Global HIV cohort studies among IDU and future vaccine trials
JPP-2015[1]
1. Journal of Pharmacy and
Pharmacology
Volume 3, Number 1, January (Serial Number 14)2015
David
David Publishing Company
www.davidpublisher.com
PublishingDavid
3. Journal of
Pharmacy and Pharmacology
Volume 3, Number 1, January 2015 (Serial Number 14)
Contents
Review
1 Vector Control—Development and Improvement of the Modern Chemical Insecticides
Ivan Popivanov, Tanya Petkova, Victoria Doycheva, Tzetza Doychinova, Ivelin Angelov and Dimitar
Shalamanov
Original articles
9 Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues
Shaikha Saif Al-Neyadi, Alaa Eldin Salem and Ibrahim Mahmoud Abdou
20 Production of Glucosamine Hydrochloride from Crustacean Shell
Martha Benavente, Selene Arias, Luis Moreno and Joaquín Martínez
27 Evaluation of Liver Function tests (AST & ALT) in Patients with Hepatitis B and C in
Tabriz-Iran (2013)
Navid Sarakhs Asbaghi, Kazem Ghahreman Zadeh, Taher Faraj Zadeh, Javid Lotfi Attari, Zahra Javan
Masoomi, Rana Faraj Zadeh, Mohammad Reza Tarmohammadi, Alireza Bakhtarai, Behzad Bahram
Zadeh and Babak Morshed Zadeh
33 Evaluation of Glycemic Control with a Pharmacist-Managed Post-Cardiothoracic Surgery
Insulin Protocol
Andrew Fung, Jeffrey Tom and KaWan Chiang
Case report
39 Hypersexuality after Modafinil Treatment: A Case Report
Süheyla Doğan Bulut, Rıza Gökçer Tulacı, Semanur Türkoğlu, Serdar Bulut and Sibel Örsel
4.
5. Journal of Pharmacy and Pharmacology 3 (2015) 1-8
doi: 10.17265/2328-2150/2015.01.001
Vector Control—Development and Improvement of the
Modern Chemical Insecticides
Ivan Popivanov1
, Tanya Petkova2
, Victoria Doycheva3
, Tzetza Doychinova2
, Ivelin Angelov2
and Dimitar
Shalamanov2
1. Department of Military Medicine, Military Medical Academy, Sofia 1606, Bulgaria
2. Department of Infectious diseases, Epidemiology, Parasitology and Tropical Medicine, Medical University, Pleven 5800, Bulgaria
3. Department of Epidemiology, Medical University, Sofia 1431, Bulgaria
Abstract: The transition from empirical and applied approach toward a scientific approach in modern medical disinsection is a result of
the discoveries of the organic chemistry. The most intensive used substance in this field—DDT (dichlorodiphenyltrichloroethane) is
introduced during World War II and contributes to world practical epidemiology just as antibiotics in clinical medicine. However, after
the 70s, this substance was placed under a ban, because of the accumulated evidence of many adverse health and environmental impacts
globally. Improvement of the insecticides after “DDT-era” is represented by the introduction of organophosphate and carbamate
insecticides in the 1960s. Their broad application is determined by better ecotoxicological characteristics. The advance in
biotechnology after the 1980s establishes the new class of insecticides—synthetic pyrethroids. Nowadays they are basic for the insect
control. Pyrethroids are characterized by selective impact on insects with much less impact on warm-blooded animals and the
environment. Insecticides from the newest class insect growth regulators realize their mode of activity by interfering with chitin
metabolism and thus prevent an insect from reaching maturity. These substances have extremely low toxicity, which makes them very
promising for the treatment of civilian and military facilities.
Key words: Vector control, insecticides, development.
1. Introduction
The endemic nature of a part of the infectious
diseases is associated with mandatory involvement of
live vectors in the epidemiological chain. General
assumptions about this date back to ancient times, but
the first reasoned link between insects and disease was
made in 1717 by Lancisi, a court physician to Popes
Innocent XI, Clement XI and Innocent XII. In his work
“De noxiis paludum effluviis eorumque remediis il (On
the noxious emanations of swamps, and their remedies)”
he recognized swamp areas as a breeding ground for
malaria and assumed the role of mosquitoes in the
disease transmission [1]. In 1897, the English
physician Ronald Ross reported in Calcutta, India, his
Corresponding author: Ivan Popivanov, Ph.D., assistant
professor, reserch fields: epidemiology of communicable
diseases, and preventive medicine. E-mail:
drpopivanov@gmail.com.
discovery of Anopheles mosquitoes as vectors of
disease [2]. Later, in 1900 in Cuba, the American
military physician Major Walter Reed confirmed
through experiments the transmission of yellow fever
via mosquitoes [3, 4]. One of the remarkable
discoveries in this field was made by Bacot and Martin
in 1914 [5]. They developed a hypothesis about
blocked fleas formed in the proventriculus of the
infected flea due to enormous multiplication of plague
bacilli. Thus, infections generated during subsequent
bloodsucking by regurgitation into the bite wound. The
figures, showing the obstruction and blood congestion
in the flea proventriculus, have not ceased to be
included in textbooks for 100 years. Today, this
classical description is successfully complemented by
multiple lines of scientific evidence for infection,
carried out by insects of medical importance, and for
pathogens in their particular organs. Such data is
DDAVID PUBLISHING
6. Vector Control—Development and Improvement of the Modern Chemical Insecticides2
obtained by microscopic, immunofluorescent,
biochemical techniques and other methods, through
which the knowledge about the mode of transmission
of various infections is becoming objective and
specified [6, 7].
For some infections transmitted by live vectors,
vaccines have been developed, although the use of such
bioproducts is generally limited. Therefore, the control
of these infectious diseases is absolutely impossible
without effective vector control. Such control is mostly
chemical and is based on the achievements of organic
chemistry and biochemistry. The knowledge about the
biological vectors is important and is provided by
medical entomology, medical zoology and medical
geography. From a bio-medical point of view, the data
on the toxicological effects becomes more relevant. In
the context of globalization and the expanding use of
chemicals, the importance of environmental principles
and criteria increases. With all these scientific methods,
the strategy and tactics of the administration and
control of the effectiveness of using chemical
insecticides are being developed. The control must
meet the requirements of two main directions:
epidemiological—aiming at maximum limitation or
eradication of insects with medical importance, and
ecological—ensuring the safety of people,
warm-blooded animals and the environment.
2. History—DDT-era
The beginning of modern chemical disinsection is
associated with boost of the development of organic
chemistry, and especially with the discovery of DDT
(dichlorodiphenyltrichloroethane). The Swiss chemist
Paul Müller discovered DDT’s contact-insecticidal
action in 1939 (awarded the 1948 Nobel Prize in
Medicine), but in practice it was first synthesized in
1873 by Othmar Zeidler in Strasbourg as its
insecticidal activity was not suspected for 66 years.
The fame of DDT began during World War II
(December, 1943). The city of Naples was
overcrowded with troops of Allies and refugees.
Against that background, there was a dramatic
outbreak of typhus fever with high mortality. The
Allied Medical Services applied a DDT-containing
powder to treat pediculosis in 673,094 militaries and
civilians. Households throughout the city and
surrounding villages were also treated systematically.
As a result, after February 1944, the number of new
patients declined significantly. This was the first
example in history, when a major epidemic of typhus
(2,020 cases and 429 deaths), spread among troops,
population and prisoners, with high pediculosis and
poor living conditions, was brought under control in
such a short time [8]. The situation of malaria in British
Ceylon is another demonstrative example. The
application of DDT was launched in 1944 and from 2.8
million cases with 13,000 deaths in 1946, they had
been reduced to 7,300 with non-fatal cases in 1963 [9].
The observed anti-epidemic effects of DTT in different
parts of the world were numerous and unprecedented.
Accelerated implementation in the practice and
production of large number of products based on DDT
in all possible forms started in many countries. Only in
the USA about 675,000 tons were used for 30 years.
World production in the early 1970s has reached up to
about 400,000 tons annually and the total production so
far is estimated to be 1.8 million tons. A significant part
(about 80%) has been applied in agriculture [10, 11]. In
Bulgaria, the use of DDT started in 1948.
With the widespread use of DDT and other
organochlorine insecticides a qualitative leap in
medical disinsection was achieved. The empirical
approach of this activity remained in the history.
Considering the rapidly achieved excellent results in
limiting malaria, leishmaniasis, typhus and yellow
fever in the 1940s and 1950s, many researchers
compared the importance of organochlorine
insecticides for preventive medicine with the
importance of using antibiotics in clinical medicine.
However, in the 1960s, sufficient evidence for the
serious toxic or adverse environmental effects of DDT
was accumulated [12]. Scientists warned the society,
7. Vector Control—Development and Improvement of the Modern Chemical Insecticides 3
“the euphoria passed”, and the accelerated pace of
implementation in the practice were remarkably
delayed. In the early 1970s, on the recommendation of
WHO, the industrially developed countries suspended
its usage. Many other countries followed the ban of
DDT, at different times thereafter. The Stockholm
convention on persistent organic pollutants (2001)
strongly restricted the use of DDT and allowed its use
only for vector control of cutaneous leishmaniasis and
malaria in countries that are highly endemic [13]. The
United Nations Environment Programme (2008)
predicted its total phase-out by 2020. The production in
the last years was reduced to about 5,000 tons of active
substance per year and only in three countries—China,
India and North Korea. Currently, DDT is still used for
vector control in several countries in Africa and Asia
that are highly endemic for cutaneous leishmaniasis
and malaria and for plant protection [14, 15].
