ARIZONA STATE UNIVERSITY
Application of Hydroxyapatite/Camphor Composites as Catalysts Spirooxindole-
Chromene Derivative
JEFF. M
Background
The shells discarded after consuming the meat of mollusks produce shell
waste. Shells make up about 55% of the total weight of the mollusk. Blood cockle
shells contain between 89 and 99% calcium carbonate, which can be used as a
calcium precursor for the synthesis of hydroxyapatite compounds. Hydroxyapatite
(HAp), with the general formula Ca10(PO4)6(OH)2, can act as a heterogeneous
catalyst due to its acid-base sites in its structure, which is related to the Ca/P ratio
in HAp compounds. In this study, hydroxyapatite will be combined with camphor to
enhance its catalytic capabilities. Camphor, with the chemical formula C10H16O, is
obtained from the distillation of the bark of the camphor tree, Cinnamomum
camphora. One benefit of camphor is its role as a catalyst, classified as an
organocatalyst. Organocatalysts are organic compounds with catalytic activity that
do not contain heavy metals, making them environmentally friendly. The use of
catalysts aligns with green chemistry principles, aimed at improving chemical
reaction efficiency. One group of organic compounds currently being studied for
their bioactivity is spirooxindole derivatives. Spirooxindole is a compound
characterized by its various rings fused to an oxindole core. It has a wide range of
applications, including as an anti-cancer, anti-inflammatory, antimicrobial,
antioxidant, antiviral, and anti-malarial agent. Heterocyclic compounds with a
combination of benzene and pyran rings are known as benzopyran or chromene.
Chromene is an important structural component of natural compounds and is
biologically significant due to its various activities such as anti-vascular,
antimicrobial, anti-cancer, estrogenic, and anti-inflammatory effects. The
integration of chromene with spirooxindole provides rigidity to the compound
through conformational constraints, potentially influencing biological activity. The
field of green chemistry addresses environmentally friendly methods for chemical
reactions by minimizing waste and by-products. One-pot multicomponent
reactions (MCRs) are a green chemistry approach where multiple reactants are
combined in a single step to yield products, offering advantages like reduced
reaction time, lower waste, simplified separation, and higher yields compared to
multistep synthesis. Energy efficiency is a key principle of green chemistry, and
microwave-assisted heating provides benefits such as reduced reaction times due
to simultaneous heat generation with reactants or solvents. Combining microwave-
assisted methods with one-pot MCRs presents a cost-effective and efficient strategy
for synthesizing a variety of products. This study utilizes the microwave-assisted
one-pot MCR method with hydroxyapatite/camphor catalyst to synthesize
spirooxindole-chromene derivatives.
Literature Review
Heterocyclic
Heterocyclic compounds are organic cyclic substances that include all carbon
atoms within the ring structure, and are referred to as carbocyclic compounds.
When at least one atom other than carbon is part of the ring system, it is termed a
heteroatom. The most common heteroatoms are nitrogen, oxygen, and sulfur,
though heterocyclic rings containing other heteroatoms are also widely recognized.
Heterocyclic compounds represent the largest and most diverse group of organic
substances. They feature structural motifs present in a vast number of natural and
synthetic compounds and play significant roles in biological activity, agrochemicals,
and pharmaceuticals.
Spirooxindole compounds
Spirooxindole compounds are polycyclic molecules containing at least two rings
joined by a single atom, typically carbon, which were previously referred to as
spirans. These compounds are now known as spiro or spirocyclic compounds, and
the central atom is termed the spiro atom. Although carbon is often the spiro atom,
other elements like nitrogen, phosphorus, and arsenic can also fulfill this role.
Spirooxindoles are characterized by various rings fused at the C3 position of the
oxindole core. They have broad applications, including anticancer, anti-
inflammatory, antimicrobial, antioxidant, antiviral, and antimalarial properties. The
class of spiro compounds, in general, has been relatively underexplored, though
recent efforts have focused on their synthesis and characterization. Many spiro
compounds exhibit promising biological activities, such as acting as anticancer,
antibacterial, anticonvulsant, antituberculosis, anti-Alzheimer, analgesic, anti-
dermatitis, and antimicrobial agents. Some spiro compounds have also found
applications in agriculture and industry, serving as antifungal agents, pesticides,
laser dyes, and electroluminescent devices, with recent uses as antioxidants.
