Novel thymol bearing oxypropanolamine derivatives as potent some metabolic enzyme inhibitors – Their antidiabetic, anticholinergic and antibacterial potentials

Article abstract of DOI:10.1016/j.bioorg.2018.08.003

A series of classical and newly synthesized thymol bearing oxypropanolamine compounds were synthesized and characterized. Their in vitro antibacterial activity on A. baumannii, P. aeruginosa, E. coli and S. aureus strains were investigated with agar well

Full text of DOI:10.1016/j.bioorg.2018.08.003

Accepted Manuscript
Novel Thymol Bearing Oxypropanolamine Derivatives as Potent Some Meta-
bolic Enzyme Inhibitors - Their Antidiabetic, Anticholinergic and Antibacterial
Potentials
Mustafa Zengin, Hayriye Genc, Parham Taslimi, Ali Kestane, Ertugrul Guclu,
Aziz Ogutlu, Oguz Karabay, İlhami Gulçin
PII:
S0045-2068(18)30564-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.08.003
YBIOO 2460
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 June 2018
1 August 2018
3 August 2018
Please cite this article as: M. Zengin, H. Genc, P. Taslimi, A. Kestane, E. Guclu, A. Ogutlu, O. Karabay, I. Gulçin,
Novel Thymol Bearing Oxypropanolamine Derivatives as Potent Some Metabolic Enzyme Inhibitors - Their
Antidiabetic, Anticholinergic and Antibacterial Potentials, Bioorganic Chemistry (2018), doi: https://doi.org/
10.1016/j.bioorg.2018.08.003
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Novel Thymol Bearing Oxypropanolamine Derivatives as Potent Some
Metabolic Enzyme Inhibitors - Their Antidiabetic, Anticholinergic and
Antibacterial Potentials
Mustafa Zengina, Hayriye Genca, Parham Taslimib*, Ali Kestanea, Ertugrul Gucluc,
Aziz Ogutluc, Oguz Karabayc, İlhami Gulçinb
aSakarya University Science and Arts Faculty Chemistry Department 54187-Serdivan Sakarya, Turkey
bDepartment of Chemistry, Faculty of Science, Atatürk University, 25240-Erzurum, Turkey
cSakarya University Faculty of Medicine Infectious Diseases and Clinical Microbiology Department
54290-Adapazarı Sakarya, Turkey
Address for Correspondences:
*Dr. Parham Taslimi
Faculty of Science
Department of Chemistry
Ataturk University
25240-Erzurum, Turkey
Phone
Fax
: +905546381920
: +904422314109
E-mails
: parham_taslimi_un@yahoo.com
parhamsmkh66@gmail.com

Abstract
A series of classical and newly synthesized thymol bearing oxypropanolamine compounds
were synthesized and characterized. Their in vitro antibacterial activity on A. baumannii, P.
aeruginosa, E. coli and S. aureus strains were investigated with agar well diffusion method.
The results were compared with commercially available drug active compounds. As well as
3a, 3b and 3c have the most significant antibacterial effect among all the tested compounds;
approximately all of them have more antibacterial activity than the reference drugs. These
novel thymol bearing oxypropanolamine derivatives were effective inhibitors of the α-
glycosidase, cytosolic carbonic anhydrase I and II isoforms (hCA I and II), and
acetylcholinesterase enzymes (AChE) with Ki values in the range of 463.85-851.05 µM for α-
glycosidase, 1.11–17.34 µM for hCA I, 2.97-17.83 µM for hCA II, and 13.58-31.45 µM for
AChE, respectively.
Key words: Oxypropanolamine; antibacterial effects; α-glycosidase; carbonic anhydrase;
acetylcholinesterase
2

1. Introduction
Through their therapeutic potential, natural products are a unique inspiration for scientists
working on drug design and synthesis [1]. Especially essential oils have a major task to
prolong the lifespan of plants by protecting them from various hazards like bacteria, viruses,
fungi or pests [2,3]. Thymol which is the most commonly used antibacterial compounds
among essential oils [4,5]. It is a naturally occurring phenolic monoterpene [6] especially
present in thyme (Thymus capitatus) [7], pepper-Rosmarin (Lippia sidoides) [8] and Labiatae
family [9]. At the present time, thymol containing compounds are continued to investigate for
various effects such as obesity [10], anti-inflammatory, cicatrizing [11], and anti-leishmanial
[12]. Oxypropanolamines are known as β-adrenoceptor antagonists for a long time [13,14].
They used in especially cardiovascular disease, and also used in the treatment of many
diseases such as hypertension, thyrotoxicosis, angina pectoris, chronic pulmonary diseases
[15], skin inflammation [16] and diuretic [17].
It may estimate that molecules containing a nucleus (thymol) that has been used for
many years as a drug and on the other side containing a group with strong antibacterial
properties (oxypropanolamine) are likely to have biological activity. Compounds 3a, 3b, and
3e are newly synthesized and compounds 3c [18] and 3d [19] has been already known as β-
adrenergic blocking agent drugs.
Carbonic anhydrases (CAs) are metalloenzymes that participate in the conservation of
pH homeostasis in the cells by catalyzing the reversible hydration of carbon dioxide (CO2) to
-
protons (H+) and bicarbonate ions (HCO3 ) [20]. CA isoforms are discovered in all three
domains of life and these enzymes belong to diverse families: α-, β-, γ-, δ-, ζ-, η- and θ-CAs.
α-CA isozymes exist in many organisms, such as fungi cells (together with β-CAs). So far,
sixteen isozymes of CA have been recorded in mammal cells that differ in their catalytic
activity, subcellular localization, and susceptibility to various types of inhibitor compounds
[21-23]. β-CA isozymes are also found in archaea, plants, and both prokaryotes and
eukaryotes [24]. γ-CA isozymes are found in bacteria cells. ζ-CAs and δ-CA isoforms exist in
some marine planktonic kinds [25]. On the other hand, η-CAs and θ-CAs are found in malaria
causative agent, Plasmodium falciparum, and in marine diatom Phaeodactylum tricornutum,
respectively [26,27]. Between these isozymes, the human cytosolic hCA I and II isozymes are
ubiquitous in the human body and are participate in the secretion of electrolyte cells in a
plenty of tissues, such as the cerebrospinal fluid, as well as maintain pH and CO2 homeostasis
-
all over the body or the HCO3 -rich aqueous humor in the anterior chamber of the eyes [28].
3