The adverse effects of DDT were clarified in detail
and led to its ban. They were generally associated with
its potential for late effects. Long-term persistence in
the environment (half-life more than 10 years), results
in a stable inclusion in the food chains, with subsequent
build-up (biomagnification) in high concentrations in
humans, mammals and birds. The accumulation in
adipose tissue and biotransformation into the extremely
resistant and toxic breakdown product DDE
(dichlorodiphenyldichloroethylene) are other side
effects. The concentrations of DDT and DDE in the fat
fraction of breast milk are important indicators of
health risk. Monitoring data in 28 countries were in
broad (values) range: the overall trend in the developed
countries showed rapid decrease from about 4,500 ng/g
in the 1970s to hardly detectable traces at the end of
20th century [12, 16], but in Poland and Greenland the
average concentrations of DDE remained over 6,000
ng/g and over 3,000 ng/g, respectively, in the year 2000
[17]. The prevalence of DDE (with definitely slower
elimination from the body compared to DDT) in these
countries is a marker for its persistence in the natural
food resources, from which it can pass into humans.
The situation in Greenland is explained by the fact that
the population consumes predominantly seafood and
this reflects the global process of contamination of the
oceans in recent decades [10]. The higher
concentrations of DDT established in Tanzania (5,500
ng/g), Zimbabwe (4,900 ng/g) and Mexico (4,700 ng/g)
are due to the fact that the use was banned recently [18].
Carcinogenicity studies are hampered by
methodological limitations and assumptions are based
mainly on experimental models [11]. The causality
with breast cancer was most widely studied. Some
studies suggest a five-fold increased risk in women of
childbearing age during the years of peak usage of
DDT [19]. There are studies for a causal link with
pancreatic cancer, leukemia, diabetes, children’s
neurological development. Although the data on the
cause-effect relationship in these cases are
controversial, studies in women and children in some
regions raise concern [20].
Another adverse effect of DDT use is resistance
development. The first observation was in 1946 for
Anopheles mosquitoes, and many other insects were
proven subsequently [21].
3. Contemporary Insecticides
The arsenal of applied epidemiology nowadays
counts mainly on organophosphate insecticides,
carbamate insecticides and synthetic pyrethroids. The
group of insect growth regulators is in the process of
being put into practice.
The group of organophosphate insecticides was
initially developed as a new generation chemical
weapon (nerve agents) during World War II and
subsequently widely used in agriculture. They were
used for medical disinsection since the 1950s [22].
Their position as an alternative to the organochlorides
was due to some advantages:
rapid decomposition to non-toxic products;
considerably shorter half-life and thereby
reducing the risk of chronic poisoning and
environmental pollution;
8. Vector Control—Development and Improvement of the Modern Chemical Insecticides4
a well known mode of action (as inhibitors of
acetylcholinesterase);
availability of effective antidote—atropine;
ability to monitor the staff working with them
through periodic control of the enzyme
acetylcholinesterase [23].
The growing resistance of insects to organochlorine
insecticides was another important reason for the
replacement of DDT with organophosphate
insecticides.
Carbamate insecticides have biological activity and
mode of action similar to organophosphates—they
inactivate acetylcholinesterase, but do not undergo
metabolism. This difference makes their effect
somewhat reversible.
Synthetic pyrethroids were preceded by
pyrethrins—natural organic compounds with active
substances chrysanthemic acid and pyrethric acid,
extracted from the seed cases of some sorts of
Chrysanthemum. In China, they were known even B.C.
and the commercial usage referred to the 19th century,
when in some Middle East, East Asian and Latin
American countries plantations for industrial
production were created. The difficulties in the
standardization of flower extracts, the dependence of
yield on climatic conditions and especially the
increased demand for insecticides during World War II,
without the possibility to be covered with only natural
products, made this type of production ineffective. All
this pushed scientists to the search of new chemical
substances and led to the aforementioned putting of
DDT into practice in large-scale.
When the cumulative health and environmental
negatives necessitated the elimination of
organochlorine insecticides and limitation of the
organophosphate insecticides, pyretroids (synthetic
analogues of pyrethrins) became dominant. Their
insecticidal activity is 10 times more powerful than the
natural substances and comes down to blocking the
axonal membrane permeability with subsequent
excitation of the muscle fibers and paralysis. The
selective toxicity is due to a 100 times higher
sensitivity of the voltage-gated sodium channels in the
nerve structures of insects as compared to the
analogous ones in mammals [24]. Furthermore, the
insecticidal activity of synthetic pyrethroids is more
pronounced at low temperature (the so called “negative
temperature effect”) [25]. This is associated with the
breakdown of the pyrethrin molecule at a high
temperature and with its more intensive biodegradation
in the microsomes of warm-blooded animals, than in
the microsomes of the insects. The insects are
poikilotherms and their body temperature is variable
and dependent on the ambient temperature. The effect
of synthetic pyrethroids is quick and strong
(“knock-down effect”) and the expenditure of the
active substance is more than 10 times lower than the
natural derivatives. Their very low oral toxicity to
humans, rapid breakdown in the organism and the
environment, multipurpose usage (as insecticides and
repellents) are important properties, determining the
growing levels of use in the world after the 1980s. In
the developed countries, they have a share of 70-80%
of all insecticides [26]. Slower trend of increase of their
relative share is established in Africa [14].
Neonicotinoids are a class of neuro-active
insecticides. They are analogous to natural tobacco
alkaloids and developed through chemical synthesis.
The class was widely used in the agricultural practice,
but did not have a large share in the medical
disinsection. The first insecticide formulation from this
group for domestic purposes, authorized for use in
Bulgaria a few years ago, is now banned along with the
entire class. In 2013 Bulgaria joined the EU
Commission restrictions of use of neonicotinoids [27],
which were suspected to be a contributing factor of bee
colony collapse disorder.
4. New Technologies
One of the directions for improvement is targeting
immature forms of insects. For that purpose biological
insecticides were used for a long time (e.g., Bacillus
9. Vector Control—Development and Improvement of the Modern Chemical Insecticides 5
thuringiensis), and after the 1960s, some
organophosphate insecticides were used. New and
perspective agents in this field are IGRs (insect growth
regulators), meeting the increased environmental
requirements and extremely unlikely to cause acute
poisoning in humans and animals. They affect insect
structures that are not typical for humans. The mode of
activity of IGRs is larvicidal by interfering with chitin
metabolism and thus preventing the insect from
reaching maturity. The chemical substances were
known since 1960s. At the end of 1970s plant
protection products were registered and products for
medical disinsection were developed after the year
2000. The main substances are diflubenzuron (chitin
synthesis inhibitor) and novaluron (synthetic analogue
of the juvenile hormone). IGRs treated larvae cannot
release the old chitin exoskeleton, or the new one is
fragile and thus they don’t have a normal exoskeleton.
If IGRs are applied on larvae in the last phase of
development, the pupae or imagoes become
underdeveloped and deformed. The result is death at
the stage of metamorphosis or shortly thereafter. If
transformed to imago forms (e.g., mosquito), they have
visible abnormalities and cannot fly out due to
deformed wings or fragile limbs. IGRs can also have an
effect on the eggs, causing sterility [28]. These effects
are due to blocking of the membrane transport of chitin
precursors [29] which occurs after affecting the
expression of multiple genes involved in the chitin
metabolism [30]. Diflubenzuron has low levels of oral
toxicity—4,640 mg/kg, and has extremely low toxicity
to mammals and fish, accordingly. Recent results from
a study on an insecticide product with
diflubenzuron-granular formulation in mosquito
habitat in Bulgaria ascertained progressively
increasing number of killed larvae and after the 4th day
all larval forms were non-viable [31]. In other studies
on the effect of granules and water-soluble powder
with an active substance diflubenzuronon mosquito
populations in large natural ponds in the Mediterranean
and some other regions of the world, complete loss of
viability of larvae was established between 6th and
12th days after treatment [32]. This was due to the fact
that the dilution of insecticide formulation was more
pronounced, and the density of the mosquito
population was significantly higher.
Nanotechnologies are another contemporary field of
pest control. They are used to improve the conventional
/classical chemical insecticides. The recently
developed nanosuspensions and nanoemulsions are
new, more effective forms of some of the so far
implemented chemical insecticides [33]. They are used
to achieve higher efficiency, because the nanoparticles
penetrate more easily through the ion channels [34-36].
Thus, reduction of the concentration and amount of the
insecticide substance is achieved.
5. Vector Control: Present State from
Medical and Environmental Perspective.
