Synthesis and SAR studies of potent H+/K+-ATPase and anti-inflammatory activities of symmetrical and unsymmetrical urea analogues

Article abstract of DOI:10.1007/s00044-017-1878-x

A sequence of symmetrical and unsymmetrical urea derivatives 1–24 were synthesized and characterized by standard spectroscopic techniques. The synthesized analogues were tested for their in vitro H⁺/K⁺-ATPase and anti-inflammatory activities. The majority of the compounds showed outstanding activity, compared to that of omeprazole and indomethacin, usual standard drugs of antiulcer and anti-inflammatory, respectively. In particular, hydroxy, methyl, and methoxy derivatives 13–24 were the most active compounds possessing a significant amplify for diverse substituents on the benzene ring thus, contributing positively to gastric ulcer inhibition. Compounds 1–3 and 22–24 showed excellent anti-inflammatory activity due to the presence of electron-withdrawing groups (Cl and F) on the molecule.

Full text of DOI:10.1007/s00044-017-1878-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1878-x
ORIGINAL RESEARCH
Synthesis and SAR studies of potent H+/K+-ATPase and anti-
inflammatory activities of symmetrical and unsymmetrical urea
analogues
Kadalipura P. Rakesh1 Nanjudappa Darshini1 Sunnadadoddi L. Vidhya2
●
●
●
Rajesha2 Ningegowda Mallesha1
●
Received: 27 December 2016 / Accepted: 15 March 2017
© Springer Science+Business Media New York 2017
Abstract A sequence of symmetrical and unsymmetrical
urea derivatives 1–24 were synthesized and characterized
by standard spectroscopic techniques. The synthesized
analogues were tested for their in vitro H+/K+-ATPase and
anti-inflammatory activities. The majority of the compounds
showed outstanding activity, compared to that of omepra-
zole and indomethacin, usual standard drugs of antiulcer
and anti-inflammatory, respectively. In particular, hydroxy,
methyl, and methoxy derivatives 13–24 were the most
active compounds possessing a significant amplify for
diverse substituents on the benzene ring thus, contributing
positively to gastric ulcer inhibition. Compounds 1–3 and
22–24 showed excellent anti-inflammatory activity due to
the presence of electron-withdrawing groups (Cl and F) on
the molecule.
Introduction
Urea is a useful molecule that is generally present in natural
products and frequently displays a broad scope of biological
activities (Fournier et al. 1991). The substituted urea is a
most importance in medicinal chemistry and it is used for
the synthesis of various organic or heterocyclic compounds
with diversified biological activities including antimicrobial
(Umadevi et al. 2012; Gulkok et al. 2012), antiviral (Rajtar
et al. 2013), anticonvulsant (Ramesh et al. 2013), anti-
proliferative (Cho et al. 2012), antitumor (Lu et al. 2013),
antiulcernic (Vijayakumar et al. 2013), antimalaria
(Anderson et al. 2013). Some of the urea derivatives are
thiourea, phenyl urea, sulfur ureas are found to exhibit a
potent inhibiting effect on HIV-1 protease enzyme and as
anticancer and sedative-hypnotic activities. They are also
served as an extensive application as fertilizers, agro-
chemicals, and synthetic intermediates for many drugs
(Viana et al. 2013).
Keywords Ureas’s SAR H+/K+-ATPase anti-
inflammatory
●
●
●
Gastric ulcers are commonly occurring diseases and
believed to result from an imbalance between the aggressive
and defensive forces in the stomach (Gustavsson et al.
1983). Newly, agents have been recognized that totally
suppress acid secretion by inhibition of the gastric proton
pump H+/K+-ATPase. Such inhibition leads to intense and
persistent achlorhydria, which outcome in ulcer healing
rates significantly more rapid than those achievable by H2
antagonists. H+/K+-ATPase catalyzes the terminal step in
gastric acid, whereas histamine plays crucial role in acid
secretion. As a result, inhibition of H+/K+-ATPase can
supply an essentially greater reduction in gastric acid
secretion (Sachs et al. 1976; Forte et al. 1980; Lind et al.
