Exploring The Importance Of Molecular Imaging Biology Essay


Molecular imaging has enormous potential as a powerful means to monitor and diagnose a disease by mapping the anatomic locations of specific molecules of interest within living tissue. In cancer management, molecular imaging of prolification, metabolism, and other more specific targets may therefore be of additional value. To monitor the biological events, large number of specific probes with wide variety of chemical properties and emission wavelength are used during the last two decades. In present scenario, these are some characteristics that have to keep in mind before synthesis of novel fluorescent probes. Anthraquinones plays a significant role in molecular imaging field due to their magnificent fluorescent property for wide range of wavelengths. The biological activity of these drugs has been recognized to the formation of intercalation complexes. DRAQ5 is a synthetic anthraquinone, far red-fluorescing agent, with an excitation maximum approximately 650 nm and an emission spectrum ranging from 665 nm to beyond 780 nm. But DRAQ5 still have some limitations, and to overcome these limitations, it is important to synthesis its analogues having similar advantages of DRAQ5 additionally overcome the limitations.


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A generic term cancer used to describe as many as 200 malignant diseases which occur in various tissues and cause variety of illnesses. Types of cancer known are almost equal to type of cells present in body. Regarding spread, incidence, and survival rates, each cancer has its own characteristics, however they share one common characteristic; that there is uncontrolled malignant cell division, generally from a single primary site (1).

There are twenty three pairs of chromosomes present in every normal cell of human body. DNA is responsible for controlling and transmitting genetic characteristics in the chromosomes, we inherit from our parents and pass on to our children. Genes are subunit of chromosomes, single chromosome contain millions of different genes. These genes contain information on how the body should function, behave and grow. Genes determine the various aspects of the body, like colour of eyes, healing of injured tissues, secretion of gastric juice and many more. During normal condition these genes function properly and send the correct messages. As the chromosome reproduces itself, it is resulted in a cell division. Therefore there are number of opportunities for something to go wrong.

Genetic change or damage to the chromosome within the cell resulted in cancer. Alteration in genes, leads to sending improper or wrong messages or entirely different message from one it should give. This improper signaling further leads to rapid growth of cell. Further multiplication of cell occurs again and again until it forms lump, that's called tumor, malignant or cancer (2).

Generally as cancer grows in a body, it is discovered, either by a patient himself/herself as an ill feeling, or as a suspicious lump, or, more often now a days by medical practitioner in a regular diagnostic test or a standard screening like mammography.

During diagnosis and treatment of patient with cancer, imaging investigation plays an essential role. Imaging techniques are not just used for initial diagnosis but it contributes a significant part in monitoring the effectiveness of treatment. Main aim of molecular imaging is to show fine structures within the patient without need of surgical investigation (3).

Molecular imaging

Since the first microscope was built in the late sixteenth century, morphological observations have driven the course of biology (4). In early twenty first century a new discipline emerged which is intersection of molecular biology and in vivo imaging and referred as molecular imaging. Molecular imaging may refers to the combination of approaches from various disciplines like chemistry, pharmacology, physics, engineering, bioinformatics, cell and molecular biology. Major application of molecular imaging is to evaluate the specific process at the cellular and sub cellular levels in living organisms (5).

Molecular imaging has enormous potential as a powerful means to monitor and diagnose a disease by mapping the anatomic locations of specific molecules of interest within living tissue (6). Imaging can provide the potential for understanding of earlier detection and characterization of disease, integrative biology and evaluation of treatment.

2.1 Molecular imaging and cancer

Tissue sampling is one of the most common methods used for represent the biochemical or pathological process under investigation; however it may not always adequate because of tissue heterogeneity, which is especially characteristic of some tumors (7). Today with the help of molecular imaging technique, clinicians are able to see not only where a tumor is located in the body, but also to visualize biological processes (apoptosis, metastasis and angiogenesis), expression and activity of specific molecules (protease and protein kinase) that influence tumor behavior and/or response to therapy. This information have great role in drug development, individualized treatment cancer detection as well as our understanding of how cancer arises (8). Moreover, the newly developed techniques in molecular imaging allow quantification and visualization of clinically relevant physiological variables such as oxygen consumption, blood flow, proliferative activities, glucose metabolism and tissue hypoxia as they takes place in living cells an tissues.

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Furthermore, one can potentially identified molecular pathways, tumor specific receptors and altered gene products with the help of molecular imaging. It will effectively be possible to estimate at risk patients earlier pathogenesis, perhaps before a tumor has even had a chance to become malignant. For detection and elucidation of disease prognosis at microscopic level, techniques such as optical imaging hold particular promise (9).

During all phases of cancer management, starts from prediction, screening, biopsy guidance for detection, staging, prognosis, therapy planning therapy guidance, therapy response, till recurrence and palliation biomedical imaging plays a crucial role. Biomarkers which are identified from the proteome and genome can be targeted using chemistry that selectively binds to the biomarkers and amplifies their imaging signal (10).

In cancer management, molecular imaging of prolification, metabolism, and other more specific targets may therefore be of additional value. As mentioned above, molecular imaging is emerging field, for this purpose ligands can be labeled with either a fluorescent dye for optical imaging, contrast agent for magnetic resonance imaging (MRI), gamma emitting radionuclide for single photon emission computed tomography (SPECT) imaging or a positron emitting radionuclide for positron emission tomography (PET) (11).