Implementation Strategies
The overall global environmental assessment of the
situation concerning chemical insecticides now is the
following: the burden of people and environment with
DDT is progressively decreasing and the trend of
clearing up the nature from some other organochlorides,
from organophosphate and carbamate insecticides is
slow. Synthetic pyrethroids are the most promising, but
their importance should not be overemphasized. Their
incorporation as a primary means in the mosquito
control programs in South Africa in 1996 did not bring
the desired results. Moreover, because of their short
biocidal effect and resistance development in
mosquitoes, in some places the growth of anopheles
mosquitoes’ population was ascertained (particularly
Anopheles funestus). This necessitated a revision of the
regulations on the use of DDT. Now DDT is authorized
for use in several countries, with application only on
the inside walls of homes (indoor residual
spraying—IRS), and with a total ban on treatment of
body, clothing or bedding. Concerning the class of
insect growth regulators, it is assumed that they have a
great potential to reduce the overall environmental risk
10. Vector Control—Development and Improvement of the Modern Chemical Insecticides6
from insecticides. Nanotechnologies are in the early
stages of use. Studies on their side effects are
forthcoming, in view of their high penetration through
membrane ion channels that could create risks for
humans and animals [37, 38].
The selection of insecticide formulations is complied
with the specific circumstances and characteristics of
the active substance. During the treatment of objects,
affected by the floods in Bulgaria in 2005, the
specialized units of Military Medical Academy used
insecticide formulations with an active substance
malation for open areas and synthetic pyrethroids for
indoor application [39]. According to the NATO
standardization agreements, the insecticides for
individual protection are on synthetic pyrethroid base
[40]. The phthalic compounds, recently recommended
as the most suitable for repelling insects, already are
replaced by the synthetic pyrethroids. Carbamate
insecticides have a better effect on crawling insects.
The insect control strategy provides treatments early in
the spring with IGRs to suppress the formation of
initial population, followed by periodical treatments to
maintain a safe level. Pyrethroids and other insecticides
in this case are supplemental to treatment. When mass
reproduction of insects is established, treatments with
synthetic pyrethroids are recommended to limit the
number of imago forms and then proceeding to use of
IGRs (e.g., diflubenzuron).
The optimal combination of larvicides and
insecticides for imago forms in medical disinsection
can reduce aggressive to humans and animals
organophosphate and carbamate insecticides.
6. Conclusions
In the 85 years history of chemical insecticides use
for vector control two stages are outlined. In the initial
stage the main purpose was to respond only to the need
for management of severe epidemic situations.
Currently, serious issues concerning the ecotoxicology
of chemical insecticides are raised. The improvement
of chemical insecticides through new concepts and
technologies is a fact. In this context, the group of
synthetic pyrethroids has a great potential and the IGRs
are approaching the concept of the “ideal insecticide”.
Considering that chemical insecticides are used in
agriculture and veterinary medicine, even in much
higher concentrations and quantities, achievement of
adequate interaction and coordination within these
scientific and applied fields is essential for effective
control of public health.
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13. Journal of Pharmacy and Pharmacology 3 (2015) 9-19
doi: 10.17265/2328-2150/2015.01.002
Synthesis and Antimicrobial Studies of New
Trifluoromethylpyrimidine Analogues
Shaikha Saif Al-Neyadi, Alaa Eldin Salem and Ibrahim Mahmoud Abdou
Department of Chemistry, University of United Arab Emirates, Abu Dhabi, Al-Ain 15551, United Arab Emirates
Abstract: This paper describes the synthesis of a new series of trifluoromethylpyrimidine and their potential antimicrobial evaluation.
We have prepared 10 novel pyrimidine derivatives in high yields and short reaction time using microwave irradiation. Compounds 4a,b
and 6a,b showed good antimicrobial activity against Gram-positive bacteria; Clostridium perfringens, Bacillus pumilus and
Enterococcus faecalis. These compounds were found to be the most potent antimicrobials compared to ampicillin, tetracycline, and
streptomycin that showed no activity against Enterococcus faecalis. Compounds 4a,b and 6a,b exhibited great antimicrobial potency
against all tested bacteria strains at a MIC of 3.125-100 μg/mL whereas only 4a showed the antimicrobial activity against
Gram-negative bacteria Klebsiella pneumonia with a MIC value 12.5 μg/mL.
Key words: Synthesis, microwave, fluoropyrimidine, antimicrobial.
1. Introduction
In spite of the remarkable growth in human
medicines, infectious diseases caused by bacteria,
fungi, viruses and parasites are still representing major
threats to public health. Their impact is particularly
large in developing countries due to the relative
unavailability of medicines, while the excessive use of
antimicrobial drugs has led to the emergence of
widespread bacterial resistance [1]. In 2003, a number
of substituted pyrimidines were synthesized and
intensively studied as potent and selective inhibitors of
Gram positive bacterial DNA polymerase IIIC [2].
Over the last decades, development of drug resistance
as well as the appearance of undesirable side effects of
some antibiotics [3] has initiated the search for new
antimicrobial agents to overcome some of the
disadvantages of the existing drugs [4]. Fluorinated
pyrimidine derivatives have attracted more attention
especially in biological and medicinal chemistry fields
because of the unique features of fluorine compounds
and their physiological activity [5,6]. The introduction
Corresponding author: Ibrahim Mahmoud Abdou, Ph.D.,
associate professor, research field: organic chemistry. E-mail
i.abdou@uaeu.ac.ae.
of fluorine atoms into organic compounds often
permits dramatic changes in their chemical and
pharmaceutical properties [7]. The presence of
pyrimidine nucleus in compounds containing fluorine
atoms was found enhancing the biological activities,
such as anti-viral [8], anti-malarial [9], adenosine
receptor [10], anti-cancer agents [11], as well as
compounds targeting delayed-type hypersensivity
agents [12].
Hence, there is a never lasting demand for synthesis
of novel antimicrobial agents with high potency,
efficacy and minimum side effects; this work aims to
synthesize novel fluoropyrimidine derivatives with
high potency and efficiency against different bacterial
strains. Our synthetic approach is based on microwave
protocols to enhance the yields in shorter times for the
targeted compounds 3-5a,b and 6a-f. The antimicrobial
activities of the newly synthesized fluoropyrimidine
derivatives 4a,b and 6a,b will be tested against different
bacteria strains such as Gram-positive bacteria,
Clostridium perfringens, Bacillus pumilus and
Enterococcus faecalis and Gram-negative bacteria,
Klebsiella pneumonia. Results out of this work will
establish a new structure-activity relationships based
DDAVID PUBLISHING
14. Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues10
on substitutions at C-2 and C-4 of the pyrimidine ring.
2. Materials and Methods
General: Microwave synthetic protocol was done
using CEM Microwave system. Melting points were
determined on (Pyrex capillary) Gallenkamp
apparatus. Infrared spectra was recorded with a
Thermo Nicolet Nexus 470 FT-IR spectrometer in the
range 4,000-400cm-1
on samples in potassium
bromide disks. 1
H-NMR spectra, 13
C-NMR spectra
were obtained on Varian Gemini 400 MHz FT NMR
spectrometer in CDCl3 and DMSO-d6; chemical shifts
were recorded in (ppm) units, relative to Me4Si as an
internal standard. All exchangeable protons were
confirmed by addition of D2O. Thin-layer
chromatography (TLC) was carried out on precoated
Merck silica gel F254 plates and UV light was used for
visualization. Column chromatography was performed
on a Merck silica gel. The reagents were purchased
from Aldrich and used without further purification.
Elemental analysis performed on Leco Model
CHN-600 elemental analyzer.
2.1 Microwave Synthesis of 2-Hydroxypyrimidine
Analogous (3a,b)
A mixture of 1,3-diketone 1a,b (2.0 mmol), urea
(120 mg, 2.0 mmol) and 2 drops of HCl (6.0 M) in
ethanol (8 mL) was mixed in 10 mL CEM-microwave
vial. The vial was sealed and irradiated in
CEM-microwave reactor at 135C for 5-10 min. The
reaction was verified for completion by TLC and
recrystallized from a proper solvent to give 3a,b in
yields 89% and 84% respectively.
4-(Thien-2-yl)-6-(trifluoromethyl)pyrimidin-2-ol
(3a): yellow crystals; yield 89%; mp 230 C; IR (KBr,
cm-1
): 3,459 (br, OH), 3,088 (C-H aromatic), 1,680
(СОNH); 1
H-NMR [DMSO-d6, 400 MHz]: (δ, ppm)
7.29 (s, 1H, H-5 pyrimidine), 7.30 (t, 1H, thien-2-yl
H-4, J = 4.0 Hz), 7.93 (d, 1H, thien-2-yl H-5, J = 5.0
Hz), 8.29 (d, 1H, thien-2-yl H-3, J = 4.0 Hz), 12.88
(1H, s, OH exchangeable with D2O); 13
C-NMR
[DMSO-d6, 100 MHz]: (δ, ppm) 103.6 (C-5), 120.6
(CF3, q, J = 274 Hz), 129.1, 131.1, 132.8 (C-3, C-4,
C-5 thien-2-yl), 140.5 (C-2 thien-2-yl), 160.7 (C-6
pyrimidine), 163.5 (C-4 pyrimidine), 165.1 (C-2
pyrimidine). Anal. Calcd for C9H5F3N2OS: C, 43.90; H,
2.05; N, 11.38; S, 13.02. Found: C, 44.35; H, 2.12; N,
11.66; S, 13.30.