1983).
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1878-x) contains supplementary material,
which is available to authorized users.
* Rajesha
rajeshamg7@gmail.com
* Ningegowda Mallesha
research@sriramchem.com
1
SRI RAM CHEM, R & D Centre, Plot No. 31, JCK industrial
Park, Belagola Industrial Area, Mysuru 570016 Karnataka, India
2
Non-steroidal anti-inflammatory drugs (NSAIDs) are
most commonly used in therapeutics, mainly for the action
Bharathi College PG and Research Center, Bharathinagara,
Maddur, Mandya district, Karnataka 571422, India

Med Chem Res
of pain and inflammation in arthritis for decades. NSAIDs
reduce the pain and swelling related with arthritis by
blocking the metabolism of arachidonic acid by cycloox-
ygenase enzyme (COX) thereby the production of pros-
taglandin (Vane 1971). COX activity originates from two
diverse and separately regulated enzymes, termed COX-1
and COX-2 (Yokoyama and Tanabe 1989; Hla and Neilson
1992). COX-1 is nominated as housekeeping enzyme
because of its physiological and homeostatic functions,
whereas COX-2 is expressed in pathological state and
triggers inflammatory signals, thus selected as an adaptive
enzyme. Both of these enzymes share more than sixty
percent of homology with respect to their structure (Kur-
umbail et al. 1996). Overall, these explanations recommend
that COX-1 and COX-2 serve up different physiological
and pathophysiological functions. Therefore, although there
are a number of anti-inflammatory drugs are available in the
market, development of novel compounds having anti-
inflammatory agents with improved profile is still a
necessity.
to the solutions, after completion of the reaction (monitored
by TLC), reaction mass was poured in to ice cold water and
then extracted with EtOAc, the organic layer was washed
with water and dried over anhydrous sodium sulfate. The
organic solvent was removed under vacuum to get crude
products (3a-r). The solid product was further purified by
column chromatography by using hexane and ethyl acetate
(95:5) as eluent to give pure products.
Biological activity
Gastric H+/K+-ATPase activity
Isolation of parietal cells from sheep stomach The fundic
stomach slice of sheep quickly after sacrificing was col-
lected and was rinsed with Krebs ringer buffer (250 mM
sucrose, 2 mM MgCl2, 1 mM EGTA, and 2 mM Hepes-Tris
of pH 7.4). The top layer was pinned with the help of
needles on the dissection table. Mucosal scrapings were
hanging in 10 volumes of Krebs ringer buffer (pH = 7.4)
containing sucrose (250 Mm) and homogenized with
20 strokes of a mortar driven Teflon pestle homogenizer.
The tissues were discarded and the filterate was subjected to
sub cellular fractionation. The pellets so obtained were
dissolved in 2 mL of sucrose-EGTA buffer and was used as
enzyme sample.
Hence, with this observations and earlier investigations
from our research group (Rakesh et al. 2015a, 2016a,
2016b, 2015b), the present work was undertaken with a
view to assess the H+/K+-ATPase and anti-inflammatory
agents of the title compounds.
Experimental
Materials
Protein estimation
Protein was calculated by using Lowry method (Lowry
et al. 1951) and bovine serum albumin as standard (0–75
µg). Eight clean and dry test tubes were taken and aliquots
of various concentrations of the synthesized derivatives
were made. To the 7th and 8th test tube, the unknown
sample (5 and 10 µL of the cells isolated from the sheep
stomach) for which the protein content was added. In every
test tube, the solution was made up to 1 mL by the addition
of distilled water followed by the addition of 5 mL of
Lowry’s reagent. All the test tubes are incubated at room
temperature (rt) for 8–10 min and 0.5 mL of Folin-Ciocalteu
reagent was added and again incubation at rt for 30 min.
