H), 6.83 (d, J=10.28 Hz, 2H; Ar–H),
6.73 (d, J =7.84 Hz, 1H; Ar–H), 5.97 (s, 2H; O–CH2–O),
4.17 (d, J = 5.32 Hz, 2H; Bn CH2), 3.36 (s, 2H; piperonyl
CH2), 3.32 (br.s, 4H; piperazine CH2), 2.49 (br.s, 7H; pip-
erazine CH2 & CH3); positive ion HRMS (C21H25N3O3) cal-
culated 368.1974 found 368.1910.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑butylpiper‑
azine‑1‑carboxamide (61)
methyl]‑N‑(pentan‑3‑yl)piperazine‑1‑carboxamide
(58)
The title compound was synthesized using butylamine car-
bamate following the general procedure for synthesis of
49 (isolated yield 49.33%); it was found by HPLC to be
95.87% pure; IR (KBr): v/cm−1 = 3307.32 (NH stretch),
2923.56 and 2855.1 (CH stretch of CH2), 1625.7 (C=O
stretch), 1548.56 (C=C stretch), 1368.25 (CH bend for CH3),
The title compound was synthesized using pentan-3-ylamine
carbamate following the general procedure for synthesis of
49. (Isolated yield 66.75%); IR (KBr): v/cm−1 = 3334.32
(NH stretch), 2922.59 and 2853.17 (CH stretch of CH2),
1619.91 (C=O stretch), 1547.59 (C=C stretch), 1254.47
(C–N stretch), 1039.44 (C–O stretch); 1H NMR (400 MHz,
DMSO-d6) δ 6.84 (s, 1H, overlapping; Ar–H), 6.81 (s,
1H, overlapping; Ar–H), 6.74 (d, J=8.00 Hz, 1H; Ar–H),
5.97–5.93 (m, 3H; NH & O–CH2–O), 3.39–3.35 (m, 3H;
piperonyl CH2 & pentyl CH), 3.31 (br.s, 4H; piperazine
CH2), 2.24 (br.s, 4H; piperazine CH2), 1.42–1.29 (m, 4H;
pentyl CH2), 0.82–0.75 (m, 6H; pentyl CH3); positive ion
HRMS (C18H27N3O3) calculated 334.2131 found 334.2039.
1
1250.61 (C–N stretch), 1038.48 (C–O stretch); H NMR
(400 MHz, DMSO-d6) δ 6.83 (d, J=8.00 Hz, 2H; Ar–H),
6.73 (d, J = 7.80 Hz, 1H; Ar–H), 6.38 (app.t, 1H; NH),
5.97 (s, 2H; O–CH2–O), 3.35 (s, 2H; piperonyl CH2), 3.23
(br.s, 4H; piperazine CH2), 2.97 (dd, J=12.62 Hz, 6.85 Hz,
2H; NH–CH2), 2.24 (br.s, 4H; piperazine CH2), 1.36–1.20
(m, 4H; CH2), 0.84 (t, J=7.24 Hz, 3H; CH3); positive ion
HRMS (C17H25N3O3) calculated 320.1974 found 320.1974.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑cyclopen‑
tylpiperazine‑1‑carboxamide (59)
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑hexylpiper‑
azine‑1‑carboxamide (62)
The title compound was synthesized using cyclopentylamine
carbamate following the general procedure for synthesis of
49. (isolated yield 67.91%); it was found by HPLC to be
98.01% pure; IR (KBr): v/cm−1 = 3359.39 (NH stretch),
2957.3 and 2840.63 (CH stretch of CH2), 1627.63 (C=O
stretch), 1538.92 (C=C stretch), 1250.61 (C–N stretch),
The title compound was synthesized using hexylamine car-
bamate following the general procedure for synthesis of 49.