4-Phenyl-6-(trifluoromethyl)pyrimidin-2-ol (3b):
white powder; yield 84%; mp 234 C, from hexane; IR
(KBr, cm-1
): 3,489 (br, OH), 3,076 (CH-aromatic),
1,676 (СОNH); 1
H-NMR [DMSO-d6, 400 MHz]: (δ,
ppm) 7.22 (s, 1H, H-5 pyrimidine), 7.52-7.61 (m, 3H,
aromatic), 8.16-8.17 (m, 2H, aromatic), 12.88 (s, 1H,
OH; exchangeable with D2O); 13
C-NMR [DMSO-d6,
100 MHz]: (δ, ppm) 109.4 (C-5), 122.8 (CF3, q, J = 274
Hz), 129.0, 130.4, 133.4, 139.8 (phenyl carbons), 161.7
(C-6 pyrimidine), 165.3 (C-4 pyrimidine), 167.8 (C-2
pyrimidine). Anal. Calcd. for C11H7F3N2O: C, 55.01; H,
2.94; N, 11.66; Found: C, 55.46; H, 3.01; N, 11.94.
2.2 Microwave Synthesis of 2-Pyrimidine Benzoyl
Esters (5a,b)
To a solution of 3a,b (2 mmol) in 15 mL pyridine,
p-fluorobenzoyl chloride (5 mmol, 0.59 mL) was
added gradually with stirring in ice bath. After the
addition is completed, the reaction mixture was heated
under microwave irradiation at 100C for about 10 min.
The progress of the reaction was monitored by TLC,
The solid obtained was washed with water and
crystallized from ethanol to give the desired
compounds 4a,b.
4-(Thien-2-yl)-6-(trifluoromethyl)-2-pyrimidinyl-4
-fluorobenzoate (4a): white crystals, yield 85%; mp
105-7 °C; IR (KBr, cm-1
): 3,116 (C-H, aromatic), 1,757
(C=O), 1,603 (C=C), 1,429 (C=N); 1
H-NMR [CDCl3,
400 MHz]: (δ, ppm) 7.17-7.22 (m, 3H, phenyl &
thien-2-yl H-4), 7.64 (dd, 1H, thien-2-yl H-5, J = 4.9
Hz), 7.79 (s, 1H, H-5 pyrimidine), 7.92 (dd, 1H,
thien-2-yl H-3, J = 3.7 Hz), 8.24 (m, 2H, aromatic);
13
C-NMR [CDCl3, 100 MHz]: (δ, ppm) 111.9 (C-5
pyrimidine), 115.41 (phenyl carbon), 122.8 (CF3, q, J =
15. Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues 11
274 Hz), 127.2, 127.3, 127.4, 128.9, 129.0
(Ar-carbons), 148.3 (C-2 thien-2-yl), 161.5 (C-6
pyrimidine), 163.2 (C-4 pyrimidine), 165.4 (CO),
165.5 (C-F, aromatic), 165.7 (C-2 pyrimidine). Anal.
Calcd for C16H8F4N2O2S: C, 52.18; H, 2.19; N, 7.61; S,
8.71; Found: C, 52.63; H, 2.26; N, 7.89; S, 8.99.
4-Phenyl-6-(trifluoromethyl)pyrimidin-2-yl
4-fluorobenzoate (4b): white powder; yield 83%; mp
114 °C; IR (KBr, cm-1
): 3,118 (C-H, aromatic), 1,758
(C=O), 1,602 (C=C), 1,428 (C=N); 1
H-NMR
[DMSO-d6, 400 MHz]: (δ, ppm) 7.26 (1H, s, H-5
pyrimidine), 7.53-7.61 (m, 5H, aromatic), 7.93-7.95 (m,
2H, aromatic), 8.25-8.27 (m, 2H, aromatic); 13
C-NMR
[DMSO-d6, 100 MHz]: (δ, ppm) 109.2 (C-5
pyrimidine), 115.1 (aromatic carbons), 122.8 (CF3, q, J
= 274 Hz), 127.2, 127.3, 128.9, 129.0, 129.8, 133.4
(aromatic carbons), 148.8 (C-6 pyrimidine), 161.5 (C-4
pyrimidine), 163.2 (C=O), 165.3 (C-F, aromatic),
165.8 (C-2 pyrimidine). Anal. Calcd for C18H10F4N2O2:
C, 59.68; H, 2.78; N, 7.73; Found: C, 60.13; H, 2.85; N,
8.01.
2.3 Microwave Synthesis of 2-Chloropyrimidine
Derivatives (5a,b)
In 10 mL CEM-microwave vessel, two drops of
pyridine were added to a mixture of 3a,b (2.0 mmol)
and POCl3 (4.0 mmol, 0.37 ml). The vial was sealed
and the mixture was heated under microwave
irradiation at 100C for 25 min. The reaction mixture
was cooled to room temperature then poured into an
ice-cold water (10 mL) under vigorous stirring. The pH
was adjusted to pH-8 and the resulting mixture was
stirred for 15 minutes. The obtained light brown solid
was filtered, washed with water (2 ×10 mL) and dried
under reduced pressure for 2 hours.
2-Chloro-4-(thien-2-yl)-6-(trifluoromethyl)pyrimid
ine (5a): brown crystals; yield 96%; mp 109 C;
1
H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 7.30-7.33 (t,
1H, thien-2’-yl H-4, J = 4.0 Hz ), 7.84 (1H, s, H-5
pyrimidine), 7.92 (d, 1H, thien-2’-yl H-5, J = 5.0 Hz),
8.21 (d, 1H, thien-2’-yl H-3, J = 4.0 Hz); 13
C-NMR
[DMSO-d6, 100 MHz]: (δ, ppm) 105.2 (C-5
pyrimidine), 122.0 (CF3, q, J = 274 Hz), 129.7, 131.5,
133.3 (C-3, C-4, C-5 thien-2-yl), 140.1 (C-2
thien-2-yl), 163.6 (C-6 pyrimidine), 164.7 (C-4
pyrimidine), 165.1 (C-2 pyrimidine).
2-Chloro-4-phenyl-6-(trifluoromethyl)pyrimidine
(5b): brown crystals; yield 95%; mp 105C, from
ethanol; 1
H-NMR [DMSO-d6, 400MHz]: (δ, ppm)
7.58-7.67 (m, 3H, aromatic), 7.75 (s, 1H, H-5
pyrimidine), 8.16-8.17 (m, 2H, aromatic); 13
C-NMR
[DMSO-d6, 100MHz]: (δ, ppm) 104.9 (C-5
pyrimidine), 122.5 (CF3, q, J = 274 Hz), 129.7, 131.4,
132.9, 136.4 (aromatic carbons), 157.5 (C-6
pyrimidine), 162.7 (C-2 pyrimidine), 165.1 (C-4
pyrimidine).
2.4 Microwave Amination Procedure (6a-f)
To a solution of 4-aryl-2-chloro-6-(trifluoro
methyl)pyrimidine 5a,b in toluene (15 mL), an excess
of amine was added at room temperature in 35 mL
CEM microwave vial. The vial was sealed and the
reaction mixture was heated under microwave
irradiation at 80-100C for 5-10 min. The progress of
the reaction was monitored by TLC and after
completion; the reaction mixture was quenched with
water (0.5 mL) and a solution of sodium carbonate (0.1
g, 2 mmol) was added with stirring at room temperature.
The reaction mixture was extracted in ether and the
organic layer was dried over anhydrous MgSO4. The
product purified by silica gel column chromatography
with ethylacetate: hexane (6:4) to give pure products
6a-f.
2-(N-Cyclopentyamino)-4-(thien-2-yl)-6-(trifluoro
methyl)pyrimidine (6a): pale yellow crystal, yield 86 %;
mp 99 °C; IR (KBr, cm-1
): 3,544 (-NH), 3,089 (C-H,
aromatic), 2,931 (aliphatic C-H), 1,478 (CN);
1
H-NMR [CDCl3, 400 MHz]: (δ, ppm) 1.47-1.73 (m, 8
H, cyclopentyl), 4.30-4.36 (m, 1H, cyclopentyl), 5.35
(d, 1H, -NH, exchanges with D2O, J = 6 Hz ), 7.08 (1H,
s, H-5 pyrimidine), 7.12-7.15 (m, 1H, thien-2-yl H-4),
7.49-7.51 (m, 1H, thien-2-yl H-5), 7.70 (m, 1H,
17. Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues 13
pyrimidine), 161.8 (C-4 pyrimidine), 167.2 (C-2
pyrimidine). Anal. Calcd. for C18H20F3N3: C, 64.46; H,
6.01; N, 12.53; Found: C, 64.91; H, 6.08; N, 12.81.
2-(N-Methylpiperazin-1-yl)-4-phenyl-6-(trifluorom
ethyl)pyrimidine (6f): white powder, Rf = 0.15
(ethylacetate:hexane 1:1), yield 82%; mp 114-16 °C;
IR (KBr, cm-1
): 3,083 (C-H, aromatic), 2,924 (aliphatic
C-H), 1,583 (CC), 1,457 (CN); 1
H-NMR [CDCl3,
400MHz]: (δ, ppm) 2.35 (s, 3H, methyl), 2.49-2.50 (m,
4H, piprazine ring), 3.98 (m, 4H, piprazine ring), 7.19
(1H, s, H-5 pyrimidine), 7.46-7.48 (m, 3H, aromatic),
8.03-8.05 (m, 2H, aromatic); 13
C-NMR [CDCl3, 100
MHz]: (δ, ppm) 43.7 (CH3), 46.2 (C-2, C-6
methylpiprazine), 54.9 (C-3, C-5 methylpiprazine),
100.6 (C-5 pyrimidine), 120.9 (CF3, q, J = 274 Hz),
127.2, 128.8, 131.2, 136.7 (aromatic carbons), 156.4
(C-6 pyrimidine), 161.7 (C-4 pyrimidine ), 166.8 (C-2
pyrimidine). Anal. Calcd. for C16H17F3N4: C, 59.62; H,
5.32; N, 17.38; Found: C, 60.07; H, 5.39; N, 17.66.
