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Summer Research Fellowship Programme of India's Science Academies

Large scale synthesis of immunomodulatory compounds

Shristy Jindal

Department of Chemistry, Kirori Mal College, University of Delhi, Delhi 110007, India

Dr. Deepak B. Salunke

Department of Chemistry and Centre of Advanced Studies in Chemistry, Punjab University, Chandigarh 160014, India

Abstract

Immunomodulators alters the immune response either by suppression (immunosuppressive) or enhancement (immunostimulant) mode. Immunomodulators generally categorized viz. corticosteroids, cytotoxic agents, thymoxin, and immunoglobulins. Several functionalised immunomodulators are naturally occurred in the body, and some of these are available in pharmacological preparations. TLRs (Toll like receptors) are key targets for the design of small immunomodulatory molecules for its use as vaccine adjuvants and anticancer/antiviral agents. The substituted imidazoquinolines are potent agonists of TLR7/8. Imiquimod is a synthetic compound with potent antiviral, antitumor, and immunoregulatory properties that stimulates both the innate and acquired immune pathways through activation of TLR7. US FDA approved imiquimod for topical treatment of external anogenital warts, actinic keratosis, and superficial basal cell carcinoma. Among the known TLR7/8 agonistic imidazoquinolines, only few are commercially available due to their tedious synthetic procedure, expensive reagents, low yield etc. Therefore, the synthesis of substituted imiquimod derivatives is required and will be optimized on large scale. Several synthetic routes are reported in the literature for the preparation of imiquimod. The present work is related to the sustainable and efficient synthesis of Imiquimod. The synthesis and biological evaluation of Imiquimod derivatives will be performed in future.

Keywords: imiquimod, TLR, agonists, immunomodulators

Abbreviations

Abbreviations
TLR Toll-like receptor
Conc. Concentrated
EtOH Ethanol
DCM Dichloromethane
MeOH Methanol
Rf Retention factor
RT Room temperature
TLC Thin layer chromatography
NaOH Sodium hydroxide
Liq. Liquid
HCl Hydrochloric acid

INTRODUCTION

Background/Rationale

In order to treat external genital and perianal warts, US Food and Drugs Administration accepted Imiquimod as an efficient immune response modifier (Mahto et al., 2010)[21]. In animal models, imiquimod is considered to have potency of antitumor and antiviral activity but no direct antiviral or antiproliferative activity in cell culture systems. (Miller et al., 1999)[24]. It enhances both innate as well as acquired immune system (particularly T helper cell type 1-mediated immune responses).

Action on innate immunity

Innate immune system triggered by imiquimod through the induction, production and secretion of cytokines such as interferon (IFN), interleukin-6 (IL-6), tumor necrosis factor-A (TNF-α), and other cytokines. It directly causes multiplication of non-specific B-cell. In animal models, it increases natural killer cell activity, activating macrophages in order to secrete cytokines which results in proliferation and distinction of B lymphocytes. On oral intake of imiquimod, the levels of serum 2¢,5¢-oligoadenylate synthetase get increased which is essential for the antiviral effects of interferon against some cytoplasmic RNA viruses (Sauder et al., 2000)[34]. On applying imiquimod 1% or 5% cream to the hairless mice, there found a substantial high level of IFN-α messenger RNA (mRNA), increased IFN-α, and increased TNF-α (Imbertson et al., 1998)[15]. There is an enlargement, activation and migration of Langerhans cells to the regional lymph node, which increase antigen exposure to T cells. Cytokine formation (including IFN-α, TNF-α, IL-1, IL-6, IL-8, IL-10, IL-12, granulocyte stimulating factor (G-CSF), macrophage inflammatory protein 1-A, and macrophage chemotactic protein (MCP-1)) are induced in human peripheral blood mononuclear cells. As a result of binding of activation units to the promoter areas of IFN-α, cytokines get induced with simultaneous activation of transcription. Due to this induction ability of imiquimod, it is having such an acute antiviral and antitumor effects.