Current role of imaging in cancer management

(Molecular oncology, (2008) 115-152

Moreover, number of different techniques like X-ray imaging (mammography and X-ray CT), ultrasound imaging (colour doppler imaging), and nuclear medicine (gamma camera, positron emission tomography and intra-operative probes) are used for molecular imaging of cancer (12).

Molecular imaging in combination with structural and functional imaging is fundamental to achieve gene expression and molecular processes within cells and tissues. Wide variety of targeted agents for cancer markers including avb3 integrin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) receptors, carcinoembryonic antigen (CEA), , MC-1 receptor, somatostatin receptors, prostate stimulating membrane antigen (PSMA), transferrin receptors and folate receptors have been developed. Except ultrasound which is based on the reflection, scattering, and frequency shift of acoustic waves, most clinical imaging systems are based on the interaction of electromagnetic radiation with body tissues and fluids.

Non-ionizing electromagnetic radiation imaging techniques such as electrical impedance spectroscopy, infrared spectroscopy and tomography, photoacoustic and thermoacoustic imaging and microwave imaging spectroscopy have been investigated mainly for breast imaging.

Future role of molecular imaging

Molecular oncology, (2008) 115-152

Nuclear medicine and positron emission tomography (PET) are the most sensitive clinical imaging techniques with between nanomole/kilogram and picomole/kilogram sensitivity. MR has about 10 mmol/kg sensitivity whereas X-ray systems including CT have millimole/kilogram sensitivity.

In cancer research institutes preclinical fluorescence and bioluminescence-based optical imaging systems are in routine use. Nanoparticals targeted tumor biomarkers and Raman spectroscopy are showing promise for future development (10).

Molecular oncology, (2008) 115-152


The major motive of molecular imaging techniques is to assess specific processes at the cellular level, including gene expression, dynamic cell tracking throughout the entire organism, protein-protein interaction and drug action analysis in living cell or tissue. Molecular imaging contributes a key role in understanding of the physiology of living organisms and offer new means for drug target identification and pre-clinical testing to improve drug discovery. These goals can be achieved rapidly, non-invasively, quantitatively, and repetitively in the same animal, under different conditions and stimuli with the help of molecular imaging (13).

For breast cancer molecular imaging can potentially be used for screening, staging, restaging, response evaluation and guiding therapies. Optical imaging, single photon emission computed tomography (SPECT) or radionuclide imaging with positron emission tomography (PET) and magnetic resonance imaging (MRI) are the major techniques used for molecular breast cancer imaging. Several tumor characteristics are candidates for development of tumor specific tracers in case of breast cancer imaging.

Schematic presentation of the potential targets for breast cancer molecular imaging

T.H.Oude Munnink et al. / The Breast 18 (2009) S66-S73

DNA synthesis or tumor cell glucose metabolism is higher in tumor cell as compared to normal cells, and by targeting these general phenomena one can visualize the tumor cell. For visualization of glucose metabolism in tumor cell, [18F] fluorodeoxyglucose (FDG) is most used PET-tracer. FDG is phosphorylated by hexokinases to FDG-6-phosphatase after transported across the cell membrane by glucose transporter proteins. Unlike glucose-6-phosphate, FDG-6-phosphate lacks a hydroxyl group at the 2-position, and therefore it is not further metabolized and thus 'trapped' in the cell. This leads to accumulation of FDG in tumor cell, which is regulated by the activity of the glucose transporters and hexokinase. In 45-90 minutes after injecting FDG into body, the tumor uptake can be detected with a PET camera (14).

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Moreover, expression of hormone receptors in tumor cell is found in most of the breast cancer, and these receptors are interesting targets for imaging in these subsets of patients. During diagnosis of breast cancer, 70% of patients have tumors positive for hormone receptors, of which the majority are positive for estrogen receptor (ER). The PET tracer 16-a-[18F] fluoro-17-b-estradiol (FES) was used as a receptor ligand for ER that binds to both subtypes ERa and ERb, with a preference for ERa (15).

Furthermore, receptors present at tumor cell membrane, such as Insulin-like Growth Factor-1 Receptor (IGF-1R), Epidermal Growth Factor Receptor (EGFR), Platelet Derived Growth Factor b Receptor (PDGF-bR), and Human Epidermal growth factor Receptor 2 (HER2) may be of interest for imaging. Additionally, expression of growth factors by tumor cells like Transforming Growth Factor b (TGF-b) and Endothelial Growth Factor (VEGF) in tumor microenvironment and therefore are tracer target candidates.

In around 25-30% of breast cancer patients, it is found that there is overexpression of HER2 due to HER2 gene amplification. Fab-fragments, F(ab_)2-fragments, full length monoclonal antibodies, minibodies, diabodies, affibodies and peptides are currently available HER2 targeted ligands (16,17).

Fluorescence spectroscopy

Fluorescence spectroscopy is a sub type of electromagnetic spectroscopy which is used to analyzes fluorescence from a sample by using a beam of light, usually ultraviolet light. A molecule that is capable of fluorescing is called fluorophore.

3.1 Principles of fluorescence

Fluorescence is the result of a three-stage process that occurs in certain molecules (mostly heterocycles or polyaromatic hydrocarbons) called fluorescent dyes or fluorophores. Fluorescence is the result of a three-stage process that occurs in certain molecules (mostly heterocycles or polyaromatic hydrocarbons) called fluorescent dyes or fluorophores. This process is illustrated by electronic stage diagram or "Jablonski diagram" (18).

Jablonski diagram illustrating the phenomenon involved in creation of excited electronic singlet state by optical absorption and subsequent emission of fluorescence.