2.5 Determination of MIC (Minimum Inhibitory
Concentration)
2.5.1 Microdilution Method
The 96-well microtitre assay using resazurin as the
indicator of cell growth [13] was employed for the
determination of the minimum inhibitory
concentration. Resazurin is an oxidation-reduction
indicator used for the evaluation of microbial growth.
The blue non-fluorescent dye turned into pink color
with fluorescent when reduced to resorufin by
oxidoreductase within cells. A 50 µL sterile deionized
water was added to each well. A 50 µL purified test
compound was added in the first well of horizontal row
and double diluted horizontally in each well. Last well
was added with 100 µL of sterile deionized water
without test compound, used as control. A 100 µL
double strength nutrient broth was added in each well
then 10 µL test organisms (OD at 600 nm ~1) added to
each well. This was followed by the addition of 1 µL
resazurin (1% stock prepared). The microtitre plate was
incubated at 37 °C for 18-24 h. The well with blue
color (no viable bacteria) just before the pink well
(viable bacteria) was taken as MIC value. The
inoculated plates incubated. MIC was defined as the
lowest concentration of the tested plant extracts that
prevented resazurin color change from blue to pink.
2.5.2 Determination of Zones of the Inhibition
All the synthesized compounds were tested for their
in vitro growth inhibitory activity against a panel of
standard strains of pathogenic microorganism
including Gram-positive and Gram-negative bacteria.
Gram-positive bacteria are Clostridium perfringens,
Bacillus pumilus and Enterococcus faecalis and
Gram-negative bacteria’s are Klebsiella pneumonia
and Pseudomonas aeruginosa. The efficacy was
determined by zone of inhibition values using disk
diffusion technique [14]. To each petri-plate, 20 mL of
sterilized medium was added. After the agar had set,
10% of inoculum of each microorganism culture was
added to each petri-plate and spread thoroughly.
Sterilized Whatmann no. 1 filter papers discs
(diameter 6 mm) were thoroughly moistened with the
synthesized compounds of specific concentrations 100
μg/mL in DMSO and placed on seeded agar plates.
Paper discs moistened with DMSO were considered as
negative control. Discs saturated with Ampicillin,
Tetracycline and Streptomycin at the same
concentrations were taken as standard (positive
control). The plates were incubated at 37 °C for 24 h.
The clear zone of inhibition around disc-paper
demonstrated the relative susceptibility towards the
synthesized derivatives.
3. Results and Discussion
Fluorine has played a pivotal role in novel drug
discovery for modulating physical and biological
properties of molecules [15-18]. Incorporation of one
or several fluorine atoms into an organic molecule may
enhance its biological activity, bioavailability,
metabolic stability and lipophilicity due to intrinsic
properties of fluorine atoms such as high
electronegativity and small atomic radius [19].
18. Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues14
3.1 Chemical Synthesis
All of the synthetic steps described in this paper
were carried out under controlled microwave
irradiation. The conversion of diketone to the targeted
substituted pyrimidine 3-6 involving cyclization,
chlorination and amination carried out under
microwave irradiation yielded 3a,b (84-89%), 5a,b
(95-96%) and 6a-f (77-89%) respectively (Table 1).
Synthetic methodologies began by the reaction
between trifluorobutane-1,3-dione 1a,b and urea via
nucleophilic substitution at the vinyl carbon atom,
followed by cyclization to form 6-trifluoromethyl
pyrimidin-2-ol 3a,b. The structure and properties of the
final products obtained have been established by their
melting point, elemental analysis, IR, 1
H-NMR and
13
C-NMR spectroscopy. The structure of
2-hydroxy-4-(thien-2-yl)-6-trifluoromethyl
pyrimidine 3a was confirmed using IR-spectroscopy
which revealed by the appearance of broad band at υ =
3,459 cm-1
corresponding to a tautomeric hydroxyl
group (N=C-OH). While, a sharp band appeared at υ =
1,680 cm-1
assigned for the keto-group (CONH) in 3a.
The 1
H-NMR (400 MHz, DMSO-d6) spectrum of
compound 3a showed a sharp signal at δ = 7.29 ppm
assigned to the H-5 of pyrimidine. The thiophene
protons showed the following splitting pattern: the H-4
appeared as triplet at δ = 7.30 ppm with coupling
constant J = 4.0 Hz, the H-5 resonated as a doublet of
doublet at δ = 7.93 ppm with coupling constant JH5,H4 =
5.0 Hz while the H-3 appeared as doublet of doublet at
δ = 8.29 ppm with coupling constant JH3,H4 = 4.0 Hz.
The hydrogen proton of the hydroxyl group resonates
as singlet at δ = 12.88 ppm. 13
C-NMR (100 MHz,
DMSO-d6) showed a signal at δ = 103.6 ppm assigned
for the C-5 of pyrimidine ring. The C-4 of pyrimidine
resonates at δ =163.5 ppm and C-6 appeared at δ =
160.7 ppm, while the signal appeared at δ = 165.1
assigned to C-2 of pyrimidine ring. The CF3 group split
as quadratic at 120.6 ppm. Thiophene carbons resonate
at δ = 129.1, 131.1, 132.8 and 140.5 ppm.
The synthetic pathway of the new p-fluorobenzoyl
pyrimidine analogues 4a,b is shown in Scheme 1.
p-Fluorobenzoyl chloride allowed to react with
2-hydroxypyrimidine analogues 3a,b in present of a
catalytic amount of pyridine under microwave protocol
to give the final products 4a,b in 85% and 83% yields,
respectively. The structure of
4-(thien-2-yl)-6-trifluoromethyl pyrimidin-2-yl
4-fluorobenzoate 4a was confirmed using IR
spectroscopic analysis. The IR spectrum of compound
4a showed a new absorption bands at 1,757 cm-1
due to
the carbonyl of the newly formed ester group. The band
at 1,603 cm-1
accounted for CC stretch in the aromatic
system. While the ether linkage (C-O-C) appeared as
two sharp signals at 1,056 cm-1
and 1,246 cm-1
. In
addition, the 1
H NMR spectrum revealed the
appearance of new two doublets of doublets at δ = 7.64
and 7.92 ppm with coupling constant J = 4.9 and 3.7 Hz
respectively assigned to the thiophene protons (H-3 &
H-5). The 13
C-NMR spectrum proved the proposed
structure due to the appearance of a signal at δ = 165.4
ppm corresponding to the carbonyl carbon of the newly
formed ester group at pyrimidine C-2, as well as the
Compound R1 Yield %
4a 2-thienyl 85
4b phenyl 83
Scheme 1. Microwave synthetic pathways of compounds 4a,b.
Reagents and conditions: (a) i- MW; 135 C, ethanol-HCl, ii- MW; 135 C, acetic acid; (b) p-fluorobenzoylchloride, pyridine, MW;
100 C.
19. Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues 15
change in the chemical shift of pyrimidine C-5 to
resonate at δ = 111.9 ppm. Moreover, the elemental
analysis of compound 4a with chemical formula
C16H8F4N2O2S showed the Anal. Calcd. C, 52.18; H,
2.19; N, 7.61; S, 8.71. found: C, 52.63; H, 2.26; N, 7.89;
S, 8.99.
A 35 mL CEM Microwave reactor containing
6-trifluoromethyl pyrimidin-2-ol 3a,b and 2
equivalents of phosphorus oxychloride (POCl3) in
ethanol with a catalytic amount of pyridine during 25
min furnished the 2-chloro-6-trifluoromethyl
pyrimidine 5a,b in ~95% yields (Scheme 2). Under
conventional conditions, this substitution reaction is
typically carried out using POCl3 as a solvent (100°C,
3-5 h) yielded 5a,b in 65-71% respectively. The
structure of the obtained products 5a,b was confirmed
using 1
H, and 13
C-NMR. The formation of
2-chloro-4-(thien-2-yl)-6-trifluoromethyl pyrimidine
5a was confirmed by shifting the signal corresponding
to the pyrimidine H-5 from δ = 7.29 ppm to δ = 7.84
ppm. Also, the 13
C-NMR showed the same shift of the
pyrimidine C-5 shifted from δ =103.6 ppm to a low
field at δ = 105.2 ppm.
Amination of 2-chloro-6-trifluoromethyl pyrimidine
5a,b was performed in CEM Microwave using
commercially available amines (2 equiv.) in toluene
(15 mL) at 100 °C and 150 W for 10 min. The reaction
produced the desired 2-(N-cycloalkylamino)-
6-trifluoromethyl pyrimidine 6a-f in 77-89 % isolated
yields (Scheme 2).
The formation of 2-(N-cycloheptylamino)-4-(thien-
2-yl)-6-trifluoromethyl pyrimidine 6b was confirmed
by elemental analysis, IR, 1
H NMR, and 13
C-NMR.