(isolated yield 68.65%); it was found by HPLC to be 98.84%
pure; IR (KBr): v/cm−1 = 3328.53 (NH stretch), 2924.52
and 2855.1 (CH stretch of CH2), 1621.84 (C=O stretch),
1548.56 (C=C stretch), 1367.28 (CH bend for CH3), 1255.43
(C–N stretch), 1041.37 (C–O stretch); 1H NMR (400 MHz,
DMSO-d6) δ 6.83 (overlapping, s, 1H); 6.82 (overlap, d,
J = 8.07 Hz, 1H; Ar–H), 6.72 (d, J= 7.76 Hz, 1H; Ar–H),
6.40 (t, J=5.08 Hz, 1H; NH), 5.97 (s, 2H; O–CH2–O), 3.35
(s, 2H; piperonyl CH2), 3.22 (br.s, 4H; piperazine CH2),
2.95 (dd, J=12.85 Hz & 6.66 Hz, 2H; aliphatic NH–CH2),
2.24 (br.s, 4H; piperazine CH2), 1.36–1.32 (m, 2H; CH2),
1
1039.44 (C–O stretch); H NMR (400 MHz, DMSO-d6)
δ 6.83 (d, J = 8.04 Hz, 2H; Ar–H), 6.73 (d, J = 7.80 Hz,
1H; Ar–H), 6.17 (d, J = 6.84 Hz, 1H; NH), 5.97 (s, 2H;
O–CH2–O), 3.88–3.83 (m, 1H; cyclopentyl CH), 3.35 (s,
2H; piperonyl CH2), 3.23 (br.s, 4H; piperazine CH2), 2.24
(br.s, 4H; piperazine CH2), 1.76–1.70 (m, 2H; cyclopentyl
CH2), 1.61–1.54 (m, 2H; cyclopentyl CH2), 1.47–1.41 (m,
2H; cyclopentyl CH2), 1.39–1.31 (m, 2H; cyclopentyl CH2);
1 3

Molecular Diversity
1.25–1.22 (m, 6H; CH2), 0.84 (t, J = 6.76 Hz, 3H; CH3);
positive ion HRMS (C19H29N3O3) calculated 348.2287
found 348.2353.
4.17 (d, J = 5.20 Hz, 2H; Bn CH2), 3.36 (s, 2H; piperonyl
CH2), 3.32(br.s, 2H, piperazine CH2), 2.48–2.24 (m, 7H;
piperazine CH2& CH3); positive ion HRMS (C21H25N3O3)
calculated 368.1974 found 368.1906.
4‑[(Benzo[d] [1, 3] dioxol‑5‑yl)methyl]‑N‑cyclohep‑
tylpiperazine‑1‑carboxamide (63)
Molecular modeling
The primary sequence for M. tuberculosis was obtained from
the Uniprot database (ID: P9WP37). Template proteins were
identifed using the BLAST search with E-value of 10100
from the protein data bank. The BLAST search retrieved
two X-ray crystal structures of M. Smegmatis QcrB (PDB
id 6HWH and 6ADQ) as suitable templates with > 82%
sequence identity. The tertiary structure of M. tuberculosis
was predicted using Modeller v9.2 [20] with UCSF Chimera
as the graphical user interface, this exercise yielded 5 mod-
els. The best model was selected based on the DOPE score.
Subsequently, the coordinates of a QcrB Q0 site inhibitor
(trifoxystrobin) bound to chicken cytochrome bc1 complex
(PDB id 3L70) were copied into the predicted model by
superimposing the backbone atoms. The f14SB force feld
was used to obtain parameters for the protein atoms, whereas
the general AMBER force feld (GAFF) was used to obtain
parameters for the atoms of trifoxystrobin. The QcrB model
bound to trifoxystrobin was minimized in vacuum by steep-
est descents for 1000 steps followed by 100 steps of conju-
gate gradients using AMBER14. After minimization, the
coordinates of the heme moiety were copied from M. Smeg-
matis QcrB X-ray structure into the model by superimposing
the protein backbone atoms.