During at its ground state, a flurophore molecule is stable, with relatively low energy, and it does not fluoresce. When it absorb light from external source, and if the energy absorbed is sufficient the molecule reaches higher energy state called excited state and the process called excitation state. Depending on the wavelength and energy of external light source, the flurophore can attain multiple excited states or energy levels. Since the fourophore is unstable at high energy configurations, it ultimately adopts the lowest energy excited state which is semi stable. Time period in which the flurophore is in excited state is called the excited life time. The flurophore is then rearranges itself from the semi stable excited state to ground state, and the excess energy is released and emitted as a light. The emitted light is of lower energy, and thus longer wavelength, than the absorbed light. Therefore the colour of light that flurophore emitted is different from the colour of the light that has been absorbed. As after, flurophore emits light it returns to the ground state from semi stable excited state.

Theoretically a flurophore can repeatedly undergo the fluorescence process indefinitely. This means that a single flurophore molecule can generate a signal multiple times. This property makes fluorescence a very sensitive technique for visualizing microscopic samples, even a small amount of the stain can be detected. However, in reality the flurophores structurally instability during the excited lifetime makes it vulnerable to degradation. Due to high illumination can cause the flurophore to change its structure so that is can no longer fluoresce, this phenomenon is called photobleaching. Therefore, after a certain period of time, the flurophores are no longer promoted to an excited state, even when the required energy is supplied (19).

Wavelength of visible spectrum is ranging from approximately 400 nanometer to 700 nanometer. Light waves with shorter wavelengths have high energy and high frequency, whereas light waves with longer wavelengths have lower energy as well as lower frequency. As mentioned above, an excited flurophore emits lower energy light than it absorbed. As a result, there is always a shift along the spectrum between the colour of the light absorbed by the flurophore during excitation and the colour emitted. A flurophore molecule absorbs light over a range of wavelengths, and every dye has a characteristic excitation range. On the other hand, some wavelengths within a specific range are more effective for excitation than other wavelengths. Therefore, for each fluroscent dye, there is a particular wavelength, the excitation maximum, that most effectively induces fluroscence (20).

3.2 Fluorescent probes

Flurophore which is designed specifically to localize within a specific region of biological specimen or to respond to a specific stimulus are called fluorescent probes (29). To monitor the biological events, large number of specific probes with wide variety of chemical properties and emission wavelength are used during the last two decades.

In present scenario, these are some characteristics that have to keep in mind before synthesis of novel fluorescent probes.

It should have low molecular weight and it will less likely to affect biological activity.

It allows acess to specific sites on biomolecules where larger molecules will not fit.

To improve small organic probes, with increased sensitivity, wider spectral range and increased stability.

All fluorescent probes are composed of two fundamental units that have to be properly engineered by organic synthesis to form final useful fluorescent unit: the core flurophore body and the subunits or R-groups, attached to the main body to make it reactive, a good DNA intercalator, water soluble, lipid soluble and so forth. On the other hand, the core flurophore determines the spectral properties of the flurophore in different microenvironments where the probe might be located spectral. Spectral properties include quantum yield, extinction, excited state lifetime, fluorescence as a function of wavelength and absorbance as a function of wavelength (31).

The R-group can be isothiocynate, succinimidyl ester or maleinde; or they may be sulfonic acid groups for aqueous solubility or long chains in the case of fluorescent membrane labels. Fluorescent probe should have sufficient fluorescence at tagged site while minimizing background and have the fluorescent signal generated immediately.

One of the major advantages of infrared fluorescent probe is deep tissue imaging. Due to long wavelength light travels farther in tissues. There is a spectral window between the limit imposed by increased water absorbance and the end of major components of hemoglobin absorbance (approximately 650 nm). Fluorescent probes under such spectral window are more likely to be useful for imaging the vascular system, tumors and the other cells and tissues in living organisms (30).

3.3 Application of fluorescent probes in cancer

Ovarian cancer is the fifth leading cause of cancer death for women after lung, breast colorectal and pancreatic cancer. In 2007, 22,430 new cases of ovarian cancer were diagnosed, which is approximately 3% of all the cancers in women in United States of America. Early detection of ovarian cancer is very difficult because there are few warning signs and symptoms, and malignant cells can escape from the ovarian capsule and disseminate throughout the peritoneal cavity. Diagnosis of cancer at the early stage is extremely challenging and has been an active research area these days in order to reduce the mortality and morbidity rate (33).

Key cornerstone in the treatment of metastatic ovarian cancer is Cytoreductive surgery. "Current guidelines define an "optimal" debulking procedure as one in which the surgeon leaves the abdomen with less than 1 cubic cm of residual tumor". Number of imaging techniques has been applied towards more sensitive exposure of diffuse peritoneal carcinomatosis, with near infrared (NIR) optical imaging showing particular promise. Detection of ovarian cancer can be achieved by targeting an enzyme widely present across most ovarian cancers (34).

As compared to other detection methods for evaluation of potential residual disease, optical imaging with molecularly targeted probes has numerous advantages. Optical imaging lacks ionizing radiation, focally highlights disease, and has a low barrier to incorporation in the operating room.

I case of breast cancer, deoxyhemoglobin and oxyhemoglobin are the main light absorbers of the breast cancer in the NIR window. Hence, for quantifying and imaging vascularization, and especially oxygen saturation of breast tumors, the optical technique is a unique noninvasive technology. These features are associated with two correlates of breast malignancy; hypoxia and angiogenesis.