The IR spectrum of compound 6b showed absorption
bands at 3,531, 3,054, 2,884 cm-1
corresponding to the
stretching vibration of NH, C-H aromatic and C-H
aliphatic respectively. The 1
H-NMR spectrum of
compound 6b showed three multiplets resonated at δ =
1.55-1.64 ppm, 2.05-2.06 ppm and 4.09 ppm
corresponding to 13 protons of cycloheptyl ring. A
doublet observed at δ = 5.36 ppm with coupling
constant J = 4.0 Hz was attributed to the -NH proton.
While, a singlet corresponding to pyrimidine H-5
shifted to δ = 7.07 ppm. The 2-thienyl protons (H-4,
H-5 and H-3) resonated at δ = 7.13, 7.50 and 7.73 ppm
respectively. 13
C-NMR (100 MHz, DMSO-d6) showed
that the cycloheptyl carbons resonated as four signals at
δ = 24.2, 30.9, 34.6, 53.2 ppm. The pyrimidine C-5
shifted to δ = 101.0 ppm, and signals assigned for C-2,
C-4 and C-6 of the pyrimidine ring resonated at 167.2,
161.7 and 156.9 respectively. Thiophene C-3, C-4 and
C-5signals appeared at δ = 127.1, 128.8 and 131.2 ppm
respectively while, the thiophene C-2' resonated at δ =
141.9 ppm.
3.2 Antibacterial Screening
Among the antimicrobial agents, derivatives
containing thiophene like cephalothin, cephalorodine
and cefoxitin exhibit high antimicrobial potency [19].
It is also noticed that the antibacterial activities
enhance by the present of 2-thienyl ring at position-4
and this might be based on the fact that 2-thiophene has
shown an array of biological activities ranging from
antibacterial [20-24], antifungal [25,26], antioxidant
[27], and anti-inflammatory activity [28].
The antibacterial assay is based on the comparison of
growth inhibition of micro-organisms by measured
known concentrations of test compounds with that
Scheme 2. Synthetic pathway of compounds 5a,b and 6a-f.
Reagents and Conditions: a) i- MW; 100 C, POCl3, Pyridine, or ii- reflux, excess of POCl3; b) 2 equiv. of cyclopentylamine,
cycloheptylamine or N-Methylpiprazine in toluene, MW; 112 C.
20. Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues16
produced by known concentrations of standard
antibiotics [29]. All of the newly synthesized
compounds evaluated against Gram-positive bacteria
(Clostridium perfringens, Bacillus pumilus and
Enterococcus faecalis), and Gram-negative bacteria
(Klebsiella pneumonia). A total of eight compounds
were screened for in vitro antibacterial activity. The
screening results (Table 2) showed that compounds
4,6a,b were found to possess appreciable antibacterial
activity with a zone of inhibition greater than 10 mm
against the Gram-positive bacteria compared to the
results obtained from three standard drugs, whereas
compound 4b showed no activity against Bacillus
pumilus. Surprisingly, our synthesized analogues with
4-fluorobenzoyloxy 4a,b or N-cycloalkylamine 6a,b
substituted at pyrimidine C-2 displayed great
antibacterial enhancement against Enterococcus
faecalis with an inhibition zones ranging between
11-12 mm while, the standard drugs used showed zero
activity (Fig. 1). When compounds 4a,b tested against
Gram-positive bacteria (Bacillus pumilus), 4a showed
better activity with inhibition zone of 16 mm (MIC
12.5 µg/mL) while, 4b gave zero activity when tested
against the same bacteria stream. Interestingly,
compound 4b was found to have two folds more active
than 4a when both derivatives 4a and 4b tested against
Gram-positive bacteria (Clostridium perfringens) with
inhibition zones of 11 mm observed from both
compounds 4a,b. The MIC result (6.25 µg/mL) for
compound 4a indicates that an enhancement in the
activity observed (MIC = 3.125 µg/mL) when 4b was
used. Results obtained from the screening against
Gram-negative bacteria (Klebsiella pneumonia)
indicate that only compound 4a showed activity with a
zone of inhibition of 11 mm (MIC 12.5 µg/mL). The
inhibition zones of the newly synthesized compounds
are shown in Figs. 2 and 3.
Individual minimum inhibitory concentration (MIC,
μg/mL) values of active compounds 4a,b and 6a,b
against the test microbes listed in Table 3. The data
derived from the MIC test can be correlated with the
data obtained from in vitro to estimate the efficacy of
the new synthesized derivatives 4a,b and 6a-f.
4. Conclusion
In conclusion, novel fluoropyrimidine analogues
3-6a-f have been synthesized using microwave
protocols. The antimicrobial screening of the newly
synthesized compounds bearing 2-thienyl group
substituted at the pyrimidine ring 4a,6a,b showed better
Table 1 Yields obtained for compounds 5,6a-f under microwave and conventional methods.
Compound R1
Yield % (t)
Compound R1 R2
Yield %
Microwave Conventional Microwave
5a 2-thienyl 96 (25 min) 65 (3.5 h) 6a 2-thienyl cylopentylamine 86
5b phenyl 95 (25 min) 71 (4 h) 6b 2-thienyl cycloheptylamine 77
6c 2-thienyl N-methylpiprazine 89
6d phenyl cylopentylamine 78
6e phenyl cycloheptylamine 83
6f phenyl N-methylpiprazine 82
Table 2 Inhibition zones (mm) as a criterion of antibacterial activity of the active compounds 4,6a,b.
Bacteria
Inhibition zones (mm)
Compounds
DMSO Ampicillin Tetracycline Streptomycin
4a 4b 6a 6b
Clostridium perfringens 11 11 10 13 0 20 31 32
Bacillus pumilus 16 NA 11 18 0 33 30 32
Enterococcus faecalis 11 11 11 12 0 NA NA NA
Klebsiella pneumonia 11 NA NA NA 0 27 32 30
NA: no activity observed.
21. Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues 17
Fig. 1 Comparison of inhibition zones’ values of the synthesized compounds 4,6a,b against Enterococcus faecalis vs standards
antimicrobial drugs: ampicillin, tetracycline, and streptomycin.
(a) (b)
Fig. 2 (a) zone of inhibition by 4a,b, and 6a,b (b) zone of inhibition by standard antimicrobials.
Fig. 3 Inhibition zones (mm) of the newly synthesized compounds 4,6a,b.
0
2
4
6
8
10
12
Zoneofinhibition(mm)
0
5
10
15
20
25
30
35
Inhibitionzones(mm)
Clostridium perfringens
Bacillus pumilus
Enterococcus faecalis
Klebsiella pneumoniae
22. Synthesis and Antimicrobial Studies of New Trifluoromethylpyrimidine Analogues18
Table 3 Minimal inhibitory concentration (MIC) for compounds 4,6a,b.
Compound
MIC (µg/mL)
Enterococcus faecalis Bacillus pumilus Clostridium perfringens Klebsiella pneumoniae
4a 50 12.5 6.25 12.5
4b 50 - 3.125 -
6a 25 25 100 -
6b 12.5 100 50 -
antimicrobial activities than other derivatives. For
instance, compounds 4,6a,b were found to be more
effective than the reference drugs, ampicillin,
tetracycline and streptomycin, when tested against
Gram-positive bacteria (Enterococcus faecalis).
Compound 4a was found to be the only active
compound when tested against Gram-negative bacteria
(Klebsiella pneumonia). Thus, in the future, this class
of compounds can be used as template to design new
derivatives that might help to enhance the activity
when used to defeat the bacterial infection.
Acknowledgments
The authors gratefully acknowledge UAE
University, Research Affairs Sector for providing
financial support (grant no. 31S030-1156-02-02-10).
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24. Journal of Pharmacy and Pharmacology 3 (2015) 20-26
doi: 10.17265/2328-2150/2015.01.003
Production of Glucosamine Hydrochloride from
Crustacean Shell
Martha Benavente1,2
, Selene Arias1
, Luis Moreno2
and Joaquín Martínez2
1 Department of Chemical Engineering, National University of Engineering, Managua 5595, Managua, Nicaragua
2 School of Chemical Science and Engineering, KTH Royal Institute of Technology, SE-100 44, Stockholm, Sweden
Abstract: The use of chitin as raw material to obtain glucosamine hydrochloride at laboratory level was investigated. Chitin was
extracted from shrimp shells by deproteinization, demineralization and depigmentation. Afterwards, glucosamine hydrochloride was
produced in four main stages: (1) acid hydrolysis of chitin with 12 M hydrochloric acid using the reflux technique; (2) filtration of
the solution to discard solid impurities; (3) recrystallization of the product using 95% ethyl alcohol as solvent, and (4) filtration,
washing and drying of final product at 50 °C. The FTIR spectrum of the product was compared to a commercial glucosamine
hydrochloride of 99.86% purity, and a coincidence between 96.90% and 99.66% was obtained. The influence of temperature,
solid/liquid ratio (g/mL), and agitation (with-without) on acid hydrolysis was studied. The best correlation corresponds to the
hydrolysis product obtained at solid/liquid ratio of 1:20, temperature of 85 °C, and with agitation. The yields of glucosamine
hydrochloride with respect to chitin were 42, 58, 36 and 48% for solid/liquid ratios of 1:10, 1:20, 1:30, and 1:40 respectively, at high
hydrolysis reaction temperature and with agitation. These results showed that in the range examined, glucosamine hydrochloride with
high quality is produced with solid/liquid ratio of 1:20.