Hence, perturbation of activity of hCA I and II in tissues give rise to pathologic situations,
such as edema or glaucoma. Therefore, hCA I and II became goals for anti-glaucoma,
diuretic, and anticonvulsant drugs [29,30].
To date, Alzheimer’s disease (AD) has no cure, and the illness is an outcome of
dementia caused by the reduction in the transition of nerve impulses due to neurodegeneration
[31,32]. Thus, the real therapies are based on drug compounds that leverage the transition of
electrical impulses. One method to improve nerve transition is to decrease or inhibit the
activity of the acetylcholinesterase (AChE) enzyme [33]. This important enzyme is
accountable for reducing the levels of acetylcholine (ACh) in the synaptic gap. Reducing the
activity of AChE, keeps most ACh levels in the gap, thus raising the transmission of the
electric impulses [34]. Indeed, the AChE inhibitors (AChEIs) are capable of improving the
advance of the AD, increasing patient’s quality of life, and improving the cognitive capability
of the patient, without, however, hampering the development of the disease [35].
The α-glycosidase enzyme is essential for carbohydrate digestion because these
molecules must be hydrolyzed by α-glycosidase enzyme in order to be absorbed in the small
intestine [36]. The inhibition act of α-glycosidase enzyme reduces the mechanism of
carbohydrate digestion and avoids postprandial hyperglycemia, which is a main reason of
chronic diabetes-associated complication [37]. Hence, many endeavors have been made to
extend an α-glycosidase inhibitor as a therapeutic factor for the suppression of metabolic
disturbance, like obesity, hyperglycemia, and non-insulin-dependent type II diabetes mellitus
(T2DM) [38].
In this study, we have performed the facile synthesis of well-defined novel thymol
bearing oxypropanolamine derivatives (2 and 3a-e) and also we investigated their
antibacterial properties. In addition of this study, we have investigated their inhibition
potential of hCA I and II isoenzymes, and to discover the most favorable and potent AChE,
and α-glycosidase inhibition properties of these compounds to give directions to further
studies.
2. Results and Discussion
2.1. Chemistry and Biology
4

Different strains susceptibility from each bacteria (A. baumannii, P. aeruginosa, E. coli, and
S. aureus) against thymol containing compounds was investigated. All strains except some E.
coli strains were multidrug resistance bacteria. Antibiotic resistance profiles of these strains
are shown in Tables 1-4, respectively. Each synthesized compound showed varied levels of
antibacterial activity against the tested bacterial strains (Figure 1).
The intermediate epoxide structure 2 has low antibacterial effect against A. baumannii.
On the other hand, the target products, 3a-e have a quite high antibacterial effect. When the
results obtained from bacteria strains are evaluated according to their average inhibition zone
diameter values, 3a-e molecules have inhibition zone diameter of 26, 30, 26, 23, and 17 mm
respectively. It was seen that commercial drugs used to treat A. baumannii bacteria did not
have any inhibition effect (Figure 1A).
Figure 1
The intermediate epoxide derivative, 2 did not show any effect against P. aeruginosa.
On the other hand, compounds 3b and 3c had > 15 mm inhibition zone diameter for all P.
aeruginosa strains. Similarly, compound 3a had > 15 mm inhibition zone diameter for P.
aeruginosa strains except for one strain. Compounds 3a-e had an average inhibition zone
diameter of 14-26 mm. As with A. baumannii, 3b showed the highest activity and 3d showed
the lowest activity (Table 2).
The measurable inhibition zone diameter was detected in the compound 2 for 13 of E.
coli strains. Compound 3a and 3c have ≥ 20 mm inhibition zone diameter in all tested E. coli
stains (Figure 1B). The compounds 3a-e had average inhibition zone diameter of 26, 20, 25,
21 and 13 mm respectively. Unlike other bacteria, the most active compound in E. coli strains
is 3a with inhibition zone diameter of 26 mm and 3c with inhibition zone diameter of 25 mm.
The least effective compound is 3e. Thirteen isolates of S. aureus strains were studied with
compounds 3a-e. All oxypropanolamine derivatives, 3a-e demonstrate ≥ 20 mm inhibition
zone diameter. However, 3b and 3a showed the best activity.
Antibacterial resistance has become an increasingly serious medical problem for the
world [39,40]. There are even some types of bacteria such as Pseudomonas spp, Klebsiella
spp, Escherichia coli, Acinetobacter spp, Enterobacter spp, and Enterococcus spp. that are
out of treatment options. Nowadays, we need more new antibacterial agents than in the past
[41]. Mean inhibition zone diameters of the synthesized compounds against A. baumannii
were determined as in the range of 22-30 mm except the intermediate 2 and bearing pyridine
5