Optical imaging and breast cancer detection is not a novel idea, Cutler in 1929 used optical imaging technique by shined light through the pendant breast to observe the absorption pattern on the other side. This method was known as 'diaphanography' or 'transillumination'. During 1990s transillumination was revisited, with further advances in source and detection technology. Photon pulses and laser light in femptosecond or picoseconds range range have been employed in breast imaging (35).

3.4 Anthraquinone based fluorescent probes

Number of anthraquinone derivatives is used as anticancer agents from a long period of time in medical history (21). Hydroxy and Amino-substituted quinones are having enormous significance in biology, pharmaceutical chemistry and dye industries. Presence of the quinone moiety, makes these molecules good electron acceptors, and has been used quite extensively to investigate the dynamics and mechanism of electron transfer processes both in homogeneous and heterogeneous media. Reasonable fluorescence quantum yields are often shown by amino and hydroxyl derivatives of quinine (25). Moreover anthraquinone derivatives have been used as organogelators, dyes, mesogens, chemical sensors, anticancer agents, as well as precursors of peripherally substituted anthracenes (22).

Anthraquinones plays a significant role in molecular imaging field due to their magnificent fluorescent property for wide range of wavelengths. The biological activity of these drugs has been recognized to the formation of intercalation complexes. Noticeable changes in the resonance Raman spectra and electronic spectra are resulted by formation of intercalation complexes. Therefore, in order to obtain structural information on the interaction a detailed knowledge of the vibrational structure of both ground and excited electronic states of the chromophore is necessary. Information on nature of the excited states can be obtained by optical absorption spectral studies of the chromophore (23). Additionally, it provides an outstanding potential probe for monitoring the excited states interaction with the environment (24).

3.5 DNA Binding agents

Principles for targeting of specific DNA in order to control gene expression are studied for several decades. DNA plays a crucial role in pathological process and cell life. Nucleic acids are known for their diverse structures together with loops, knots, pseudoknots, hairpins, triple helices, helical junctions and bulges (26). Design of specific DNA probes is a significant research area for the development of new therapeutic agents and tools in biochemistry (27)

To investigate the properties of new DNA binding agents, it is important to understand the binding mode of available DNA binding agents. Researchers are now been interested to design agents that are capable of sequence specific binding interaction with double-stranded DNA. Intercalation and groove binding are the most possible binding modes for noncovelent binding to DNA (28). During intercalation binding in which a planar ligand moiety is inserted between adjacent base pairs, results in a substantial change in DNA structure. In contrast, groove binding characteristically results in only subtle changes in structure. Intercalation binding may cause unstacking, unwinding and distortions of the helix (29).

Basically there are three different ways of drug-DNA binding. Primarily, through control of polymerases and transcription factors. In this type of interaction, the drug first interacts with protein that binds to DNA. Secondly, sequence specific binding which is RNA binding to DNA double helices to form nucleic acid triple helical structures. Finally, the third way of drug DNA binding in which small aromatic ligand molecules binds to DNA double helical structure or non covalent interaction. This type of interaction is further divided into subcategories (i) minor groove binders or (ii) intercalating between stacked base pairs thereby distorting the DNA backbone conformation and interfering with DNA-protein interaction. Minor groove binding cause little distortion of DNA back bone. Synthetic compounds work similar to the pharmacological ligand for cell surface receptors in excitable tissue, and appear to be more readily delivered to cellular targets than large RNA or protein ligands.

Due to lack of sequence specificity for intercalating molecules, nevertheless, does not allow to target specific genes. However, rather certain physiological and pathological conditions or cellular states , like rapid cell growth and division that can be selectively suppressed as compared to non growing or slowly growing healthy tissue.

Role of base pair sequence, degrees of freedom, role of solvent ligand-receptor binding and counter ion effects are some properties have been identified as important for the successful modeling of ligand-DNA interaction.

Degree of freedom

This problem is similar to that of protein ligand interaction. The planar aromatic ring structure is major requirement for intercalating agents. This aromatic ring structure is supposed to fits in between two adjacent base pair planes. Moreover this structure is much restricted to rotate freely within the plane. However as per rule, binding affinity is directly proportional to intercalating side chains linked with a single ligand structure.

Counter ion effect

Positively charged Na+, or Ca++ and Mg++ ions as well as basic residues of proteins are attracted to negatively charged polyanion of DNA. During drug binding, the counter ions can screen and shield the negative backbone surface allowing non electrolytes as well as positively charged ligand to interact more strongly with the DNA. However, non covalent interaction mediated by hydrogen bonds and electrostatic interactions is reduced by high ionic strength (33).

DRAQ5: (1.5-bis{[2-(methylamino) ethyl]amino}-4,8-dihydroxy anthracene-9,10-dinone): DRAQ5 is a synthetic anthraquinone, far red-fluorescing agent, with an excitation maximum approximately 650 nm and an emission spectrum ranging from 665 nm to beyond 780 nm. DRAQ5 is DNA interactive, highly cell permeable agent, with fluorescence mark extending into the infra-red region. It is ideally compatible with GFP and FITC based fluors due to its far-red emission it is spectrally and it will work with most benchtop confocal systems. As it does not require a UV laser source for excitation, it is became a choice for nuclear staining of live cells. DRAQ5 is stable at normal lighting condition, at room temperature, and it is soluble in water at biologically compatible pH (39).

It has been recently introduced as a new way to visualize and label DNA chromatin.