Key words: Acid hydrolysis, chitin, glucosamine, reflux technique, shrimp shells.
1. Introduction
D-glucosamine (C6H13NO5) or
2-amino-2-deoxy-D-glucose is an amino sugar
(hexosamine) with a molecular weight of 179.17,
naturally present in human body and crustacean shells.
It is a precursor of biochemical synthesis of the GAGs
(glycosaminoglycans) found in cartilage. Premature
loss of cartilage is part of the clinical syndrome
recognized as OA (osteoarthritis) [1].
Glucosamine in the form of glucosamine sulphate,
glucosamine hydrochloride, or N-acetyl-glucosamine
is extensively used as a dietary supplement in the
treatment for osteoarthritis, knee pain, and back pain
[2, 3], and a critical evaluation indicated that
glucosamine is safe under current conditions of use
and does not affect glucose metabolism [4].
Glucosamine can be prepared by acid hydrolysis [5,
Corresponding author: Martha Benavente, LicEng in
chemical engineering, research field: process and
environmental engineering. E-mail: bena@kth.se.
6] using strong mineral acids or by enzymatic
hydrolysis [7] using bacterial chitinase. Different
methods for acid hydrolysis of chitin to produced
glucosamine hydrochloride (G-HCl) have been
studied. Leite et al. [5] used the reflux technique to
hydrolyze chitin with 37% hydrochloric acid (1:5 S/L
ratios) at 100 °C and under different reaction times.
Novikov [6] carried out the acid hydrolysis of chitin
and chitosan with 36.5% HCl at 50 and 70 °C. Li et al.
[8] increased the temperature from 60 to 90 C to
optimize the preparation process of G-HCl.
Although glucosamine can be produced from
different natural sources, e.g., chitin and fermentation
of corn and wheat, the most effective one is derived
from chitin of shellfish [9]. Chitin is a natural
polysaccharide, no toxic, biodegradable and is part of
the structural material of the fungal cell walls, insect
exoskeletons and crustacean shells. Chitin and its
derivative chitosan have a wide range of applications
in different areas such as agriculture, drinking water
DDAVID PUBLISHING
25. Production of Glucosamine Hydrochloride from Crustacean Shell 21
and wastewater treatment, food and beverages,
cosmetics and toiletries, biomedics and pharmaceutics,
fibres and textiles, and paper technology [10].
Nicaragua produces thousands of tons of
crustaceans such as shrimps and prawns annually [11]
and the waste shells, the raw material to produce
chitin constitute approximately 40%-50% of the total
weight [12]. Currently, the wastes from seafood
factory operations are available in large quantities.
The use of these waste shells can be a low cost
alternative to obtain valuable products such as chitin,
chitosan and G-HCl.
The aim of this work is the use of crustacean shell
as raw material to obtain glucosamine hydrochloride
at laboratory level. For this purpose, chitin was
extracted from shrimp shells using a chemical
treatment, and glucosamine hydrochloride was
produced by acid hydrolysis of chitin with 12 M
hydrochloric acid using the reflux technique.
2. Materials and Methods
2.1 Materials
Shrimp waste (head, legs, shell, and tail) was
provided by Camanica (PESCANOVA Group), a
shrimp processing facility located in Chinandega,
Nicaragua.
Chitin and D–Glucosamine HCl was purchased
from Jining Green Group Co. Ltd, Shandong, China.
D–Glucosamine HCl is a white crystalline free
flowing powder with a 99.86% purity and -400 mesh
particle size.
2.2 Chitin Extraction
Chitin was extracted from shrimp waste by
desproteinization, demineralization and
depigmentation at laboratory level. The Fig. 1 shows a
block diagram of the overall process. The raw material
was first submerged in 10% (wt) NaOH solution for 2
h with constant agitation to remove proteins. The
desproteinized material was then demineralized using
1.8 mol/L HCl solution for 12 h and subsequently, the
product was submerged in 0.38 wt% NaClO solution
for 1 h with agitation to remove pigments. Chitin was
washed several times using distilled water until pH
between 6 and 7 was achieved and dried at 50°C
during 10 h. Finally, chitin was milled and screened to
select the fraction of particles with a size lower than
0.22 mm.
2.3 Glucosamine Hydrochloride Production
According to block diagram in Fig. 1, the principal
stages for G-HCl production from chitin were: (1) acid
hydrolysis of polysaccharide; (2) filtration of the
solution, (3) recrystallization of the product, and (4)
filtration, washing and drying of final product at 50°C.
The acid hydrolysis procedure was performed with
12 M hydrochloric acid using the reflux technique
according to the procedure in Ref. [4]. However, in
preliminary experiments under these conditions
(hydrolysis for 1 h at 100°C) it was observed that the
results were not satisfactory because HCl was
consumed very rapidly in the first minutes of the
reaction. Based on this experience, the acid hydrolysis
was carried out in two experimental sets at different
conditions (Table 1): (1) two different solid/liquid
ratios, at different temperatures and agitation (with
and without) and (2) different solid/liquid ratios, at
temperatures range of 68-85 °C and with agitation.
HCl was previously heated to 60°C before the addition
of chitin, according to procedure in Ref. [7]. All
experiments were carried out in duplicate.
The process for G-HCl production was performed
as follows: 1 g of chitin and an amount of 12 M HCl
(depending on solid/liquid ratio) were introduced into
a 150 mL reflux vessel. The mixture was kept at the
given temperatures until the solid was completely
dissolved. The resulting hydrolyzed was filtered by
gravity through a VWR Grade 474 filter paper to
remove the solid particles present in solution, and it
was left to crystallize at room temperature (25 ± 2°C)
by 25 days. In order to increase the crystallization rate,
ethyl alcohol (15 mL, w = 95%) was added, and the
26. Production of Glucosamine Hydrochloride from Crustacean Shell22
Fig. 1 Block diagram for the extraction of chitin from shrimp shells and production of glucosamine hydrochloride.
Table 1 Work conditions in each experiment set for acid hydrolysis of chitin.
Experiment set 1 Experiment set 2
Exp No. Solid/liquid ratio Temperature (°C) Agitation Exp No. Solid/liquid ratio
1.1 1:10 40 Without 2.1 1:10
1.2 1:10 68 With 2.2 1:20
1.3 1:20 58 Without 2.3 1:30
1.4 1:20 85 With 2.4 1:40
resulting mixture was cooled to 5°C for 15 days. The
mixture was once more filtered, and the solid crystals
were washed with ethyl alcohol and dried at 50 °C in
an oven for 12 h.
2.4 Collection of FTIR Spectra
The spectra of chitin and G-HCl obtained in these
experiments were compared with the commercial
references. A Bruker Optics ALPHA FT-IR
spectrometer was used to determine their spectra. A
spectral range between 400 and 4,000 cm-1
was used.
3. Results and Discussion
3.1 Chitin Characterization
The principal components of shrimp shells such as
proteins, calcium minerals, and pigments were
0.38%
NaClO
Effluent
(pigments)
Demineralization
(0.50 kg/L)
Shrimp waste
1.8 M HCl
Effluent
(CaCl2)
Depigmentation
(0.15 kg/L)
Chitin
Washing
Washing Drying
Recrystallization
(5°C)
95% Ethyl
alcohol
Filtration
Solid impurities
Drying of crystals at
50°C
Ethyl alcohol
Acid Hydrolysis (Reflux
technique)
12 M HCl
Glucosamine
hydrochloride
Grinding & sieving at
particle size < 0.22mm
Washing
Effluent
(proteins)
10% NaOH
Deproteinization
(0.34 kg/L)
Washing
Filtration
Filtrate (Ethyl alcohol +
HCl + acetic acid)
27. Production of Glucosamine Hydrochloride from Crustacean Shell 23
removed to get chitin as final product. The average
content of chitin in the raw material (dry basis) was
25 %. The identification of chitin by comparing its
FTIR spectrum with that of a reference sample
showed a correlation coefficient of 95%.
Fig. 2 shows FTIR spectrum of product in the
4,000-400 cm-1
region where the different
characteristic bands of the molecular structure of
-chitin can be identified. The fundamental vibrations
are due to O–H and N–H stretching band (in the range
3,700-3,000 cm-1
), C–H stretching band (in
3,000-2,850 cm-1
) and carbonyl group (in 1,830-1,650
cm-1
). In last range, two peaks are displayed: one
which is attributed to the occurrence of intermolecular
hydrogen bond COHN at 1,660 cm-1
and another
due to the intramolecular hydrogen bond COHOCH2
at 1,625 cm-1
. The band split is used to distinguish
between -chitin and -chitin since a single band is
observed in case of the -chitin at 1,656 cm-1
[13].
3.2 Glucosamine Production and Characterization
The influence of temperature, solid/liquid ratio
(g/mL), and agitation (with-without) on acid
hydrolysis was studied. During the hydrolysis process,
it was observed that chitin was completely dissolved
in a period of 3 to 18 min, and immediately after that
the solution darkens, becoming a brown colour. The
dissolution of chitin was influenced by temperature
and agitation. The hydrolysis reaction was faster when
the mixture was agitated and temperature was high.
The change to brown colour in solutions can be
caused by the Maillard reaction which involves amino
groups reacting with an aldehyde and can lead to
colour change associated with hydrolysis of chitin
[14].