Chromenes are heterocyclic compounds with a benzene ring fused to a pyran ring.
2H-chromene (2H-1-benzopyran) and 4H-chromene (4H-1-benzopyran) are bicyclic
heterocyclic compounds with oxygen as the heteroatom, formed from the
combination of a benzene ring with a 2H-pyran or 4H-pyran ring. In 2H-chromene,
the carbon atom at position two participates in a double bond with oxygen,
nitrogen, or sulfur, or alternatively, forms two single bonds with different
substituents.
Multicomponent reactions
Multicomponent reactions are integral to organic synthesis within green chemistry,
which aims to minimize waste and byproducts at each stage of synthesis. Ideally,
the synthesis should produce the desired product in as few steps as possible, with
high overall yield and by using environmentally friendly reagents. Multicomponent
reactions (MCRs) are designed to generate a single product in a one-step procedure,
utilizing three or more reactants. The one-pot MCR method often shortens reaction
time, results in a higher overall chemical yield compared to multi-step synthesis,
and consequently reduces energy consumption. Due to these advantages, the
development of new MCRs using eco-friendly methods remains a significant and
ongoing challenge in green chemistry.
Microwave-assisted methods
Microwave-assisted methods utilize microwaves, which have frequencies between
0.3 GHz and 300 GHz in the electromagnetic spectrum, positioned between radio
frequencies (RF) and infrared (IR). Most microwave instruments operate at 2.45 GHz,
corresponding to wavelengths between 1 mm and 1 m. When molecules with
permanent dipoles are placed in an electric field, they align with the field. As the
electric field oscillates, the orientation of the molecules changes in response to
each oscillation. This causes the molecules exposed to the electromagnetic
radiation's wavelength to become highly agitated and align with the oscillating field,
generating intense internal heat that can increase by as much as 10°C per second.
Nonpolar molecules like toluene, carbon tetrachloride, diethyl ether, and benzene
are inactive toward microwaves, while polar molecules such as DMF, acetonitrile,
CH2Cl2, ethanol, and H2O are microwave-active, meaning they can align with the
electric field. Traditional heating methods for organic synthesis at high
temperatures rely on conductive heating from an external heat source. This method
is relatively slow and inefficient in transferring energy into the system, as it depends
on the thermal conductivity of various materials that must be penetrated, resulting
in the vessel's temperature being higher than the reaction mixture's temperature.
In contrast, microwave radiation generates efficient internal heating by directly
coupling microwave energy with the molecules (e.g., solvents, reagents, catalysts)
present in the reaction mixture.
Homogeneous catalysts
A catalyst is a substance that increases the rate of a chemical reaction without
being permanently involved in the reaction. It accelerates reaction kinetics by
providing an easier pathway for molecules to follow. In this way, a small amount of
catalyst can transform a large quantity of reactants, often under milder conditions
than those required by the stoichiometric reaction pathway. Catalysts are generally
classified as either homogeneous or heterogeneous. Homogeneous catalysts are
typically well-defined chemical compounds or coordination complexes that are
molecularly dispersed in the reaction medium along with the reactants. In
heterogeneous catalysis, the reactants and the catalyst are in different phases, with
solid catalysts most commonly used alongside gas or liquid reactants, or
sometimes both. The advantage of using a heterogeneous catalyst is that it can be
easily separated from the reaction mixture by simple filtration at the end of the
reaction, ensuring that the product is not contaminated by the catalyst and
allowing the catalyst to be reused in subsequent reactions.