The title compound was synthesized using cycloheptylamine
carbamate following the general procedure for synthesis of
49. (Isolated yield 53.37%); IR (KBr): v/cm−1 = 3364.25
(NH stretch), 1627.63 (C=O stretch), 1537.95 (C=C stretch)
1
1250.61 (C–N stretch), 1040.41 (C–O stretch); H NMR
(400 MHz, DMSO-d6) δ 6.83 (s, 2H, overlapping; Ar–H),
6.81 (s, 1H, overlapping; Ar–H), 6.74 (d, J = 7.84 Hz,
1H; Ar–H), 6.12 (d, J = 7.68 Hz, 1H; NH), 5.97 (s, 2H;
O–CH2–O), 3.56 (br.s, 1H; cycloheptyl CH), 3.35 (s, 2H;
piperonyl CH2), 3.22 (br.s, 4H; piperazine CH2), 2.24 (br.s,
4H; piperazine CH2), 1.71–1.33 (m, 12H; cycloheptyl CH2);
positive ion HRMS (C20H29N3O3) calculated 360.2287
found 360.2190.
4‑[(2‑(Benzo[d] [1, 3] dioxol‑5‑yl)ethyl]‑N‑benzylpip‑
erazine‑1‑carboxamide (64)
The title compound was synthesized using phenethylamine
carbamate following the general procedure for synthesis of
49 (isolated yield 71.67%); it was found by HPLC to be
97.33% pure; IR (KBr): v/cm−1 = 3320.82 (NH stretch),
1614.13 (C=O stretch), 1531.2 (C=C stretch), 1244.83
(C–N stretch), 1042.34 (C–O stretch); 1H NMR (400 MHz,
DMSO-d6) δ 7.28 (t, J=7.8 Hz, 2H; Ar–H), 7.18–7.15 (m,
3H; Ar–H), 6.84–6.82 (m, 2H; Ar–H), 6.74 (d, J=7.88 Hz,
1H; Ar–H), 6.56 (t, J = 5.32 Hz, 1H; NH), 5.97 (s, 2H;
O–CH2–O), 3.35 (s, 2H; CH2), 3.23–3.16 (m, 6H; Pipera-
zine CH2 & piperonyl CH2), 2.70–2.66 (t, J=7.60 Hz, 2H;
CH2), 2.24 (br.s, 4H; piperazine CH2); positive ion HRMS
(C21H25N3O3) calculated 368.1974 found 368.1974.
The binding site for docking was defned using the center
of mass of trifoxystrobin. The protein and ligands were pre-
pared for docking using the MGLTools v1.5.6. Autodock
VINA [21] was used as the docking engine with a grid of
size 25 Å3 grid, and with exhaustiveness for conformational
sampling of 25. The protocol was validated by redocking
trifoxystrobin, following which molecules I and Q203 were
docked.
4‑[(2H‑1,3‑Benzodioxol‑5‑yl)methyl]‑N‑[(3‑methyl‑
phenyl)methyl]piperazine‑1‑carboxamide (65)
Molecular similarity calculation
Molecular similarity values were calculated using the
KNIME platform. KNIME is a free, open source platform
for data analysis and integration. A workfow was gener-
ated using few nodes (a node is small programming unit in
KNIME which is functionalized for specifc tasks) available
in the node repository. A set of 54 compounds in SMILES
format was loaded into the fle reader node; compound I was
entered into the workfow by the structure sketcher node.
Both data were converted into CDK format (an internal for-
mat where it is regarded as a molecule for further calcula-
tions) using the Molecule to CDK node. Fingerprints were
generated of these CDK data sets by the Fingerprints node
The title compound was synthesized using 3-methylbenzyl
amine carbamate following the general procedure for the
synthesis of 49 (isolated yield 69.45%); it was found by
HPLC to be 99.87% pure; IR (KBr): v/cm−1 =3435.56 (NH
stretch), 1627.63 (C=O stretch), 1546.63 (C=C stretch),
1365.35 (CH bend for CH3), 1237.11 (C–N stretch), 1040.41
(C–O stretch) 777.17 (C-H stretch for aromatic with three
adjacent hydrogen atoms); 1H NMR (400 MHz, DMSO-d6)
δ 7.11–7.06 (m, 4H; Ar–H), 6.98 (app.t, 1H; NH), 6.84 (s,
1H, overlapping; Ar–H), 6.82 (s, 1H, overlapping; Ar–H),
6.72 (d, J =7.84 Hz, 1H; Ar–H), 5.97 (s, 2H; O–CH2–O),
1 3

Molecular Diversity
(using Estate method); this was followed by comparison of
the fngerprints of each molecule in the set of 54 compounds
against compound I using fngerprint similarity node. The
calculated values were processed into an excel fle by the
Excel writer node.