derivatives 3e. Antibacterial superiority of synthesized molecules against commercially
available drugs has been seen in Figure 1A. The mean zones of inhibition observed by the
synthesized compounds ranged between 14-24 mm for P. aeruginosa except the intermediate
2 and 3e bearing pyridine derivative (Figure 1C). The similar lower inhibition zone diameter
of the intermediate 2 and pyridine bearing derivative 3e also determined in the inhibition of E.
coli. Apart from this other derivative have in the range of 18-26 mm inhibition zone diameter
against E. coli (Figure 1B).
The average inhibition zone diameter of all phenoxypropanolamine derivatives on S.
aureus is > 19 mm except the intermediate 2. Three phenoxypropanolamine derivatives
having the strongest antibacterial effects on S. aureus with 27, 26 and 24 mm inhibition zone
diameter are 3b, 3a, and 3d respectively (Figure 1D).
Herein, the antibacterial activities of oxypropanolamines containing tymol structure
were investigated. Thymol is already known as antibacterial compounds [42,43]. To synthesis
oxypropanolamine derivatives, firstly the oxirane derivative, 2 were synthesized by reacting
epichlorohydrin with Thymol. However, the antibacterial activity of intermediate 2 is low
against all tested bacteria. This actually was an expected result because a phenolic structure
with high antibacterial activity turns to an epoxide structure with lower antibacterial activity.
In the second step, the oxirane ring of 2 was opened with an amine derivative to synthesis
oxypropanolamine derivatives. These oxypropanole amines have shown powerful
antibacterial effects on Gram-negative and Gram-positive bacteria. 3b which is
phenoxypropanolamine derivative bearing cyclohexenyl group have the highest antibacterial
activity among other derivatives on all of bacteria except E. coli strains. It created about 30
mm inhibition zone diameter on A. baumannii > 25 mm inhibition zone diameter on P.
aeruginosa and S. aureus strains.
The second most powerful antibacterial is 3a compound consisting of allylamine
structure. Although it has a moderate activity on P. aeruginosa with 19 mm inhibition zone
diameter, it has > 25 mm inhibition zone diameter on S. aureus and A. baumanni.
Additionally, this compound has the highest activity on E. coli with 26 mm inhibition zone
diameter. Both 3a and 3b are molecules containing an isolated double bond. The strong
antibacterial effects of 3a and 3b can be related to this double bond. The compound 3c
carrying tertbutyl amino group also has approximately similar effects like 3a and 3b. It was
the second effective molecule on P. aeruginosa and E. coli while the third on A. Baumannii
and forth on S. aureus. The oxypropanolamine derivative containing diethyl amine 3e has an
average antibacterial effect on S. aureus, A. baumannii and E. coli stains with 20 mm
6

inhibition zone diameter while having a bit low activity on P. aeruginosa with < 15 mm
inhibition zone diameter. On the other hand, the molecule having the lowest antibacterial
effect was 3e containing 3-picolylamine. While any antibacterial effect of 3e was not
observed against P. aeruginosa, it also has very low antibacterial effect on E. coli (inhibition
zone diameter < 15 mm). According to these results, the newly produced products 3a, 3b, and
3c have significant antibacterial effect. However, in vivo tests are required for the
effectiveness and adverse effects of these products. These materials can be used locally and
parenterally for their natural structure.
2.2. Biochemical Studies
The heterocyclic/aromatic sulfonamide compounds and their bioisosters such as sulfamides
and sulfamates have been developed as CA inhibitors (CAIs) due to their high affinity for
most of the CA isoenzymes [44,45]. Some of the sulfonamides comprising drugs, which are
utilized clinically for many years as antiglaucoma and diuretics agents such as,
methazolamide, acetazolamide, dichlorphenamide, dorzolamide, ethoxzolamide and
brinzolamide [46]. All the synthesized molecules (novel thymol bearing oxypropanolamine
derivatives (2, and 3a-e)) were tested to investigate their inhibitory activity towards the
slower cytosolic hCA I, the more rapid cytosolic hCA II isoenzymes, α-glycosidase, and
AChE enzymes. The chemical formula of novel thymol bearing oxypropanolamine
derivatives (2, and 3a-e) is given in Scheme 1 and their AChE, α-glycosidase, and CA I and II
isoforms inhibition data are summarized in Table 1. Novel thymol bearing oxypropanolamine
derivatives (2, and 3a-e) showed effective inhibition profiles against the enzymes mentioned
above. The following results are presented in Table 1.
Cytosolic hCA I, and II isoenzymes are both existed in erythrocyte cells, which are
indispensable items for regulating the physiological pH of the blood via manufacturing of
‒
HCO3 [47]. The cytosolic hCA I isoenzyme was inhibited by the novel thymol bearing
oxypropanolamine derivatives (2, and 3a-e), with Ki values were found between 1.11±0.16
and
17.34±0.94
µM.
In
addition,
1-(tert-butylamino)-3-(2-isopropyl-5-
methylphenoxy)propan-2-ol (3c), and 1-((2-(cyclohex-1-en-1-yl)ethyl)amino)-3-(2-isopropyl-
5-methylphenoxy)propan-2-ol (3b) recorded the most powerful hCA I isoform inhibition
properties with Ki values of 1.11±0.16 and 1.51±0.23 µM, respectively. The clinically and
standard used drug acetazolamide (AZA) demonstrated a Ki value of 46.17±4.17 µM. Thus,
7