By conventional flow cytometer equipped with an argon ion laser, DRAQ5 permit analysis of DNA content of a cell. As permeabilization and/or no fixation is required, antigen expression and light scatter are completely preserved. Moreover DRAQ5 can be used in combination with frequently used fluorochromes such as phycoerythrin (PE) and fluorescein isothiocyanate (FITC), without spectral compensation (37).

In case of DRAQ5, its deep red Ex_max/Em_max (Ex_max 646 nm; Em_max 681; Em_ range 665→800 nm) does not interfere with other fluorescent species used for tagging proteins and at the same time avoids the use of toxic UV irradiation as excitation source, it have several advantages for visualizing DNA in living cells as compared to other DNA dyes such as Hoechst 33258 and 4,6-diamidino-2-phenylindole (DAPI). Furthermore, penetration power of DRAQ5 into nucleus is high and it stains the DNA stochiometrically without apparent severe cytotoxic effects. Additionally, DRAQ5 can be detectable in cells even after 24 hours after its addition.

On the other hand, DRAQ5 possibly distorts the helical structure of DNA because it intercalates into the DNA-helix (38).

TFIIH is a heterodecameric complex which is involved in both nucleotide excision repair and transcription. Studies suggest that of the effect of after treatment with DRAQ5, the sub-cellular localization of TFIIH is substantially changed as DRAQ5 DNA intercalation progressed. Furthermore, TFIIH became progressively excluded from the nucleoli and more heterogeneously distributed.

During examination of effect of DRAQ5 on specific DNA binding proteins involved in transcription, effect of DRAQ5 intercalation on a specific transcription activator, the

androgen receptor was measured. After one hour treatment with DRAQ5, measurable increase was found in the speed of fluorescence recovery, compatible with a strong reduction of the GFP-AR binding pool. It shows that GFP mobility is not altered after incubation with DRAQ5, therefore it is assumed that, GFP mobility is not altered after incubation with DRAQ5, ), supporting the idea that DRAQ5 mainly affects the mobility of chromatin-associated proteins implicated in transcription such as AR and RNAPII and suggests that DRAQ5 interferes with binding of these factors to their DNA substrates (39).

During investigating the potential of DRAQ5 to trigger the genotoxic stress response, it was found that intercalation of DRAQ5 into the DNA does not activate cellular stress responses, even though DRAQ5 alters the structure of the DNA molecule and possibly its organization in the nucleus.

In a comparative study of DRAQ5 with propidium iodide, it was found that multiparameter DRAQ5 assay has a superior sensitivity and specificity compared to the conventional propidium iodide based method.

Rationale of making DRAQ5 analogues

DRAQ5 is one of the most preferable fluorescent probe for DNA staining now a days.

On the other hand, with all these advantages, DRAQ5 still have some limitations, which are mentioned below:

Emission should be collected with far-red filters (780/60) to obtain optimal CVs, for cell cycle analysis.

Due to broad emission/excitation spectra, it has a limited use in multi-colour flow cytometry.

It may be necessary to label cells for a longer period of time and with a higher concentration to obtain optimal CVs, for cell cycle analysis (40).

In terms of toxicity, DRAQ5 appears to be fairly toxic, so the long-term viability of the stained cells is limited (41).

Therefore to overcome these limitations, we need to synthesis DRAQ5 analogues which have all the positive properties of DRAQ5 but free from all the limitations mentioned above.

General chemistry of anthraquinone

In 1868 with the elucidation of the structure of the naturally occurring compound alizarin (1,2-dihydroxyanthraquinone) by C. Graebe and C. Liebermann there is a revolutionary began in chemistry of anthraquinone.

Large numbers of steps are required for synthesis of anthraquinone dye. For example, CI Disperse Blue requires six steps and CI Disperse Red requires five steps starting five steps starting from anthraquinone. Furthermore, synthesis of vat dyes are more complicated, and in extreme case eleven steps are require of synthesis of CI vat blue 64 starting from phthalic anhydride.

Synthesis of anthraquinone dyes or probes are divided into two categories. Primarily by introduction of substituent or substituent's onto the anthraquinone nucleus or secondarily synthesis of anthraquinone nucleus with desired substituent, starting from naphthalene or benzene derivatives. Sulfonation or nitration is the principle reactions which are very important in preparing alpha substituted anthraquinonessuch as 2-chloroanthraquinone and 2-methylanthraquinone. Nucleus synthesis plays an important role in production of beta substituted anthraquinones such as 2-chloro anthraquinone and 2-methylanthraquinone.

Ultraviolet-visible spectrum of anthraquinone due to p-p* transition shows an absorption maximum at 323 nm whereas very weak absorption in the visible range, 405 nm due to n-p* transition. Anthraquinone itself is almost colourless. Charge-transfer band from the lone pair of amino or hydroxyl groups to the oxygen atom of the carbonyl group causes a biometric shift (because of electron donating substituents). Biometric shift can be enhanced by increasing the electron donating ability of substituents. In the condition of same substituent, bathochromic shift is larger when the substituent is in the 1-position rather than in the 2-position. Absorption maximum of the spectrum is affected a little due to introduction of an electron withdrawing group.

In terms of hydrogen-bonding of the substituent with the adjacent carbonyl group that promotes the conjugation of the lone pair of electrons of the donor, methylamino group is more effective than a dimethylamino group.

Table 1

Spectral Data for Some Mono substituted Anthraquinones in Methanol




lmax, nm e

lmax, nm e


378 5200

363 3950


402 5500

368 3900


400 5600

367 4200


475 6300

440 4500


325 4300

323 5200


333 5000

325 3900

Dyes Anthraquinone, vol. 9, 305

In the case of disubstituted anthraquinone, absorption maximum is greatly depends on the substituents and their positions.