Hydroxyapatite
Calcium phosphate forms a unique class of biomaterials due to its excellent
biocompatibility, potential biodegradability, and possible bioreactivity. Depending
on the Ca/P ratio, several families of calcium orthophosphates can be defined, such
as pyrophosphate (Ca/P=1), octacalcium phosphate (Ca/P=1.33), tricalcium
phosphate (Ca/P=1.5), hydroxyapatite (Ca/P=1.5; C/P=1.67), and tetracalcium
phosphate (Ca/P=2). Hydroxyapatite (HAp) [Ca10(PO4)6(OH)2] is a biocompatible
polycrystalline ceramic material with a chemical composition and crystal structure
similar to human bone and teeth. HAp possesses unique properties such as low cost,
chemical and thermal stability, and extreme water insolubility (Kps = 10
−
59 at room
temperature). These attributes have generated significant interest in its
applications in the field of catalysis. In heterogeneous catalysis, the acid-base
properties of solid catalysts are known to play a crucial role in their catalytic
performance, with the quantity and strength of acid/base sites being key factors
driving the activity and selectivity of many catalytic reactions, not only in acid-base
transformations but also in reduction and oxidation reactions. Hydroxyapatite
exhibits the rare property of containing both acid and base sites within its structure,
a characteristic closely correlated with the Ca/P atomic ratio in calcium phosphate
compounds. Thus, it can be suitably modified to achieve desired acid and/or base
functions. In fact, the higher the Ca/P ratio, the lower the density of acid sites and
the higher the density of base sites. At a Ca/P ratio of 1.50, HAp acts as an acidic
catalyst, but when Ca/P = 1.67, hydroxyapatite displays basic behavior. Therefore,
when the Ca/P ratio is between 1.50 and 1.67, HAp develops both acidic and basic
properties.
Camphor (C10H16O)
Camphor (C10H16O) is a white crystalline substance with a strong odor and a
pungent taste, derived from the plant *Cinnamomum camphora* and other related
trees in the laurel family. It is obtained through the steam distillation, purification,
and sublimation of the wood, branches, and bark of these trees. Camphor has
numerous pharmaceutical applications, including as an analgesic, antiseptic, anti-
inflammatory, anti-infective, and cough suppressant. It serves as an intriguing
starting material for the synthesis of various useful synthetic building blocks and
biologically significant heterocyclic compounds due to its several reactive sites. A
range of C-C and C-heteroatom bond-forming reactions, such as Knoevenagel,
Michael, Diels-Alder, cycloaddition, and others, are catalyzed by camphor
derivatives as organocatalysts. Camphor-10-sulfonic acid functions as a Brønsted
acid catalyst, camphor sulfonyl hydrazine acts as a Lewis base catalyst, and
camphor pyrrolidine and camphor thiourea derivatives are considered the most
important camphor derivatives used in organocatalytic reactions.
Thin Layer Chromatography (TLC)
Thin Layer Chromatography (TLC) is a separation method that can be used to
assess the purity of a mixture. TLC is a type of liquid chromatography where the
mobile phase is a liquid, and the stationary phase is a thin layer on a flat plate. The
working principle of TLC is based on partitioning, where the components of a
compound are separated according to the liquid phase. The procedure involves
spotting the sample onto a TLC plate coated with silica gel (SiO2), which is then
placed in a chamber containing the mobile phase (solvent). Once the solvent rises
near the top of the plate, the plate is removed, dried, and visualized using UV light.
UV-Vis spectrophotometry
UV-Vis spectrophotometry is an analytical technique that involves the absorption of
ultraviolet (180-390 nm) or visible (390-780 nm) radiation by a chemical molecule in
solution or gas phase. The working principle of a UV-Vis spectrophotometer is that
different compounds contain chromophore groups, which absorb specific
wavelengths of UV or visible light. A UV-Vis spectrophotometer consists of five main
components: a light source, monochromator, sample holder (cuvette), detector,
and computer. In a UV-Vis spectrophotometer, a beam of light from a suitable UV or
visible light source passes through a prism or diffraction grating monochromator.
The light then passes through the sample to be analyzed before reaching the
detector.
Fourier Transform Infrared Spectroscopy (FTIR)
Fourier Transform Infrared Spectroscopy (FTIR) is a method for determining
compounds based on the identification of functional groups within molecules.
These functional groups undergo vibrations, either stretching or bending in various
ways, when exposed to light of a specific wavelength. The resulting vibrations and
their intensities (% transmission) are plotted against the light frequency (cm-1)
exposed to the sample, producing an FTIR spectrum. The basic components of FTIR
instrumentation include an infrared light source, interferometer, sample chamber,
detector, laser, and computer. The polychromatic infrared radiation emitted from
the source enters the interferometer, where a specific signal pattern is generated.