the evaluated novel thymol bearing oxypropanolamine derivatives (2, and 3a-e) showed better
inhibitory profiles when compared to AZA molecule (Table 1 and Figure 2).
hCA II isoenzyme has been dependent on various transporters including the Na+/H+
‒
‒
exchanger, the Cl‒/HCO3 exchanger, and the Na+/HCO3 cotransporter [48]. Indeed, hCA II
isoenzyme is mostly associated with multiple diseases such as glaucoma, renal tubular
acidosis, and osteoporosis [49]. It is the most largely characterized and studied CA
isoenzyme. The reults clearly showed that hCA II was impressively inhibited by the novel
thymol bearing oxypropanolamine derivatives (2, and 3a-e). These compounds had strong
hCA II inhibition with Ki values ranging from 2.97±0.43 µM to 17.83±3.58 µM. Ki values of
novel molecules are better than those of the standard used drug AZA (Ki: 39.15±7.11 µM).
All the evaluated novel thymol bearing oxypropanolamine derivatives (2, and 3a-e) showed
potent inhibition against hCA II, but the compounds of 1-((2-(cyclohex-1-en-1-
yl)ethyl)amino)-3-(2-isopropyl-5-methylphenoxy)propan-2-ol (3b), and 1-(tert-butylamino)-
3-(2-isopropyl-5-methylphenoxy)propan-2-ol (3c) showed significant inhibition profile
against hCA II with Ki values of 2.97±0.43 and 5.48±2.04 µM (Table 1 and Figure 2).
Recently, our research group has reported the synthesis and biological evaluation of some
N,N′-bis-cyanomethylamine and alkoxymethylamine derivatives, 4-[5-aryl-3-(thiophene-2-
yl)-4,5-dihydro-1H-pyrazol-1-yl]benzenesulfonamides, pyrimidine-thiones, chalcone-imide
derivatives, phloroglucinol derivatives, pyridine compounds containing bis-1,2,4-triazole
moiety, and N-propylphthalimide and 4-vinyl benzyl substituted benzimidazole salts [49-53]
as hCA I, II, and AChE inhibitors, which showed excellent inhibition profile for both CA
isoforms.
The inhibition of AChE enzyme results in the obstruction of ACh hydrolysis. Hence, the
design of modulator or inhibitor for AChE enzyme is of major interests since it is commonly
one of the most favored aims to hamper AD, resulting in three commercial drugs approved by
FDA such as rivastigmine, galantamine, and donepezil [54-56]. Many potential inhibitor
compounds of AChE enzyme are under development such as derivative compounds from
tacrine derivatives and Zijuan tea. Some of them are under clinical evaluation such as
huperzine A and ganstigmine [57,58]. The inhibitory effects of the novel thymol bearing
oxypropanolamine derivatives (2, and 3a-e) on AChE are shown in Table 1. The AChE
inhibition profiles of the compounds evaluated here were quite interesting. Overall, the novel
thymol bearing oxypropanolamine derivatives (2, and 3a-e) had excellent inhibitory activity
with Ki values ranging from 13.58±3.47 µM to 31.45±7.37 µM. Furthermore, tacrine, used as
8