Action on acquired immunity

In mouse splenic cultures, bone marrow culture, and human peripheral blood mononuclear cell culture, there is an indirect stimulation of production of T-helper type-1 (Th1) cytokine IFN-γ. However, there is no such action of imiquimod to stimulate T cells to multiply or induce T-cell cytokines like IL-2, IL-4, or IL-5. Induction of IL-12 receptor B2 subunit on Th1 cells by IFN-α respond to IL-12 and forms IFN-γ. As a result, Th1 cells are found to be a main source of IFN-γ, although it can also be produced by cytotoxic T cells and natural killer cells followed by stimulation by imiquimod. Because of increased levels of IFN-γ, IFN-α and IL-12, there is a triggering of Langerhans cells after the topical application of imiquimod. It prevents the formation of Th2 cytokine IL-5 in human and mouse cell systems. Inhibition of IL-5 formation is performed by IFN-α and IFN-γ. in those cases where elevated Th1 response is needed, imiquimod plays a vital role.

Summary

In developing an innate and adaptive immunity, Toll-like Receptors (TLRs) are found to be a promising approach (Takeda and Akira, 2001)[39]. In case of Plasmodium infection, TLR7 was found to play a major role in inducing immunity (Baccarella et al., 2013)[5]. One of the TLR agonist namely, Imiquimod, with P. falciparum peptide vaccine developed humoral immunity to fight against sporozoite (Othoro et al., 2009)[26]. It is also used to treat external genital and perianal warts. It stimulates the innate immune system by activating TLR7, commonly involved in pathogen recognition and thereby activating immune cells (monocytes, macrophages, natural killer cells) to produce antiviral cytokines, particularly interferon- α (IFN- α) and tumor necrosis factor- α (TNF- α) and interleukin (IL)-12, IL-10, IL-1, IL-6, and IL-8.52,53. It indirectly enhances acquired immune responses by activating antigen-presenting dendritic cells, including Langerhans cells and Th1 lymphocytes. Langerhans cells subsequently migrates to local lymph nodes and activate the adaptive immune system. IFN- production from T cells stimulates cytotoxic T lymphocytes, which is important in clearance of virally infected cells. There are some other cells which get activated by imiquimod which include natural killer cells, macrophages and B-lymphocytes. Therefore, the aim of the present work is to synthesize Imiquimod on a large scale in a sustained manner.

Objectives of the Research

The main objective of this project is to synthesize imiquimod on a large scale.

LITERATURE REVIEW

Information

Earlier, AGIN was treated with surgeries and medical or photodynamic therapy which is having side-effects like irritation, less success rate and more chances of reappearance in future. (Porter et al., 2002)[29]

Moreover, around 2017, malaria has become one of the most deadly disease, which is caused by P. falciparum. (Organization 2010)[25] The most popular one is cerebral malaria which causes neurological syndrome in the affected individual with distinct features like binding of infected or non-infected RBCs and leukocytes to the cerebral vessels. Due to its similarity with cerebral malaria in humans, to study cerebral disorder in rodents, P. berghei ANKA is one of the approved representation or framework. (Engwerda et al., 2002)[10]

Because of some genetic changes in the constitution of Plasmodium, it has become unaffected to several antimalarial drugs causing a major problem in the treatment of the disease. In poverty-stricken areas, the treatment of malaria seems to be a tough task as the available drugs are very costly. By giving vaccination, one can manipulate the immune response which plays a major role in maintaining public health. With the help of vaccines, one can gain active acquired immunity to fight against a disease. For the complete elimination of malaria, there is a need to have some new articulations to enhance immunity. On the basis of mechanism of action, configuration and properties of adjuvants, there is a group of adjuvants which acts directly on the immune system to intensify the response to antigens and other group to present the vaccine antigens. (Petrovsky and Aguilar, 2004)[28] Various adjuvants have been accepted for clinical trials or evaluated in malaria vaccines. (Petrovsky and Aguilar, 2004)[28]