1,5-disubstituted anthraquinone

1,5-Dinitroanthraquinone are prepared by nitration of anthraquinone with nitric acid in sulfuric acid. It can also be pre-prepared by nitration of anthraquinone in concentrated nitric acid. One of the major advantage of 1,5- dinitroanthraquinone is they can then be easily separated from the reaction mixture by filtration, in comparison of 1,8- or other isomers than 1,5-dinitroanthraquinone, as they are completely dissolved in concentrated nitric acid.

Nevertheless, this is unsuitable process for industrial production for safety reasons, mixture of concentrated nitric acid and dinitroanthraquinone forms a detonation mixture.

During industrial production, dinitroanthraquinones are synthesized by nitration of anthraquinone in mixed sulfuric-nitric acid.

To enrich the content of 1,5-dinitroanthraquinone in solid phase, reaction mixture is then heated to a temperature slightly higher than the nitration reaction temperature. Reaction mixture is then cooled and filtered to obtain the 1,5-dinitroanthraquinone wet product. Filtrate is redistilled and precipitated isomers are filtered off and filtrate is again recycled to the nitration step.

1,5-dinitroanthraquinone is used as starting material for synthesis of 1,5-diaminoanthraquinone. By ammonolysis of 1,5-dinitroanthraquinone in aqueous ammonia, organic solvent, by reduction with sodium sulfide or by catalytic hydrogenation in an organic solvent, it is converted into1,5-diaminoanthraquinone.

Spectral Data for Some disubstituted Anthraquinones in Methanol

R1 R2*

lmax, nm

skj e




NH2 5- NH2



NH2 8- NH2



NH2 4- NH2



NH2 4-OH















Dyes Anthraquinone, vol. 9, 305

Alternatively, it can also be prepared from anthraquinone 1,5-disulfonic acid by ammonolysis in the presence of an oxidizing agent such as m-nitrobenzenesulfonic acid.

1,5-dinitroanthraquinone is also used as starting material for synthesis of 1,5-diphenoxyanthraquinone, which is precursor of 1,5-dihydroxy-4,8- dinitroanthraquinone, and is prepared from 1,5-dinitroanthraquinone and alkali metal phenoxide in an inert organic solvent or in phenol. Purity is one of the major criteria for manufacturing dyes by 1,5-dimethoxyanthraquinone. Even a small amount of unreacted starting material affects the brightness of the dye and makes it much duller (42).

Experimental Details

Methodologies and experimental data

Scheme 1: 1,5-bis (4-fluorobenzylamino)-4,8-diisopropoxyanthracene-9,10-dione (NRSC-1-A)

Method: Wolfe and Buchwald. Tet. Lett. 1997, 38,(36), 6359-6362.

Toluene (4 ml) was added to BINAP (32 mg, 0.052 mmol, 0.25 eq.) and palladium acetate (9.3 mg, 0.041 mmol, 0.2 eq.) under argon. Resulting mixture was stirred for 30 minutes at room temperature. 1,5-dibromo-4,8-diisopropoxyanthraquinone (100 mg, 0.207 mmol, 1 eq.), cesium carbonate (135 mg, 0.414 mmol, 2 eq.) and 4-flurobenzylamine (237 µl, 207 mmol, 10 eq.) were added to the reaction tube. Reaction mixture was stirred and heated at 100˚C for 24 hours under argon.After 24 hours, resulting dark purple solution was cooled at room temperature and solvents were evaporated under vacuum. Resulting product was dissolved in CH2Cl2 (30 ml) and washed with H20 (3 x 20 ml). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum. Resulting crude residue was purified by column chromatography on silica gel using gradient eluent (CH2Cl2) to yield the title compound (61.2 mg, ???? %) as a dark purple solid. Rƒ 0.54, (CH₂CI₂); δH (400 MHz, CDCl₃) 2.0 (2H,d,J= , H-3 & H-7), 1.86 (2H,d, J= , H-2 & H-6), 1.97 (2H, m, CH(CH₃)₂), 3.93(t,C6H5), 3.81(C6H5), 11.76 (d, J= ,CH(CH₃)₂), 3.87 (s, NCH₂). δc (100 MHz, CDCI₃), 186.23, 163.34, 160.91, 149.27, 144.85, 133.83, 128.73, 125.35, 119.15, 115.61, 77.36, 74.66, 47.06, 22.37. m/z (AP+) 528 (52%), 570 (100%), 572 (40%).

Scheme 2

1,5-bis(3-fluorobenzylamino)-4,8-diisopropoxyanthracene-9,10-dione (NRSC-2-A)

NRSC-2-A was prepared using the same procedure for preparation of NRSC-1-A.

Rƒ 0.41, (CH₂CI₂);, 7.24 (2H,d,J= , H-3 & H-7), 5.34 (2H,d, J= , H-2 & H-6), 1.65 (2H, m, CH(CH₃)₂), 1.35 (t, C6H5), 4.34 (m, J= , NCH₂), 1.58 (m,C6H5), 7.24 (d, J= ,CH(CH₃)₂), 2.00 (s, NCH₂). δc (100 MHz, CDCI₃), 186.34, 164.41, 161.98, 157.42, 149.05, 147.43, 141.35, 130.32, 128.75, 125.46, 122.52, 118.76, 114.32, 113.77, 77.43, 76.76, 74.74, 46.96, 30.36, 22.28. . m/z (AP+) 527 (52%), 571 (100%), 572 (40%).