a standard AChE inhibitor in this study, demonstrated Ki value of 54.18±9.37 µM toward
AChE. As these results show, the inhibition of AChE of novel thymol bearing
oxypropanolamine derivatives (2, and 3a-e) is much better than standard drugs and the newly
synthesized compounds. The compounds of 1-(diethylamino)-3-(2-isopropyl-5-
methylphenoxy)propan-2-ol
(3d),
1-(2-isopropyl-5-methylphenoxy)-3-(pyridine-3-
ylmethylamino)propan-2-ol (3e) showed excellent inhibition profile against AChE with Ki
values of 13.58±3.47 and 18.05±5.86 µM, respectively (Table 1 and Figure 2).
Therapeutic drugs such as miglitol, voglibose, and acarbose in current medications are
used as α-glucosidase and α-amylase inhibitors [59]. Intestinal α-glucosidase and pancreatic
α-amylase are significant enzymes in the digestive system and catalyze the first step in the
hydrolyzing the α-1,4-glucoside linkages and the digestion of starch [60]. The inhibition act
of these important enzymes remarkably reduces the digestion and uptake of carbohydrate
molecules, therewith reducing the postprandial blood glucose level in the nation-insulin-
dependent T2DM patients [61]. The major drawback of these drugs is their side effects such
as bloating, meteorism, abdominal distention, possibly diarrhea and flatulence [62]. For the α-
glycosidase enzyme, the novel thymol bearing oxypropanolamine derivatives (2, and 3a-e)
had IC50 values in the range of 418.13–1024.7 and Ki values in the range of 463.85±69.42-
851.05±101.2 µM (Table 1 and Figure 1). The results obviously showed that all novel thymol
bearing oxypropanolamine derivatives (2, and 3a-e) demonstrated efficient α-glycosidase
inhibitory effects than that of acarbose (IC50: 22.80 μM) as a standard α-glycosidase inhibitor
[63,64]. However, the most effective Ki values were obtained by 1-(2-isopropyl-5-
methylphenoxy)-3-(pyridine-3-ylmethylamino)propane-2-ol (3e), and 1-(diethylamino)-3-(2-
isopropyl-5-methylphenoxy)propan-2-ol (3d), with Ki values of 463.85±69.42 and
481.35±94.58 µM, respectively.
Table 1 and Figure 2
3. Conclusions
As a result, this study contains the synthesis of the novel thymol bearing oxypropanolamine
derivatives (2, and 3a-e). Also, all of the compounds synthesized have been characterized by
using the spectroscopic methods of 1H NMR, 13C NMR, and elemental analysis techniques. All
novel synthesized molecules effectively inhibited some metabolic enzymes such as hCA I,
hCA II, α-glycosidase, and AChE enzymes at the nanomolar levels. As we characterized
above, novel thymol bearing oxypropanolamine derivatives (2, and 3a-e) can be acceptable
candidate drugs, the same as CAIs, for therapy of some diseases such as glaucoma, epilepsy,
9

gastric and duodenal ulcers, osteoporosis, mountain sickness, or neurological disturbances. On
the other hand, T2DM is a metabolic difficulty created by high blood glucose content and can
reason other health disturbances, such as neuropathy, weakness, nephropathy retinopathy, high
blood pressure, cardiovascular disease, gangrene, and other dysfunctions. Also, these novel
compounds can be selective inhibitor AChE and α-glycosidase enzymes.
4. Materials and Methods
4.1. Synthesis of 2-((2-isopropyl-5-methylphenoxy)methyl)oxirane (2)
To a solution of thymol (0.5 g, 6.6 mmol) with epichlorohydrin (10 mL) in 85°C was added to
dropwise sodium hydroxide (0.24 g, 6.6 mmol) in water (10 mL) over a period of 0.5 h under
vigorous stirring. The mixture was continued to stir and maintain temperature for until
complete conversion of the initial thymol. The organic phase was mixed with ethylacetate (20
mL) and washed with brine (2x15 mL), dried over MgSO4, and filtered. Organic phase with
excess epichlorohydrin was distilled off under reduced pressure (20-25 mmHg) gave an oily
residue 2 (1.28 g, 94%).
4.2. General synthesis of propan-2-ol amine derivatives (3a-e)
To a solution of 2 (1 mmol) and aqueous saturated solution of K2CO3 (2 mL) was added
amine (3 mmol) in one portion, at room temperature under vigorous magnetic stirring for 5-24
h. The reaction was monitored with thin layer chromatography (TLC). The mixture was
extracted with ethylacetate (10 mL) and washed with water (2x10 mL). The organic layer was
separated and dried over anhydrous MgSO4. The solvent was removed under reduced
pressure. Almost pure product was analyzed by 1H and 13C NMR.
4.2.1.1-(allylamino)-3-(2-isopropyl-5-methylphenoxy)propan-2-ol (3a)
1
Yield 0.25 g, 96%; H NMR (300 MHz, CDCl3) δH 1.21 (d, 6H, J 6.92 Hz), 2.33 (s, 3H),
2.80-2.95 (AB, 2H (Diastereotop), J 12.08, 3.4 Hz), 2.86 (s,OH), 2.90 (s, NH), 3.22-3.35 (m,
1H, J 6.92 Hz), 3.32-3.35 (dd, 2H, J 6.73, 2.90 Hz), 3.95-4.05 (m, 2H, J 9.37, 5.2 Hz), 4.10-
4.17 (m, 1H, J 9.95 Hz), 5.12-5.26 (dd, 2H J 11.12, 9.98, 4.39 Hz ), 5.86-5.99 (m, 1H, J 11.3
13
Hz), 6.5 (s 1H), 6.76-6.79 (d, 1H, J 7.61 Hz ), 7.107.11 (d, 1H, J 7.61 Hz); C NMR (75
MHz, CDCl3) δc, 21.5, 22.9, 23.0, 26.8, 51.7, 52.5, 68.8, 70.8, 112.6, 116.6, 121.8, 126.1,
134.2, 136.6, 136.7, 155.8.
4.2.2. 1-((2-(cyclohex-1-en-1-yl)ethyl)amino)-3-(2-isopropyl-5-methylphenoxy)propan-2-ol
(3b)
10