Absorbance of each solution was read at 670 nm against the
blank solution. A graph was plotted by using concentration
of protein on x-axis and OD on y-axis. From the standard
graph obtained, the unknown concentration of protein
sample was calculated and found to contain 21 mg of pro-
tein per 8 g of tissue homogenate.
All chemicals and reagents obtained from Sigma Aldrich
(India), Merck (India), and Avra Synthesis (India) were
used without further purification. Melting points were
determined on a Superfit melting point apparatus (India) and
are uncorrected. 1H nuclear magnetic resonance (NMR)
(400 MHz) and 13C NMR (100 MHz) spectra were recor-
ded on a Agilent Technologies (USA) using dimethyl
sulfoxide (d6) or CDCl3 as solvent. High-resolution mass
spectroscopic analysis was performed on a Bruker Micro-
TOF QII mass spectrometer in positive mode. Progress of
the reaction was monitored by TLC using silica gel 60
F254, with the solvent system comprising hexane and
ethylacetate in the ratio 04:01 and the compounds on the
TLC plates were detected by under ultraviolet light and
iodine vapor.
General synthesis procedure of N’-substituted ureas
(1–24)
Inorganic phosphorus estimation
The ureas (1–24, 1 mmol) was separately dissolved in 5 mL
of THF and added anhydrous sodium hydride (1 mmol) to
the solution, after 5 min, isocyanates (1 mmol) were added
Inorganic phosphorus was estimated according to literature
reported method (Fiske and Subbarow 1925). Aliquots of
working standard solution (40 µg/mL) were added into eight

Med Chem Res
fresh and dry test tubes in the volume of 0 to 1 mL; 5 and
10 µL of the enzyme sample were taken in test tube 7th and
8th, respectively. The volumes of all test tubes were made
up to 8.6 mL using 10% trichloroacetic acid (TCA).
Ammonium molybdate (1 mL) and 8-Anilino-1-naphthale-
nesulfonic acid ammonium salt, reagent (0.4 mL) were
added to all test tubes. All test tubes were allowed to stand
for 10 min at rt. Then the color was developed and read at
its λmax of 660 nm.
solution alone. The mixtures were incubated for 10 min at
room temperature, centrifuged for 10 min at 3000 rpm and
supernatant was measured by spectrophotometrically at 540
nm. The % inhibition of haemolysis was calculated from the
following formula.
ꢀ
ꢁ
A1 ꢀ A2
% Inhibition of haemolysis ¼
ꢁ 100
A1
Where:
A1 = Absorbance of hypotonic buffered solution alone
A2 = Absorbance of test /standard sample in hypotonic
ATPase activity
solution
ATPase activity was determined as described by using
reported method (Im et al. 1985). Basal Mg2+ dependent
ATPase activity was calculated in 1 mL of the reaction
includes of 2 mmol/L ATP and 50 mmol/L Tris-HCl buffer
Results and discussion
Chemistry
−
(pH = 7.5). K+ stimulated and HCO3 stimulated ATPase
activity in the basal medium. The ATPase reaction was
started by the addition of the substrate (ATP), carried out at
37 °C for 10–15 min and closed with 1 mL cold 20% TCA.
Liberated inorganic phosphate from ATP was estimated by
using literature method (Fiske and Subbarow 1925).
Activity of the enzyme sample in the presence of ATP is
0.066 µmoles.
All the urea are prepared as per our recently reported pro-
tocol (Rakesh et al. 2016a, 2016b), amides are reacted with
isocyante in presence of sodium hydride at room tempera-
ture for 1–2 h. Corresponding urea obtained in reasonable to
excellent yield (Scheme 1) and obtained products are well
characterized with NMR and mass spectroscopic techni-
ques. Obtained derivatives are tabulated Table 1.
Activity of the enzyme sample in the absence of ATP is
0.042 µmoles.
100% activity of the enzyme is 0.066–0.042 µmoles =
0.024 µmoles.