Scheme 3

1,5-bis(benzylamino)-4,8-diisopropoxyanthracene-9,10-dione (NRSC-3-A)

NRSC-3-A was prepared using the same procedure for preparation of NRSC-1-A.

Rƒ 0.52 (CH₂CI₂); δH (400 MHz, CDCl₃) 2.0(2H,d,J= , H-3 & H-7), 2.0 (2H,d, J= , H-2 & H-6), 2.35 (2H, m, CH(CH₃)₂), 8.37(t,C6H5),4.34 (t, J= , NCH₂), 2.11(m, C6H5), 12.96 (d, J= ,CH(CH₃)₂), 4.31 (t, NCH₂) δc (100 MHz, CDCI₃), 186.05, 139.13, 129.78, 129.27, 128.66, 126.57, 125.79, 121.53, 118.02, 117.59, 110.63, 77.29, 76.09, 74.93, 73.23, 45.47, 35.68, 29.73, 22.26, . m/z (AP+) 565 (47%), 564 (100%).

Scheme 4

1,5-bis (2-fluorobenzylamino)-4,8-diisopropoxyanthracene-9,10-dione (NRSC-4-A)

NRSC-4-A was prepared using the same procedure for preparation of NRSC-1-A.

Rƒ 0.54 (CH₂CI₂); δH (400 MHz, CDCl₃) 1.97 (2H,d,J= , H-3 & H-7), 2.91 (2H,d, J= , H-2 & H-6), 2.00 (2H, m, CH(CH₃)₂), 2.06 (t,C6H5), 4.94 (m, J= , NCH₂), 2.11(m, C6H5), 12.60 (d, J= ,CH(CH₃)₂), 4.46 (t, NCH₂). δc (100 MHz, CDCI₃), 186.26, 161.89, 159.37, 157.39, 149.18, 147.42, 145.05, 139.41, 136.08, 128.93, 126.82, 125.49, 121.28, 118.79, 115.72, 77.35, 76.73, 74.74, 40.91, 22.35, . m/z (AP+) 5573 (47%), 572 (100%), 527 (30%).

Scheme 5

1,5-bis-benzylamino-4,8-diisopropoxy anthraquinone (NRSC-5-A)

NRSC-5-A was prepared using the same procedure for preparation of NRSC-1-A.

Rƒ 0.52, (CH₂CI₂); δH (400 MHz, CDCl₃) 1.87 (2H,d,J= , H-3 & H-7), 3.58 (2H,d, J= , H-2 & H-6), 2.00 (2H, m, CH(CH₃)₂), 1.85 (t,C6H5), 4.34 (m, J= , NCH₂), 2.11(m, C6H5), 11.62 (d, J= ,CH(CH₃)₂), 3.86 (s, NCH₂).δc (100 MHz, CDCI₃), 186.13, 137.80, 128.57, 127.38, 126.91, 125.20, 119.92, 77.19, 76.74, 74.61, 73.24, 48.14, 22.37, 22.08. . m/z (AP+) 536 (100%), 537 (41%).

Scheme 6

1,5-diisopropoxy-4,8-bis(4-methoxybenzylamino)anthracene-9,10-dione (NRSC-6-A)

NRSC-6-A was prepared using the same procedure for preparation of NRSC-1-A.

Rƒ 0.62, (CH₂CI₂); δH (400 MHz, CDCl₃) 4.14 (2H,d,J= , H-3 & H-7), 2.00 (2H,d, J= , H-2 & H-6), 2.12 (2H, m, CH(CH₃)₂), 4.02 (t,C6H5), 6.18 (s, NCH₂), 1.78 (m, C6H5), 11.92 (d, J= ,CH(CH₃)₂), 3.92 (s, NCH₂).δc (100 MHz, CDCI₃), δc (100 MHz, CDCI₃), 186.01, 159.04, 129.45, 128.71, 128.06, 125.06, 120.30, 114.14, 77.37, 77.05, 76.74, 74.52, 55.31, 22.34, 22.23, . m/z (AP+) 597 (42%), 596 (100%).

Deprotection of alcohol group

Method: Wolfe and Buchwald. Tet. Lett. 1997, 38,(36), 6359-6362.

Scheme 1

Deprotection of NRSC-1-A to form NRSC-1X-A

Acetic acid (8.53 ml) was added to 1,5-bis (4-fluorobenzylamino)-4,8-diisopropoxyanthracene-9,10-dione (51.2 mg, 1 eq.). Sulfuric acid (0.17 ml) was then added dropwise to the solution. Resulting mixture was then stirred at 80ËšC for 2 hours. After completion of reaction, resulting solution is cooled at room temperature; volatile solvents were removed in vacuo. Residue was then extracted to collect organic layer and to remove acids with water (15 x 3) and saturated sodium bicarbonate. The combined organic layers were dried over MgSOâ‚„, filtered and concentrated under vacuum.

Resulting residue was purified by column chromatography on silica gel using gradient eluent (Pet. Eth: Et. Acetate, 8:2) to yield the title compound.

Scheme 2

NRSC-2X-A was prepared using the same procedure for preparation of NRSC-1X-A.

Scheme 3

NRSC-3X-A was prepared using the same procedure for preparation of NRSC-3X-A.

Scheme 4

NRSC-4X-A was prepared using the same procedure for preparation of NRSC-4X-A.