1
Yield 0.30 g, 92%; H NMR (300 MHz, CDCl3) δH 1.18-1.21 (d, 6H, J 6.91 Hz), 1.52-1.62
(m, 4H, J 11.55, 7.91, 6.01 Hz), 1.92-1.97 (dd, 4H, J 9.37, 6.01), 2.12-2.17 (t, 2H, 6.81, 6.90
Hz), 2.28 (broad, 1H, NH) 2.30 (s, 3H), 2.65-2.70 (dd, 2H, J 11.56, 8.40 Hz), 2.71-2.91 (dt,
2H, J 11.56, 8.58 Hz), 3.24-3.28 (m, 1H, J 6.91), 3.66 (broad, 1H, OH), 3.91-4.01 (tt, 2H, J
11.64, 8.40 Hz), 4.02-4.1 (t, 1H, J 11.64 Hz), 5.46 (d, 1H, J 1.17 Hz), 6.66 (s, 1H), 6.74-6.76
13
(d, 1H, J 7.69 Hz),7.07-7.10(d, 1H, J 7.71 Hz); C NMR (75 MHz, CDCl3) δc 21.5, 22.7,
23.0 (2C), 23.2, 25.5, 26.8, 28.4, 38.5, 47.8, 52.2, 68.6, 70.7, 112.6, 121.7, 123.1, 126.1,
134.2, 135.4, 136.6, 155.8.
4.2.3. 1-(Tert-butylamino)-3-(2-isopropyl-5-methylphenoxy)propan-2-ol (3c)
Yield 0.26 g, 95%; 1H NMR (300 MHz, CDCl3) δH 1.12 (s, 9H), 1.20 (d, 6H, J 7.02 Hz), 2.24
(broad, 1H, NH) 2.30 (s, 3H), 2.72-2.80 (dd, 2H, J 6.74, 3.8 Hz), 2.81 (broad, 1H, OH), 3.30-
3.17 (m 1H, J 7.3 Hz), 3.97-3.91 (dd, 1H, J 7.3 Hz), 4.02-3.97 (dd, 2H, J 5.85, 2.63), 6.57 (s,
1H), 6.67-6.66 (d, 1H, J 7.90 Hz), 7.10-7.05 (d, 1H, J 7.90 Hz); 13C NMR (75 MHz, CDCl3)
δc, 21.5, 23.02 (2C), 26.8, 29.3(3C), 45.0, 50.5, 68.9, 70.8, 112.6, 121.7, 126.1, 134.1, 136.6,
155.8.
4.2.4. 1-(Diethylamino)-3-(2-isopropyl-5-methylphenoxy)propan-2-ol (3d)
Yield 0.25 g, 92%; 1H NMR (300 MHz, CDCl3) δH 1.03-1.07 (t, 6H, J 7.3 Hz), 1.17-1.22 (d,
6H, J 6.73 Hz), 2.20 (broad, 1H, NH) 2.30 (s, 3H), 2.40-2.68 (q, 4H, J 7.32 Hz), 2.69-2.74
(AB, 2H (Diastereotop), J 9.37, 6.15, Hz), 3.15-3.32 (m, 1H, J 6.73 Hz), 3.91-3.99 (m, 1H, J
8.2 9.37 Hz), 4.00-4.23 (dd, 2H, J 6.15, 8.2 Hz), 6.66 (s, 1H), 6.72-6.75 (d, 1H, J 7.61 Hz)
7.04-7.09 (d, 1H, J 7.61 Hz); 13C NMR (75 MHz, CDCl3) δc, 12.2(2C), 21.5, 22.9 (2C), 23.0,
47.5 (2C), 56.3, 66.3, 70.5, 112.5, 121.6, 126.1, 134.2, 136.5, 156.0.
4.2.5. 1-(2-isopropyl-5-methylphenoxy)-3-(11yridine-3-ylmethylamino)propan-2-ol (3e)
1H NMR (300 MHz, CDCl3) δH 1.21 (d, 6H, J 6.92 Hz), 2.33 (s, 3H), 2.86-2.91 (dt, 2H, J
11.56, 8.58 Hz), 2.88-2.96 (AB, 2H (Diastereotop), J 12.08, 3.4 Hz), 3.22-3.30 (m, 1H), 3.86
(s, 2H), 3.98-4.01 (m, 2H), 4.16-4.18 (m, 1H), 6.67 (s, 1H), 6.75-6.78 (d, 1H, J 7.61Hz), 7.08-
7.10 (d, 1H, J 6.71 Hz), 7.25-7.29 (dd, 1H,J 7.75-6.85 Hz), 7.70-7.73 (d, 1H, J 7.77 Hz),
13
8.57(s, 1H); C NMR (75 MHz, CDCl3) δc 21.54, 23.01, 26.80, 51.45, 51.9, 68.96, 70.73,
112.64, 121.87, 123.76, 126.15, 134.15, 135.66, 136.23, 136.68, 148.68, 149.76, 155.72.
4.3. Strains and Growth Conditions
11