Biological activity
H+/K+-ATPase activity
Anti-inflammatory activity
We have synthesized disubstituted urea’s (1–24) from this
work and tested for their capacity to reduce H+/K+-ATPase
using standard drug omeprazole. The obtained results (IC50
values) are tabulated in Table 1 and the data represents
average values from triplicate runs. A close test of the
outcome results revealed some interesting facts with respect
to structure–activity relationship. It is evident from the
results that electron-donating and electron-withdrawing
groups is important for inhibition of H+/K+-ATPase
activity. Investigations have shown that H+/K+-ATPase
inhibition is associated with the modification of electron-
donating and electron-withdrawing groups in the benzene
ring, this could be the reason for higher activity of electron-
donating (OH, OCH3, and CH3) analogue compared to
electron-withdrawing (Cl and F) analogues. Based on our
Human erythrocyte suspension
The human blood was collected from a healthy volunteer
who had not taken any NSAIDs for 2 weeks prior to the
experiment and collected in heparinzed vacutainer. The
collected healthy human blood was washed 0.9% saline and
centrifuged for 10 min at 3000 rpm. The packed cells were
washed with 0.9% saline and 40% v/v suspension made by
isotonic phosphate buffer of 154 mM NaCl in 10 mM
sodium phosphate buffer at pH 7.4 used as Stock ery-
throcyte or RBC suspension.
Hypotonic solution-induced haemolysis
The activity of the synthesized compounds was performed
according to the reported method (Shinde et al. 1999). The
test sample consisted of stock erythrocyte (RBC) suspen-
sion 0.5 mL mixed with 5 mL of hypotonic solution (50 mM
NaCl in 10 mM sodium phosphate buffered saline at pH
7.4) containing different concentrations of sample (20, 40,
60, 80, and 100 µg/mL). The control consists of 0.5 mL
RBC suspension mixed with 5 mL of hypotonic buffered
Scheme 1 Synthesis of symmetrical and unsymmetrical urea from
sodium hydride and isocyanate

Med Chem Res
Table 1 Antiulcer activity and anti-inflammatory activities of the synthesized compounds
Entry
1
Structure
Antiulcer activitya (IC50 µg/mL)
Anti-inflammatory activitya (IC50 µg/mL)
62.4 1.23
34.4 0.12
2
3
58.5 0.45
52.3 1.24
28.3 1.24
20.7 1.74
4
5
6
7
78.8 0.75
70.4 1.44
66.1 0.58
36.1 1.47
60.6 1.07
56.3 0.12
52.0 0.73
48.3 0.43
8
9
30.0 0.33
22.9 0.14
50.1 0.92
48.2 0.47
10
11
Nil
Nil
Nil
Nil

Med Chem Res
Table 1 continued
Entry
12
Structure
Antiulcer activitya (IC50 µg/mL)
Anti-inflammatory activitya (IC50 µg/mL)
90.5 0.49
86.1 1.44
13
14
34.3 0.14
32.2 1.40
50.1 0.14
42.2 0.12
15
16
36.7 0.58
30.1 0.11
42.1 0.44
44.4 0.24
17
18
34.9 0.11
10.1 0.45
38.2 0.47
36.1 0.14
19
38.4 0.22
58.1 0.41
20
21
34.3 0.43
18.4 0.14
50.6 0.41
46.7 0.17
22
60.4 0.88
40.0 0.42

Med Chem Res
Table 1 continued
Entry
23
Structure
Antiulcer activitya (IC50 µg/mL)
Anti-inflammatory activitya (IC50 µg/mL)
56.1 0.47
36.4 0.41
24
48.5 1.54
20.3 0.61
Omerazole
38.2 0.85
–
Indomethacin
–
44.8 0.25
a
Values are mean of three determinations, the ranges of which are <5% of the mean in all cases
earlier results (Rakesh et al. 2016), compounds with
electron-donating groups on the phenyl ring showed
excellent activity. Compounds with electron-withdrawing
groups on phenyl ring showed moderate or less activity.
The same development has been noticed here also.
sides of aliphatic groups (10 and 11) are also showed very
less or poor activity.