The minimum inhibitory concentration (MIC) of the
samples against the test organisms was determined using
resazurin microplate assay. This assay was performed using
fat-bottom 96-well clear microtiter plates. The wells in
frst row of each column were flled with 100 μl of Muel-
ler–Hinton containing standard antibiotic with culture, while
second row was flled with 100 μl of the respective media
with culture. The third row was flled with culture containing
100 μl 2X media (Mueller–Hinton), and 100 μl of samples
(0.032 mg/mL) was added in each row. Then, from the fourth
row onward, each well was flled with 1X Media (Muel-
ler–Hinton) along with the culture. An identical twofold
serial dilution was made from the third row to the eighth
row. Lastly, all wells were mixed thoroughly. Final concen-
trations of sample in the third to eighth row ranged from
0.032 mg/mL to 0.001 mg/mL with 1 x 106 CFU/ml. The
cultured microplates were sealed with the lid and incubated
at 37 °C for 24 h. The MIC of samples was detected follow-
ing addition (5μL) of 2 mg/mL resazurin in all wells, and
this was incubated at 37 °C for 30 min. Microbial growth
was determined by observing the change in the color of resa-
zurin in the micro-plate wells (pinkish-red formazan when
there is growth and blue when there is no growth). MIC
was defned as the lowest sample concentration showing no
color change (clear) and exhibited complete inhibition of
microbial growth. This study was carried out in triplicate.
HPLC analysis
Analytical HPLC was done on a Agilent Quadrupole LCMS
(6120). HPLC analysis was carried out using a Inertsustain
C18 (250×4.6 mm×3µ) column with an isocratic gradient
of 0.1% formic acid in water and methanol (20:80, v/v) with
a runtime of 8 min. The fow rate was maintained at 1 mL/
min and temperature 40 °C. The test sample was prepared
using methanol as a diluent and injection volume was 20µL
(1000 ppm solution).
Biological activity studies
Determination of minimum inhibitory
concentration (MIC90)
MICs were determined against M. tuberculosis H37Rv
(ATCC 25618) grown in Middlebrook 7H9 medium contain-
ing 10% v/v OADC (oleic acid, albumin, dextrose, catalase)
supplement (Becton–Dickinson) and 0.05% w/v Tween 80
(7H9-Tw-OADC) under aerobic conditions as previously
described [8]. Bacterial growth was measured after 5 days
of incubation at 37 °C. MIC90 was defned as the concentra-
tion of compound required to inhibit growth of M. tubercu-
losis by 90% and was determined by plotting the growth,
followed by curve ftting using the Levenberg–Marquardt
least-squares method.
Cytotoxicity studies
Cellular toxicity assay was carried out on HepG2 cells by
the MTT assay. Briefy, the test compounds were incubated
with HepG2 cells for 48 h at 37 °C, at concentrations of 5,
10, 25, 50, 75 and 100 μM. After 48 h, cell viability was
measured using MTT reduction assay. Paclitaxel was used
as the positive control.
Antibacterial assay
Materials used: HEPG2 (Human liver cancer cell line),
Dulbecco`s Modifed Eagle Media (DMEM) with low glu-
cose—Cat No-11965-092 (Gibco, Invitrogen), Fetal bovine
serum (FBS)—Cat No-10270106 (Gibco, Invitrogen),
Antibiotic Antimycotic 100X solution (Thermofsher Sci-
entifc)—Cat No-15240062.