Scheme 5

NRSC-5X-A was prepared using the same procedure for preparation of NRSC-5X-A.

Scheme 6

NRSC-6X-A was prepared using the same procedure for preparation of NRSC-6X-A.


Substitution of aromatic amino group

1,5-dibromo-4,8-diisopropoxy anthraquinone was selected as a starting material provided by ICT for synthesis of 1,5-bis (4-fluorobenzylamino)-4,8,diisopropoxyanthracene-9,10-dione (1-A), 1,5-bis (3-fluorobenzylamino)-4,8,diisopropoxyanthracene-9,10-dione (2-A), 1,5-bis (pheneethylamino)-4,8,diisopropoxyanthracene-9,10-dione (3-A), 1,5-bis (2-fluorobenzylamino)-4,8,diisopropoxyanthracene-9,10-dione (4-A), 1,5-bis benzylamino-4,8,diisopropoxyanthracene-9,10-dione (5-A) and 1,5-bis (4-methoxybenzylamino)-4,8,diisopropoxyanthracene-9,10-dione (6-A).

The lone pair of electron on the nitrogen atom in side chain for example (1-A) 1,5-bis (4-fluorobenzylamino)-4,8,diisopropoxyanthracene-9,10-dione is attracted towards the + carbon in the halogenoalkane. It forms a bond with it, in process to remove the bromine as a bromide ion. Palladium acetate is used in a reaction as a catalyst to accelerate the reaction. Cesium carbonate is used to maintain the basicity of the reaction. BINAP is a chiral ligand for the amination.

Same reaction mechanism takes place for (2-A), (3-A), (4-A), (5-A) and (6-A), for the substitution of specific side chain. All these intermediate compounds are found to be pure after chromatography.

Deprotection of alcohol at 1 and 5 position.

Acetic acid and sulphuric acid are used to deprotect alcohol group at position 1 and 5. Addition of acid increases the H+ ions in the reaction mixture. Negative ion of oxygen reacts with hydrogen ion of acid resulted in alcohol.

But we did not got the expected final product for any of our six intermediate compound after this step.


Molecular imaging forms an essential role to furnish structural, functional, metabolic and morphological information. Early diagnosis of cancer by molecular imaging helps in reduction in mortality for certain cancers. As compared to other detection methods for detection of disease especially cancer, optical imaging with molecularly targeted probes shows numerous advantages. Optical imaging lacks ionizing radiation, and has a low barrier to incorporation in the operating room. In the detection of ovarian cancer peritoneal metastases by targeting an enzyme class widely present across most ovarian cancers, NIR optical imaging offers distinct advantages (43).

DRAQ5 is consider as a highly useful molecule to stain DNA living cells. Previous studies showed that it can be used in analyzing the mobility of DNA binding proteins in different chromatin domains (44).In comparison with other fluorescent dyes DRAQ5 reveals the lack of DNA content discrimination (45). Moreover DRAQ5 showed ability of DNA binding by penetrating the plasma membrane with high efficiency.

This study was designed to synthesis DRAQ5 analogues by considering all the plus points of DRAQ5 and to develop a better flurophore which lacks all the limitation of DRAQ5.

Main aim of this study is to synthesis analogues of commercially available fluorescent probe DRAQ5. We used six different aromatic side chains in order to obtain six analogues. This synthesis is designed as two step reaction for each compound; first to attach a specific aromatic side chain and second to deprotect alcohol group.

First part of synthesis is based on nucleophilic substitution of aromatic amine motility at position 4 and 8 by replacing bromine. One hour after starting reaction, colour changes from yellow to purple and getting dark with time. We can predict the progress of reaction with changing of coluor. For instance, we tried 80ËšC instead of 100ËšC for 1-A (1,5-bis(4-fluorobenzylamino)-4,8-diisopropoxyanthracene-9,10-) dione. After one hour colour changes from yellow to dark red instead of purple. After NMR study we found that, obtained product is mono substituted. This indicates that reaction is temperature dependent.

At 100ËšC all reaction shows change in colour from yellow to dark purple. After completion of reaction we analyze all the compound with the help of proton NMR, carbon NMR and mass spectroscopy. All data indicates that the product obtained is expected product.

For deprotection of protected alcohol, we used acetic acid and sulfuric acid. As per protocol, for compound 1-A and 2-A, we set reaction for three hours at 80ËšC. After purification of compound, we analyze it with proton NMR and we did not found the expected product. Moreover it was found that structure of compound 1-A is even affected after reaction.

Therefore, for compound 3-A and 4-A, we tried reaction for shorter period of time. This time we stopped reaction after 2 hours. By NMR data we found that product obtained is unexpected.

For the compound 5-A and 6-A, we started the reaction with reduced temperature at 40ËšC. At this temperature, we monitored the reaction with the help of TLC after every 15 minutes. After two hours it was found that there is still remaining starting material left. Hence we extend the time of reaction for two more hours, after completion of four hours, there is still starting material left is reaction mixture. Therefore, we increase the temperature by 20ËšC and at 60ËšC for two more hours, there is no starting material left. But with increased time period number of byproducts are found on TLC plate. By proton NMR it was found that expected product is not present in reaction mixture.

There may be number of possible reasons for failure of reaction. It may be possible that after attachment of aromatic side chain, our molecule is unstable which after reaction with acids form entirely different product from expectation. One another reason for failure of reaction may be the interaction of hydrogen ion from the acid with oxygen at position 9 and 10. Further investigations are underway to better understand this reaction and to explore its overview.