The main motive behind vaccination is to induce long-term immunity against the co-administered antigen. (Reed et al., 2013)[30] In previous times, the killed vaccines were used which had some adverse effects like fever and pain at the injection site. To overcome this problem, vaccines with more purified antigens and well-defined composition developed. There are several advantages of these “Modern Vaccines” like protection, quality assurance and easily producible. But, because of highly soluble proteins, these antigens have poor immunity and therefore, they need help of “adjuvants” which are responsible for strong and long-lasting immunity. (Pasquale et al., 2015)[27] Various natural as well as synthetic compunds were identified to have adjuvantic activity. In the 19th century, the mineral salt “Alum” (Aluminium hydroxide) was used in licensed vaccines globally. Its salts act by forming a depot at the injection site from where there is a slow release of antigen. As soon as there is an infection of the human body by pathogens, the innate immune system, which is rapid and unspecified, gets activated first and cease the infection and thereby activates adaptive immune system. (Clark and Kupper, 2005)[7] Then, the adaptive immune system clears the infection and keep it as a memory in the form of antibodies for further protection in future. TLRs recognize pathogens and activates innate immune system. They act as a link between innate and adaptive immune system. (Kawai and Akira, 2011)[18] TLRs are primarily expressed on special cells such as macrophages and Dendritic cells. Specific pathogen associated molecular patterns (PAMPs) recognition activates them and produce pro-inflammatory cytokines which generates a defence mechanism. During this, there is a maturation of dendritic cells which results in the activation of T-lymphocytes. There is an activation of T-cells and other components of adaptive immune system by cytokines, chemokines and pro-inflammatory mediators and this leads to Cellular and Humoral immunity. Since the discovery of TLR activation, there is a rapid expansion of molecular mechanism of innate immune system and several pattern recognition receptors were provided as a potential targets for immunomodulators discovery. (Takeuchi and Akira, 2010)[40] There are thirteen TLRs identified till now, out of which ten are functional in human body. TLRs 1,2,4,5,6 and 11 are present on cell surface and TLRs 3,7,8 and 9 are in the endosomes (Beutler et al., 2009[6]; Kawasaki and Kawai, 2014[19]). In case of these receptors, there is a very specific ligand-receptor combination. Lipopeptides are recognized by TLR2 in combination with TLR1 or TLR6, lipopolysaccharides by TLR4, bacteria Flagellin by TLR5 and protozoan profiling by TLR11. The double stranded viral RNA, in the endosomes, is recognised by TLR3 and ssRNA by TLR7 and TLR8. The CpG motifs of bacterial DNA are recognized by TLR9. Since few years, few initiatives are being taken to develop new TLR agonists as vaccine adjuvants. (Kaur et al., 2018[17]; Mbow et al., 2010[22]; Salunke, 2012[33], 2013[31], 2017[32]; Shukla et al., 2012[36]; Steinhagen et al., 2011[38]) After having a sufficient knowledge of its molecular structure, synthetic TLR agonists analogues can be designed and issues like toxicity, potential and production problems can also be overcome. An adjuvant should enhance the cytotoxic (T cell) and humoral (B cell) responses.

In the future, there is a need to evaluate clinical potential of imiquimod in the treatment of other cutaneous and mucosal viral infections, dysplasias and neoplasia, as well as potential vaccine adjuvants.

Summary

There are some reported schemes to synthesize Imiquimod which are having a need to be modified because of certain associated problems (Colombo et al., 2005)[8].

IMG_20190823_073424.jpg
    Scheme 1

    Problems associated: This process is time-consuming and complex. The chlorine atom at the 4-position in the intermediate (7) is replaced by an amino group by treatment with ammonia for 18 hours at 1550C in a steel autoclave. Said reaction is carried out under conditions involving temperatures and pressures which require the use of Specific industrial equipment. There is therefore the need for alternative methods for the preparation of Imiquimod, which are more industrially suitable.

    IMG_20190823_073451.jpg
      Scheme 2

      Problems associated: 4-hydroxyquinolin-2(1H)-one (9) is costly, and the yield of cyclisation step i.e. fifth step results in low yield, the process is time-consuming and complex.

      IMG_20190823_111959.jpg
        Scheme 3

        Problems associated: The process involves the preparation of intermediate (17) which requires, inter alia, the use of nitric acid and sodium azide, products known to be dangerous. Furthermore, said intermediate, like other intermediates useful for the synthesis of Imiquimod, is characterized by the simultaneous presence of a nitro group and a tetrazole ring.

        METHODOLOGY

        Concepts

        All chemicals used were Analytical. FT-IR spectra (4000-200 cm-1) of some reactants and complexes were recorded using a Nicolet iS50 FT-IR spectrometer. 1H NMR spectra were recorded on Bruker Ascend 500MHz spectrophotometer. All chemical shifts (δ) were recorded in ppm with reference to TMS. All chemicals were used as supplied without further purification. All the solvents used were of spectroscopic grade.

        Nuclear magnetic resonance spectrometer

        The nuclear magnetic resonance spectrophotometer is used to predict the chemical structure on the basis of the nuclei present in the given sample (Fig. 4,5). Common nuclei such as 1H, 13C were used to predict the structure of any organic molecule. The experimental results present in this work were recorded on Bruker Ascend 500MHz NMR spectrophotometer instrument. 