Conclusion
Among the derivatives, diphenyl urea 6 displayed
activity with IC50 = 66 μg/mL which is less potent than
omeprazole (IC50 = 38 μg). It can be inferred that com-
pounds without substitution on the aryl ring showed lesser
activity. Therefore, the effect of substituents on the phenyl
ring and different aromatic and aliphatic isocyanates roles
was further investigated. When a methoxy, hydroxyl, and
methyl groups was placed on the phenyl ring, derivatives
exhibited good activity. Compound 7–9 and 13–21 showed
good activity with IC50 values are lower than that of the
reference standard drug omperazole (38 μg/mL). Further,
the substitution of halogens (Cl and F), compounds (1–3
and 22–24) showed moderate or least H+/K+-ATPase
activity. We have also interestingly to design both sides of
aliphatic groups (10 and 11) and tested the activity, the
obtained results are nil or poor H+/K+-ATPase activity.
In the present study, we have tested for their in vitro H+/K+
ATPase and anti-inflammatory activity of disubstituted
urea’s analogues. Compounds 7–9 and 13–21 with OH,
OCH3, and CH3 groups in benzene ring showed excellent
H+/K+ ATPase activity than omperazole. Compounds 1–3
and 22–24 with Cl and F in benzene ring showed good anti-
inflammatory activity than indomethacin. Finally, we have
synthesized simple and small active analogues, which
shown potent H+/K+ ATPase and anti-inflammatory
activity.
Acknowledgements We are thankful to SRI RAM CHEM man-
agement for their continuous encouragement towards research.
Conflict of interest The authors declare that they have no competing
interests.
In vitro anti-inflammatory activity
References
All the synthesized compounds were further tested for their
in vitro anti-inflammatory activity and results are presented
in Table 1. A few numbers of compounds have been noticed
for good to moderate activity compared to standard drug
indomethacin. The compounds 1–3 and 22–24 showed
excellent activity with IC50 values are lower than the stan-
dard indomethacin (42 µg/mL). It is evident from the results
that the compounds containing electron-withdrawing groups
Cl and F (1–3 and 22–24) are good anti-inflammatory
agents and electron-donating groups OH and OCH3 (7–8
and 19–21) are less activity and methyl (CH3) groups
(13–18) in the molecule showed moderate activity. Two
Anderson JW, Sarantakis D, Terpinski J, Kumar TR, Tsai HC, Kuo M,
Ager AL et al. (2013) Novel diaryl ureas with efficacy in a mouse
model of malaria. Bioorg Med Chem Lett 23:1022–1025
Cho HJ, EI-Gamal MI, Oh CH, Lee SH, Kim G, Jun HH, Hong SC,
Kyung HY (2012) Synthesis and antiproliferative activity of new
aminoisoquinolinylurea derivatives against melanoma cell line.
Bull Kore Chem Soc 33:3635–3639
Fiske CH, Subbarow Y (1925) The colorimetric determination of
phosphorous. J Biol Chem 66:375–400
Forte JG, Machen TF, Obrink KJ (1980) Mechanism of gastric H+ and
-Cl transport. Annu Rev Physiol 42:111–126
Fournier J, Bruneau C, Dixneuf H, Lécolier S (1991) Ruthenium
catalyzed synthesis of symmetric N, N′-disubstituted ureas

Med Chem Res
directly from carbon dioxide and amines.