The resulting light passes through the sample chamber, where it is either
transmitted or reflected off the sample surface. The sample absorbs specific energy
frequencies unique to its composition. The radiation is then directed to the
detector, which continuously monitors the range of infrared wavelengths, and a
computer is needed to visualize the generated spectrum.
X-ray diffraction (XRD)
X-ray diffraction (XRD) is a technique that provides chemical information for
element and phase analysis. It is used primarily for characterizing material
properties such as crystal structure, crystal size, and strain within crystals. The
main components of an XRD instrument include an X-ray tube, a sample holder, and
an X-ray detector. The process involves X-rays striking atoms in a crystal, causing
scattering phenomena. When the scattered X-rays from one atomic plane are in
phase with those scattered from another plane, diffraction rays are produced.
These diffraction rays are then detected, processed, and analyzed. The intensity of
the diffracted rays at various angles is plotted to generate a diffraction pattern.
The Scanning Electron Microscope (SEM)
The Scanning Electron Microscope (SEM) is a technique used to capture images of
the microstructure and morphology of compounds. The SEM instrumentation
includes an electron source, column, deflection system, electron detector, sample
chamber, and computer system. In SEM, a low-energy electron beam is directed at
the sample to scan its surface. Various interactions occur as the beam hits and
penetrates the material, leading to the emission of photons and electrons from
near the sample's surface. The signals generated from these interactions are
detected by various types of detectors to create an image.
Work Procedure
Synthesize Hydroxyapatite (HAp)
To synthesize Hydroxyapatite (HAp), shell waste is first cleaned by boiling in water
for 30 minutes and then rinsed with distilled water. The cleaned shells are dried
overnight in an oven at 80°C. The dry shells are then weighed and heated in a
furnace at 900°C for 8 hours. The resulting calcium oxide (CaO) is then ground. The
yield is calculated by comparing the mass change of the sample to its initial mass.
For HAp synthesis, 50 mL of 0.1M EDTA solution and 50 mL of 0.06M Na2HPO4.2H2O
solution are required. The 0.1M EDTA solution is prepared by dissolving 1.461 grams
of EDTA in 50 mL of distilled water, and the 0.06M Na2HPO4.2H2O solution is made
by dissolving 0.534 grams of Na2HPO4.2H2O in 50 mL of distilled water. 2.8 grams
of CaO is mixed with 50 mL of 0.1M EDTA solution to form a 0.1M Ca-EDTA complex.
The 50 mL of 0.06M Na2HPO4.2H2O solution is added dropwise at a rate of 4
mL/minute to the Ca-EDTA complex. The mixture is stirred for 15 minutes at 500
rpm using a magnetic stirrer and then irradiated in a microwave (2.45 GHz, 1100W)
at 50% power for 30 minutes until dry. The resulting precipitate is washed multiple
times with distilled water and then dried in an oven at 80°C for 6 hours to obtain
HAp powder. For HAp/camphor composite synthesis, a 1:1 ratio is prepared by
reacting 2 grams of camphor dissolved in ethanol with 2 grams of HAp powder. In a
2:3 ratio synthesis, 3 grams of camphor dissolved in ethanol is mixed with 2 grams
of HAp powder. Both mixtures are stirred using a magnetic stirrer for 3 hours at 500
rpm and then dried in an oven at 40°C for 8 hours.
The HAp/camphor characterized
The HAp/camphor catalyst was characterized using FTIR to identify functional
groups, with surface morphology analyzed through SEM, and crystallinity structure
assessed by XRD. For X-ray Diffraction (XRD), the catalyst was ground and placed on
a sample plate, and crystallinity testing was performed using Cu K
α
radiation (k =
0.15418) within the angle range of 5° < 2
θ
< 90°. The resulting diffractogram
provided the crystallinity phase of the HAp/camphor catalyst. Fourier-transform
infrared spectroscopy (FTIR) analysis involved grinding 500-1000 mg of
HAp/camphor with 100-200 mg of dry KBr until fine and homogeneous. The mixture
was placed in a sample holder, and the spectrum of the hydroxyapatite/camphor
composite was recorded in the frequency range of 400-4000 cm^-1. Scanning
electron microscopy (SEM) was used to analyze the morphology of the
HAp/camphor catalyst, where electron beams interacted with the catalyst to
produce secondary electrons, revealing the surface topography of the catalyst.