The hydrolysis process involves two acid-catalyzed
hydrolysis reactions: the glycosidic linkage
(depolymerisation) and the N-acetyl linkage
(deacetylation). Hackman [15] observed that most of
the degradation of the chitin chain occurred during the
first few minutes, and that the products formed were
oligosaccharides. Fig. 3 illustrates the most accepted
reaction mechanism for the acid catalyzed hydrolysis
of a glycosidic linkage (SN1) and the reaction
mechanism for the acid-catalyzed hydrolysis of the
N-acetyl linkage (SN2 reaction) [16, 17].
It was observed that the recrystallization process
was slow at room temperature (25 ± 2 °C). Therefore,
in order to increase the crystallization rate and favour
the crystal formation, the mixture was cooled to 5°C
and 95% ethyl alcohol was added as solvent. The
results showed that the crystallization time decreased
and the crystals formed were thinner, clearer and
brighter in these conditions. According to Myerson
[18], the solvent can have a significant effect on the
solubility of the solute, the structure and crystal size, as
well as morphology and purity of the crystals.
Temperature plays an important role in crystallization
since it can influence nucleation and crystal growth via
its effects on the solubility and supersaturation of the
Fig. 2 FTIR spectra of -chitin produced from shrimp waste (head, legs, shell, and tail).
28. Production of Glucosamine Hydrochloride from Crustacean Shell24
Fig. 3 Schematic illustration of the proposed reaction mechanism for (a) the acid-catalyzed hydrolysis of the glycosidic
linkage in chitosan [16] and (b) for the acid-catalyzed hydrolysis of the N-acetyl linkage [17].
Table 2 Results of the glucosamine hydrochloride production according to experimental conditions of set 1.
Exp No. Solid/liquid ratio Yield (%) FTIR spectrum correlation* (%)
1.1 1:10 34.0 96.90
1.2 1:10 42.0 98.76
1.3 1:20 56.0 99.53
1.4 1:20 58.0 99.66
*With regard to the commercial reference.
Table 3 Glucosamine hydrochloride production at different solid/liquid ratio at 68-85 °C and with agitation.
Exp No. Solid/liquid ratio Yield (%) FTIR spectrum correlation* (%)
2.1 1:10 42 98.76
2.2 1:20 58 99.66
2.3 1:30 36 99.36
2.4 1:40 48 99.14
*With regard to the commercial reference.
sample.
Table 2 shows the results of the yields of G-HCl
and the correlation coefficient for the comparison
between the product and the reference for the
experiments of set 1. In general, the results showed
that the yield of G-HCl can increase with temperature
and the agitation of the sample. The increase of
temperature and the use of agitation lend to the result
that chitin dissolves faster and this can contribute to a
more effective hydrolysis.
Table 3 shows the results at different solid/liquid
ratios, and in a temperatures range of 68-85 °C and
with agitation. We can observe that the yields of
G-HCl with respect to chitin were 42, 58, 36 and 48%
for solid/liquid ratios of 1:10, 1:20, 1:30, and 1:40
respectively. The best yield was obtained at
solid/liquid ratio of 1:20. The production of G-HCl is
greatly influenced by the experimental conditions;
however, it is not possibly to extract a conclusive
tendency from the experiments.
The FTIR spectra were also compared and a
coincidence between 98.76% and 99.66% was
obtained. The highest correlation corresponds to the
hydrolysis product obtained at solid/liquid ratio of
1:20.
3.3 FTIR Analysis of Glucosamine Hydrochloride
(G-HCl)
The FTIR spectra provided by Bruker Optics
ALPHA FT-IR spectrometer, were used to identify
29. Production of Glucosamine Hydrochloride from Crustacean Shell 25
Fig. 4 FTIR spectra of glucosamine hydrochloride produced under Exp 2.2 conditions (Sample) and D-glucosamine HCl
from Jining Green Group Co. Ltd (reference).
the product and determine the degree of similarity.
The results of the comparison between the spectra of
G-HCl products and the commercial reference
revealed that a coincidence between 96.90 and
99.66% was obtained. The highest correlation
corresponds to the hydrolysis product obtained at
solid/liquid ratio of 1:20, temperature of 85 °C, and
with agitation (Exp 2.2). Lower values were obtained
for lower temperature, larger solid/liquid ratio and
without agitation. These results showed that in the
range examined, G-HCl with high quality is produced
with solid/liquid ratio of 1:20.
The FTIR spectra of G-HCl produced under the
work conditions of Exp 2.2 (sample) and G-HCl
commercial reference are displayed in Fig. 3. It shows
that the FTIR spectra of both materials are essentially
identical with regard to the band-positions of G-HCl
main groups. This fact is confirmed by the correlation
coefficient of 99.66% which indicates a very high
degree of similarity between sample spectrum and the
reference spectrum.
The FTIR spectrum of G-HCl produced (sample in
Fig. 4) exhibits an intense band at 3,370-3,300 cm-1
associated with the O–H and N–H stretching, a NH2
scissoring band at 1,615 cm-1
and at 1,094 cm-1
due to
secondary alcohol –OH.
4. Conclusions
In conclusion, these results showed that in the range
examined, G-HCl with good quality is produced with
solid/liquid ratio of 1:20, at high hydrolysis reaction
temperature and with agitation. Additionally, the low
temperature (5°C) and the use of ethyl alcohol support
the formation of G-HCl crystals. Although, it is
possible to convert waste materials into valuable
products such as glucosamine, more experimental
work should be carried out to optimize the process. As
well, additional information about biological and
chemical behaviour should be necessary in order to
assure if this product is suitable for dietary
supplement.
Acknowledgements
The financial support of the Swedish International
Developments Cooperation Agency (Sida) is
gratefully acknowledged.
References
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31. Journal of Pharmacy and Pharmacology 3 (2015) 27-32
doi: 10.17265/2328-2150/2015.01.004
Evaluation of Liver Function tests (AST & ALT) in
Patients with Hepatitis B and C in Tabriz-Iran (2013)
Navid Sarakhs Asbaghi1
, Kazem Ghahreman Zadeh2
, Taher Faraj Zadeh1
, Javid Lotfi Attari1
, Zahra Javan
Masoomi1
, Rana Faraj Zadeh1
, Mohammad Reza Tarmohammadi1
, Alireza Bakhtarai1
, Behzad Bahram Zadeh1
and Babak Morshed Zadeh1
1. Faculty of Basic Sciences, Department of Genetics, Islamic Azad University, Tabriz 51589, Iran
2. Clinical Central Lab of Province, Tabriz 51589, Iran
Abstract: Viral hepatitis is among the infections that primarily affect the liver and is one of the main causes of death in the world.
Every year, more than one million people worldwide die of viral hepatitis. In recent decades, the number of people with hepatitis B
and C has declined in Iran. The purpose of this study was to investigate normal and abnormal liver enzymes (AST, ALT) in patients
with chronic hepatitis B and C in a number of public and private laboratories in Tabriz. In the study conducted in 2013, of those who
had referred to clinical laboratories for various reasons or who had been reported by centers of infectious or dialysis therapy, a
sample of 1,000 patients were identified with hepatitis B and C; 693 people had hepatitis B and 307 people had hepatitis C. On a
sample of patients, liver enzymes were evaluated using standard methods. The percentage of women and men in this study were
inconsistent with global statistics. However this inconsistency could be justified by the alcohol consumption and an increase in the
number of addicted people in society as well as women’s fear due to some social issues.
Key words: Hepatitis B & C, liver function tests, AST & ALT, Tabriz.
1. Introduction
Hepatitis is the inflammation of liver and is
believed to disrupt the activity of it. Hepatitis B virus
(HBV) belongs to the family of Hepadnaviridae. It
only attends limited number of hosts. It sometimes
infects pancreas and kidneys of humans and monkeys.
This small capsidated virus has a double-stranded,
circular DNA with a single-stranded part and contains
RTase (reverse transcriptase enzyme) which is
attached to the virus genome and represents
ribonuclease activity. Hepatitis B infection causes
liver problems, kidney problems, liver cirrhosi, and
high risk of liver cancer. However, another risk that
threatens people with chronic hepatitis B, is
simultaneous infection to hepatitis D [1]. Hepatitis C
virus belongs to the Flaviviridae family. It is a small
virus coated with positive single-stranded RNA and
Corresponding Author: Navid Sarakhs Asbaghi, M.Sc.,
research field: genetic and clinical laboratory medicine. E-mail:
dr_navid_asbagi@yahoo.com.
infection with this type of hepatitis causes liver
dysfunction, and liver failure resulting in the liver
cirrhosis and liver cancer in rare cases [1, 2].
Hepatitis B infection is a major health concern of
the world and a leading cause of liver cancer. About
400 million people around the world are infected with
this disease, from which 75% are Asian. Based on an
estimation by World Health Organization, each year
about 1.2 million people affected by the hepatitis B
virus lose their lives [3, 4]. Among these people, only
10% are diagnosed with a chronic illness. The
prevalence of HBV infection in different parts of the
world can be divided into three categories: low
prevalence (less than 2%) in some parts of America,
Australia and northern Europe; medium prevalence
(2%-7%) as in many parts of Asia, North Africa and
eastern regions of South America and high prevalence
(over 8%) as in Africa, coasts of South East Asia and
Alaska. In areas with high prevalence, infection
usually occurs at birth. In other parts of the world,
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