56:4456–4458
Gulkok Y, Bicer T, Onurdag K, Ozgen S, Sahin MF, Dogruer DS
(2012) Synthesis of some new urea and thiourea derivatives and
J
Org Chem
Rakesh KP, Ramesha AB, Shantharam CS, Mantelingu K, Mallesha N
(2016a) An unexpected reaction to methodology: an unprece-
dented approach to transamidation. RSC Adv 6:108315–
108318
evaluation of their antimicrobial activities. Turk
36:279–291
J
Chem
Rakesh KP, Shantharam CS, Manukumar HM (2016b) Synthesis and
SAR studies of potent H+/K+-ATPase inhibitors of quinazoli-
none-schiff’s base analogues. Bioorg Chem 68:1–8
Rakesh KP, Suhas R, Chandan S, Manukumar HM, Gowda DC
(2015b) Quinazolinones linked amino acids derivatives as a new
class of promising antimicrobial, antioxidant and anti-
inflammatory agents. Eur J Chem 6:254–260
Gustavsson S, Adam HO, Loof L, Nyberg A, Nyren O (1983) Rapid
healing of duodenal ulcers with omeprazole: double-blind dose-
comparative trial. Lancet 322:124–125
Hla T, Neilson K (1992) Human cyclooxygenase2 Cdna. Proc Natl
Acad Sci 89:7384–7388
Im WB, Sih JC, Blakeman DP, McGrath JP (1985) Omeprazole, a
Ramesh D, Gangadhar S, Katti SA (2013) Synthesis, characterization
and anticonvulsant activity of 1-(aryl)-3-(diphenylmethyl) urea
derivatives. Int J Res Pharm Chem 3:748–752
specific inhibitor of gastric H+/K+-ATPase, is a H+-activated
oxidizing agent of sulfhydryl groups.
J
Biol Chem
260:4591–4597
Sachs G, Chang HH, Rabon E, Shackman R, Lewin M, Saccomani G
(1976) A noneletrogenetic H+ pump in plasma membranes of hog
stomach. J Biol Chem 251:7690–7698
Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ,
Saraf MN (1999) Membrane stabilizing activity a possible
mechanism of action for the anti-inflammatory activity of Cedrus
deodara wood oil. Fitoterapia 70:251–257
Umadevi P, Deepti K, Srinath I, Vijayalakshmi G, Tarakaramji M
(2012) Synthesis and in-vitro antibacterial activity of some new
urea, thiourea and thiosemicarbazide derivatives. Int J Pharm
Pharma Sci 4:379–383
Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegeman RA,
Pak JY, Gildehaus D et al. (1996) Structural basis for selective
inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nat
New Biol 384:644
Lind T, Cederberg C, Eberved G, Haglund U, Olbe L (1983) Effect of
omeprazole –a gastric proton pump inhibitor –on pentagastrin
stimulated acid secretion in man. Gut 24:270–276
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein
measurement with the folin phenol reagent. J Biol Chem
193:265–275
Lu CS, Tang K, Li Y, Jin B, Yin DL, Chen XG, Ma C, Huang HH
(2013) Synthesis and in vitro antitumor activities of novel benzyl
urea analogues of sorafenib. Yao Xue Xue Bao 48:709–717
Rajtar B, Szacon E, Swiatek L, Rzadkowska M, Matosiuk D, Mal-
gorzata PD (2013) Antiviral activity of 1-(1-arylimidazolidine-2-
ylidene)-3-(4-chlorobenzyl)urea derivatives. J Pre Clin and Clin
Res 7:104–106
Rakesh KP, Manukumar HM, Gowda DC (2015a) Schiff’s bases of
quinazolinone derivatives: synthesis and SAR studies of a novel
series of potential anti-inflammatory and antioxidants. Bioorg
Med Chem Lett 5:1072–1077
Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism
of action for aspirin like drugs. New Biol 231:232–235
Viana GM, Aguiara LC, Ferrão JA, Simas ABC, Vasconcelos MG
(2013) The use of aqueous potassium dichloroiodate for the
synthesis of ureas. Tetra Lett 54:936–940
Vijayakumar S, Bongu S, Gomathi P (2013) Anti-ulcerogenic activity
of unsymmetrically substituted urea derivative (1-benzyl-3-(4-
methylphenyl) urea. Afri J Pharm Pharmacol 7:2522–2527
Yokoyama C, Tanabe T (1989) Cloning of human gene encoding
prostaglandin endoperoxide synthase and primary structure of the
enzyme. Biochem Biophys Res Commun 165:888–894