The synthesis of spirochromene derivatives
The synthesis of spirochromene derivatives was carried out using a microwave-
assisted one-pot multicomponent reaction method. Isatin (1 mmol), malononitrile
(1 mmol), and resorcinol (1 mmol) were reacted with HAp/camphor catalyst and 5
mL solvent in a reaction tube within a microwave (100W). The reaction progress was
monitored by TLC. The catalyst was separated using filter paper, and the product
was purified through recrystallization with hot ethanol. The yield of the compound
was calculated by comparing the mass of the synthesized product to the theoretical
mass. Catalyst optimization involved varying the amount of HAp/camphor catalyst
(from 0% to 10%) and reacting the mixture in a microwave for 7 minutes at 100
watts. The optimal catalyst amount was determined by the highest yield. Solvent
optimization was performed by varying solvents (none, distilled water, and ethanol)
under optimum catalyst conditions, reacting the mixture for 7 minutes in the
microwave at 100 watts. The optimal solvent was determined by the highest yield.
Reaction time optimization involved varying reaction times (5, 6, 7, and 8 minutes)
with the optimum catalyst and solvent conditions. The optimal reaction time was
established based on the highest yield.
The characterization of spirooxindole compounds involves several analytical
techniques. Thin Layer Chromatography (TLC) is used where the sample is spotted
on a TLC plate placed in a chamber containing a mobile phase (n-hexane: EtOAc =
4:6). As the mobile phase ascends near the top of the plate, it is removed, dried, and
visualized under UV light. For UV-Vis spectrophotometry, 4 mL of ethanol serves as
a blank, and 1 mg of the spiro-coumarin derivative is dissolved in methanol and
measured in the spectrophotometer across a wavelength range of 200-600 nm.
Fourier-transform infrared spectroscopy (FTIR) analyzes functional groups where
500-1000 mg of the sample mixed with 100-200 mg of dry KBr is ground into a fine,
homogeneous powder, placed in a sample holder, and scanned over a frequency
range of 400-4000 cm-1. Liquid Chromatography-Mass Spectrometry (LC-MS)
involves dissolving the spiro-coumarin derivative in a solvent adjusted for both
mobile and stationary phases. The stationary phase used is Thermo Scientific
™
Hypersil GOLD
™
C-18, while the mobile phase consists of water and acetonitrile
with 0.1% formic acid, and the sample solution is injected into the LC-MS apparatus.
Result
The synthesis of hydroxyapatite (HAp) from blood clam shells involves using
calcium and phosphate as the primary precursors. Calcium oxide (CaO) serves as
the calcium precursor in this synthesis, and in this study, it is derived from the
calcination of blood clam shell waste. Calcination is a process that involves heating
solids at high temperatures to remove water, carbon dioxide, sulfur dioxide, or
other volatile elements, or to oxidize some or all of the substances. Blood clam
shells contain 98.7% calcium carbonate (CaCO3). After cleaning, the shells are
crushed and subjected to calcination at 900°C for 8 hours to produce CaO. During
calcination at temperatures above 700°C, CaCO3 decomposes into CaO, releasing
CO2. The calcination process yields a white CaO powder with a yield of 65.58%. The
CaO obtained is then used in the synthesis of hydroxyapatite. It is mixed with EDTA
solution, which acts as a chelating or complexing reagent, forming a Ca-EDTA
complex around Ca2+. Sodium phosphate solution (Na2HPO4.2H2O) is added
dropwise to the mixture and stirred for 15 minutes. The addition of Na2HPO4
causes the release of free Ca2+ ions from the Ca-EDTA complex. The free Ca2+ ions
combine with OH- ions from water ionization and with PO43- ions from Na2HPO4 to
form HAp crystal nuclei. Crystal growth of HAp continues under microwave
radiation until a dry precipitate is formed.