Bacterial Cultures: Staphylococcus aureus ATCC 6538,
Escherichia coli ATCC 25922 and Salmonella typhi ATCC
14028.
Sample preparation: The compounds were provided in the
form of powder (≈10 mg). This was dissolved in methanol
to prepare a stock concentration of 10 mg/mL and a working
stock of 0.5 mg/mL.
The cells were seeded in a 96-well fat-bottom micro-plate
and maintained at 37 °C in 95% humidity and 5% CO2 over-
night. Diferent concentrations (100, 75, 50, 25, 10, 5 μM) of
samples were treated. The cells were incubated for another 48 h.
The wells were washed twice with PBS, and then, 20 μL of the
MTT staining solution was added to each well and the plate was
incubated at 37 °C. After 4 h, 100 μL of DMSO was added to
each well to dissolve the formazan crystals, and absorbance was
recorded at 570 nm using a micro-plate reader [22].
Preparation of Inoculum: A loopful of cultures were inoc-
ulated in a suitable broth (Luria–Bertani Broth) and grown
for 24 h. The culture OD was adjusted was to 0.5 McFarland
standard in order to get 1.5×108 CFU/ml. The cell suspen-
sion was mixed to homogeneity to obtain a fnal density of
1×106 CFU/ml.
Surviving cells (%) = Mean OD of test compound/Mean OD of Negative control × 100
1 3

Molecular Diversity
IC50 of the compounds was calculated with Graph Pad Prism
Version 5.1.
w/v of glycerol and 10 mM EDTA, pH 7.4 (10 ml per 10 g
liver) by homogenization. The microsomes were stored at
−70 °C until further use.
(ii) CYP content determination
Metabolism studies
CYP450 content was determined by the method of Oumura
and Sato [9]. The microsomes were diluted in the ratio of 1:9
with 0.1 M potassium phosphate bufer, pH 7.4, containing
0.5% v/v Triton X 100 and 1 mM EDTA and were mixed
thoroughly. The solution was then saturated with 50–60 bub-
bles of carbon monoxide gas at the rate of 1–2 bubbles/sec.
After equilibration with carbon monoxide, the solution was
equally divided into two 3 ml quartz cuvettes and a baseline
absorbance reading was recorded. A pinch of sodium dith-
ionite was added only to the sample cuvette, mixed by inver-
sion and scanned from 400 to 500 nm to obtain a reduced
carbon monoxide v/s oxidized carbon monoxide diference
spectrum. The absorbances at 450 and 490 nm were noted
(Fig. S2). An extinction coefcient of 106 mM−1cm−1 was
used for the determination of CYP450 content.
The oxidative metabolic stability of a select set of com-
pounds (I, 27, 28, 49, and 50) was assessed in rat liver
microsomes. Briefy, rat liver microsomes were isolated
by the calcium aggregation method [23]. Stock solutions
of 10 mM of the compounds were prepared in acetonitrile.
Individual incubations to determine the metabolic stability
of each of the above compounds were carried out in a total
volume of 1 mL and had compound at a fnal concentra-
tion of 0.1 mM, NADPH (0.6 mM) in 0.1 M sodium phos-
phate bufer, pH 7.5. Control incubations without NADPH
and without microsomes were also done. All incubations
were done in duplicate. The incubations were started with
NADPH and terminated after 15, 30 and 60 min by addition
of an equal volume of ice-cold acetonitrile. After termina-
tion of the reaction, 50 μL of internal standard was added to
the incubation. Compound 50 was used as internal standard
for incubations of compounds I, 27, 28, and 49, while com-
pound 27 was used as internal standard for incubation of
compound 50. The samples were centrifuged at 13000 ×g
and the supernatant injected onto the HPLC. HPLC anal-
ysis was done on a Jasco 4000 instrument equipped with
Chromnav 2.0 software. A Sunniest ECO C18 column
(250 mm × 4,6 mm i.d., 5 micron) was used. The mobile
phase was acetonitrile: 0.1% formic acid (8:2) at a fow rate
of 1 ml/min. The column temperature was 38 °C and detec-
tion was by UV at 254 nm. (Table S1).