Each novel compound was tested with four different bacteria including A. baumannii, P.
aeruginosa, E. coli, and S. aureus strains. All bacteria were obtained from Sakarya University
Faculty of Medicine Infectious Diseases and Clinical Microbiology Department Laboratory
stocks. These stock strains were isolated from various clinics ‘hospitalized patients’ clinical
specimens, such as urine, sputum, and pus, which were sent for laboratory identification. The
antibiotic susceptibility profiles of all isolates were assessed by Kirby Bauer’s disc diffusion
method according to the recommendations of the Clinical and Laboratory Standards Institute
(CLSI) [65]. All of A. baumannii, P. aeruginosa and S. aureus and some E. coli strains were
defined as multidrug-resistant as described elsewhere [66].
The isolates were stored at -20oC in skimmed milk agar until used in this study. Prior
to the study, each strain was subcultured on 5% blood agar at 37oC for two consecutive days.
From strains obtained in the second passage, bacterial suspensions were prepared in tryptone
soy broth (TSB) (Oxoid, Basingstoke, UK), and adjusted to a turbidity equal to McFarland 0.5
(1.5 x 108cfu/mL) (DIN EN 1040, 2005). All strains were studied with an agar well diffusion
method (31) [67].
4.4. Agar Well Diffusion Method
Each Mueller-Hinton agar plate was inoculated with the microorganism by streaking the swab
over the entire sterile agar surface. This procedure was repeated by streaking 2 more times,
rotating the plate approximately 60oC each time to ensure even distribution of the inoculum.
As a final step, the rim of the agar was also swabbed. Once the agar was solidified, it was
punched with eight-millimeter diameter wells and filled with 50 μL of the test material. The
concentration of the chemical compound extracts employed was 10 mg/mL in DMSO. They
are incubated at 37oC for 18 to 24 h. Subsequently, the plates were examined for bacterial
growth inhibition and the inhibition zone diameter was measured with a ruler to the nearest
millimeter.
4.5. Biochemical Studies
4.5.1. hCA isoenzyme purification and inhibition studies
In this work, both CA isoenzymes were separated and purified by Sepharose-4B-L-Tyrosine
sulfanilamide affinity chromatography in a single stage [68]. The column chemical material of
affinity chromatography containing Sepharose-4B-L-Tyrosine-sulfanilamide was created
conforming to a former procedure [69]. The protein molecules flow in the column eluates was
12

spectrophotometrically obtained at 280 nm as explained formerly [70]. CA isoenzymes’
activity investigation was obtained using the spectrophotometric style of Verpoorte et al. [71]
as described formerly [72]. In this work, changes in absorbance were recorded for 3 min at
348 nm using p-nitrophenylacetate (PNA) was used as a substrate which converted by both
isoenzymes to the p-nitrophenolate ion [73]. For estimation of protein quantity, the Bradford
method [74] was used during the separation stages. The presence and purity of both
isoenzymes were recorded by the SDS-PAGE method [75].
4.5.2. AChE inhibition study
The inhibitory effects of novel thymol bearing oxypropanolamine derivatives (2 and 3a-e) on
AChE activity were measured conforming to the spectrophotometric procedure [76].
Acetylthiocholine iodide (AChI) was utilized as a substrate for the enzymatic reaction. 5,5’-
Dithio-bis(2-nitro-benzoic) acid (DTNB) was utilized for the measurement of the AChE
activity. Briefly, 100 μL of Tris/HCl buffer (1.0 M, pH 8.0), 780 μL of sample solution
dissolved in deionized water at different concentrations, and 20 μL of AChE solution were
mixed and incubated for 10 min at 25oC. Then, 50 μL of DTNB (0.5 mM) was added. The
reaction was initiated by the addition of 50 μL of AChI [77]. The hydrolysis of these
substrates was monitored spectrophotometrically by the formation of the yellow 5-thio-2-
nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by
enzymatic hydrolysis of AChI, with an absorption maximum at a wavelength of 412 nm [78].
For the calculation of Ki values, three different novel thymol bearing oxypropanolamine
derivatives (2 and 3a-e) concentrations were used. Finally, the Lineweaver-Burk curves were
drawn [79]. One AChE unit is defined as the amount of enzyme that hydrolyzes 1.0 mol of
AChI to choline and acetate per minute at pH 8.0 at 37oC [80].
4.5.3. Measurement of α-Glycosidase inhibitory activity
α-Glycosidase inhibitory efficacy of novel thymol bearing oxypropanolamine derivatives (2
and 3a-e) was performed using p-nitrophenyl-D-glycopyranoside (p-NPG) as the substrate,
according to the procedure of Tao et al [81]. Samples were prepared by dissolving 10 mg in
10 mL (EtOH: H2O). First, 100 µL of phosphate buffer was mixed with 20 µL of the enzyme
solution in phosphate buffer (0.15 U/mL, pH 7.4) and 10-100 µL of the sample [82]. Multiple
solutions in phosphate buffer were prepared in case of getting full enzyme inhibition. Then, it
was pre-incubated at 35ºC for 12 min previous by adding the p-NPG to the initiation of the
13

reaction. Also, 50 µL of p-NPG in phosphate buffer (5 mM, pH 7.4) after preincubation was
added and again incubated at 37ºC. The absorbances were spectrophotometrically measured at
405 nm [83]. The IC50 amount was calculated from activity (%) versus plant concentration
plots. Lineweaver-Burk graphs were used to determine Vmax and other inhibition parameters.
The Ki was calculated from these graphs.
Conflicts of interests
The authors declare that no conflicts of interests.
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19