(iii) Incubation with microsomes
The incubations were carried out at 37 °C in water bath
shaker. The bufer used for incubation was 0.1 M sodium
phosphate bufer, pH 7.5. In a test tube were taken bufer,
10 μl of a 10 mM of synthesized compound (1/27/28/49/50)
and 50 μl of microsomes (Table S4). The reaction was initi-
ated by addition of 60 μL of NADPH (10 mM in phosphate
bufer), and the incubation mixtures were kept at 37 °C in a
water bath shaker for 30 and 60 min. The incubations were
terminated by addition of 1000 μL of cold ACN and then
50 μL of internal standard added. The reaction mixtures
were then centrifuged at 9000 X g for 10 min at 4 °C. The
supernatant was then injected into an HPLC instrument for
analysis. All incubations were carried out in duplicate.
(i) Isolation of microsomes
The entire isolation procedure was carried out in an ice bath
(0–4 °C). The excised rat livers (10 g per batch) stored at
− 70 °C were allowed to thaw to room temperature. The
livers were fnely chopped with a pair of scissors, followed
by homogenization in a Potter Elvehjem glass homogenizer
equipped with a Tefon pestle. For homogenization, four
times the weight in volume (40 ml) of 10 mM tris–HCl
bufer containing 0.25 M sucrose, pH 7.4 was used. The
homogenate was centrifuged at 13,000 x g for 10 min at 4 °C
in a refrigerated centrifuge (Biofuge Stratos), and the precip-
itate was discarded. Calcium chloride was then added slowly
with stirring to the supernatant to a fnal concentration of
10 mM. The solution was stirred for 15–20 min with a glass
rod and then centrifuged again at 25,000 x g for 10 min at
4 °C. Firmly packed pellets of microsomes were obtained.
The supernatant (cytosol) obtained was stored separately at
−70 °C, and the pellet of microsomes was resuspended by
homogenization in 100 mM Tris–HCl bufer containing 20%
Acknowledgements We thankfully acknowledge the National Facility
for Biopharmaceuticals, G N Khalsa College, Mumbai, for antibacte-
rial activity results; Maratha Mandal’s Central Research Laboratory,
Belgaum, for cytotoxicity results; SAIF, Panjab University, Chandi-
garh, for HRMS analysis, Mrs. Varsha Vhadge, Ambernath Organics
Pvt. Ltd, Mumbai, for HPLC purity results and Dr. M. K Rangnekar
Memorial testing laboratory, Bombay College of Pharmacy, Mumbai,
for FTIR analysis.
Author contributions SS and AAK wrote the paper and synthesized
the molecules. RC, AK, BS synthesized the molecules. JP oversaw the
synthesis and analysis. DD, KR, KRI performed metabolism studies.
EAFM performed computational studies. AK and TP antimycobacterial
assay (H37Rv strain). ECC and SN designed and oversaw the study.
Funding Financial support for this work was provided by Ambernath
Organics Pvt. Ltd. and the Amrut Mody Research Foundation at Bom-
bay College of Pharmacy.
1 3

Molecular Diversity
analyte-induced recruiting of membrane receptors. Angew Chem
Int Ed 51(40):10060–10063
Compliance with ethical standards
13. Broggini G, Barbera V, Beccalli EM, Chiacchio U, Fasana A,
Galli S, Gazzola S (2013) Selective intramolecular palladium
(II)-catalyzed aminooxygenation vs. diamination of alkenylureas:
efcient microwave-assisted reactions to bicyclic piperazinones.
Adv Synth Catal 355(8):1640–1648
Conflicts of interest The authors declare no competing fnancial inter-
est.
14. Giofre S, Beccalli EM, Foschi F, La Rosa C, Presti LL, Chris-
todoulou MS (2019) Chemo-and regioselective palladium (II)-
catalyzed aminoarylation of N-allylureas providing 4-arylmethyl
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