Table 1. The enzyme inhibition results of novel thymol bearing oxypropanolamine
derivatives (2, and 3a-e) against human carbonic anhydrase isoenzymes I and II (hCA I and
II), achethylcholinesterase (AChE) and α-glycosidase (α-Gly) enzymes
IC50 (µM)
r2
Ki (µM)
Compounds
hCA I
13.02
8.03
r2
hCA II
20.43
13.27
3.22
AChE
52.32
38.85
34.73
40.05
r2
α-Gly
418.13
624.03
1024.7
843.55
r2
hCA I
hCA II
AChE
α-Gly
0.9744
0.9805
0.9918
0.9912
0.9628
0.9740
0.9908
0.9913
0.9744
0.9509
0.9888
0.9614
0.9917
0.9924
0.9734
0.9384
17.34±0.94
11.47±2.02
1.51±0.23
1.11±0.16
17.83±3.58
15.11±3.58
2.97±0.43
5.48±2.04
31.45±7.37
19.68±4.03
26.38±9.16
30.44±3.58
583.66±83.11
505.18±53.58
819.04±126.4
851.05±101.2
2
3a
3b
3c
1.75
1.08
4.80
4.17
6.88
0.9424
0.9755
0.9680
7.14
12.36
41.22
0.9790
0.9889
0.9778
21.48
31.55
-
0.9411
0.9649
-
495.23
509.24
-
0.9638
0.9712
-
5.38±1.02
5.94±0.92
46.17±4.17
7.04±1.00
10.36±3.58
39.15±7.11
13.58±3.47
18.05±5.86
-
481.35±94.58
463.85±69.42
-
3d
3e
34.88
AZA*
-
-
-
-
-
-
-
-
76.20
-
0.9304
-
-
-
-
-
-
-
-
54.18±9.37
-
-
TAC**
22.80
12.60±0.78
ACR***
*Acetazolamide (AZA) was used as a positive control for human carbonic anhydrase I and II isoforms (hCA I
and II).
**Tacrine (TAC) was used as a positive control for acetylcholinesterase (AChE) enzyme.
***Acarbose (ACR) was used as a positive control for α-glycosidase enzyme.63,64
.
20

FIGURE LEGENDS
Figure 1. A. The mean zones of inhibition of synthesized derivatives and known antibiotic
agents against A. baumannii. B. The mean zones of inhibition of synthesized
derivatives and known antibiotic agents against E.Coli. C. The mean zones of
inhibition of synthesized derivatives and known antibiotic agents against P.
aeruginosa. D. The mean zones of inhibition of synthesized derivatives and known
antibiotic agents against S. aureus. (CAZ: ceftazidime, CIP: ciprofloxacin, IPM:
Imipenem, AK: Amikacin, CN: Gentamicin TZP: Piperacillin / Tazobactam, SAM:
Ampicillin / sulbactam, VA: Vancomycin, TEC: Teicoplanin, OX: Oxacillin, E:
Erythromycin, P: Penicillin, CRO: Ceftriaxone, SXT: Trimethoprim
sulfamethoxazole)
-
Figure 2. Determination of Lineveawer-Burk graphs for excellent inhibitors of hCA I (3c)
and hCA II (3b) isoenzymes, achethylcholinesterase (AChE) (3d) and α-glycosidase
(α-Gly) (3e) enzymes
21

30
20
10
0
30
20
10
0
P. aeruginosa
A. baumannii
A
B
30
30
20
10
0
E.coli
S. aureus
20
10
0
C
D
Figure 1
22

120
90
60
30
0
60
45
30
15
0
CA I
CA II
-1
0
1
2
3
4
5
6
7
-2 -1
0
1
2
3
4
5
6
7
1/[S] (µM)-1
1/[S] (µM)-1
180
135
90
45
0
240
AChE
α-Gly
180
120
60
0
-5
0
5
10
15
-8
0
8
16
24
32
40
1/[S] (µM)-1
1/[S] (µM)-1
Figure 2
23

3a
3c
3b
3d
3e
Scheme 1. Synthesis procedure for 2 and 3a-e. (i) epichlorohydrin, NaOH/water, 85°C, 0.5 h; (ii)
Amine compound, saturated K2CO3 solution, room temperature, 5-24 h
24

Highlights
 The first synthesis of novel thymol bearing oxypropanolamine derivatives (2, 3a-e)
was realized.
 The compounds effectively inhibit hCA I and II isoenzymes.
 The compounds were powerful inhibitor against AChE and α-glycosidase enzymes.
 Also we investigated their antibacterial properties
25

Novel Thymol Bearing Oxypropanolamine Derivatives as Potent Some
Metabolic Enzyme Inhibitors - Their Antidiabetic, Anticholinergic and
Antibacterial Potentials
Mustafa Zengina, Hayriye Genca, Parham Taslimib*, Ali Kestanea, Ertugrul Gucluc,
Aziz Ogutluc, Oguz Karabayc, İlhami Gulçinb
aSakarya University Science and Arts Faculty Chemistry Department 54187-Serdivan Sakarya, Turkey
bDepartment of Chemistry, Faculty of Science, Atatürk University, 25240-Erzurum, Turkey
cSakarya University Faculty of Medicine Infectious Diseases and Clinical Microbiology Department
54290-Adapazarı Sakarya, Turkey
26