        IMG_20190627_150120963.jpg
          An operational view of the Nuclear Magnetic Resonance Spectrometer instrument operating at 500 MHz
          IMG_20190627_150342778.jpg
            An inlet unit of the Nuclear Magnetic Resonance Spectrometer instrument operating at 500 MHz

            FT-IR spectrophotometer

            The FT-IR instrument used to study the determination of functional group present in the sample (Fig. 6). Since, every covalent bond between the atoms has different natural frequency of vibration at the molecular level. No two molecules results in the same IR frequencies even, with the presence of same functional group at two different positions. The basic fundamental rule behinds the IR spectroscopy is that change is dipole moment. FT-IR instrument detects the entire wavelength at the same and results in the spectrum % Transmittance versus wavenumber. The IR spectroscopy has several types of stretching, bending and scissoring phenomena which absorbs different range of IR frequencies, which corresponds to the fingerprint region of 900-1400 cm-1. This instrumental technique has several advantages in the functional group determination, residing of water in the sample etc. The experimental data obtained in the present work were recorded on Nicolet iS50 FT-IR instrument.

            IMG_20190625_184440722.jpg
              A schematic view of Fourier Transform-IR instrument

              Mass spectrometry

              The LC-MS spectrometer is a hyphenated technique used to study the molecular ion peak of more than three compounds altogether present in the same sample (Fig. 7). In this technique, the sample is dissolved in any polar organic solvent, syringed and passed through the column present in the instrument, which separates each impurity and bombards the sample with the electron source of 70eV, results in the different molecular ion peak. The results then presented in the form of m/z versus abundance, the highest intense peak usually corresponds to the molecular ion peak, which corroborate the mass of the given sample. The isotopic abundance rule also validate for fewer elements result in the splitting of the molecular ion peak viz. M, M+1, M+2. The experimental results present in this work were recorded on the TSQ 8000 triple quadrupole GC-MS mass spectrometer instrument.

              IMG-20190723-WA0004.jpg
                A schematic view of Mass Spectrometer

                Synthesis of imiquimod involves a series of ten steps which are given as follows:

                STEP 1: AIM: To synthesize anthranilic acid from pthalamide

                IMG_20190823_112807.jpg
                  Chemical reaction involving synthesis of anthranilic acid from pthalamide
                  Calculations for the synthesis of anthranilic acid from pthalamide
                  S. No. Chemical Weight or Volume Molecular Weight Millimole Mole Equivalent Density
                  1. NaOH pellets (for solution A) 15g 40.00 gmol-1 375 mm 4.574 -
                  2. Bromine (liq.) 4.2ml or 13.1g 159.81 gmol-1 81.97 mm 1.005 3.119 g/ml
                  3. Pthalamide 12g 147.13 gmol-1 81.56 mm 1 -
                  4. NaOH pellets (for solution B) 11g 40.00 gmol-1 275 mm 3.354 -
                  5. Conc.HCl 40ml - - - -
                  6. Acetic acid 12.5ml - - - -
                  7. Distilled H2O 60ml (for solution A) 40ml (for solution B) - - - -

                  PROCEDURE:

                  A solution of 15g of NaOH was prepared in 500ml conical flask and named as solution A. Cooled it to 0oC or below in a bath of ice and salt. 4.2ml of bromine was added to the above solution in one portion and shaken until all bromine was reacted. The temperature got raised and was further cooled to 0oC. Meanwhile, a solution of 11g NaOH was prepared in 40ml distilled water and named as solution B. 12g of finely powdered pthalamide was added to the cold sodium hypobromite solution i.e. solution B in the form of a smooth paste with 25ml H2O with rapid stirring. Flask was removed from the cooling bath during pthalamide addition and vigorously shaken until a yellowish orange solution obtained. Solution B was added to the above solution in one slot and heated the resultant solution to 80oC for two minutes and filtered, if necessary. Cooled the solution in ice and 40ml conc. HCl was added slowly with stirring until the solution got neutralised which was detected by pH paper. The anthranilic acid was precipitated completely by the gradual addition of 12.5ml glacial acetic acid. Foaming occurred and the contents were transferred to 1000ml beaker. The prepared acid was filtered off at vacuum pump and washed with water. Recrystallised from hot water with the addition of little decolourising carbon and dried.

                  IMG_20190823_112327.jpg
                    Mechanism showing synthesis of anthranilic acid from pthalamide 

                    STEP 2: AIM: To synthesize 2-(2-nitroethylidene amino) benzoic acid from 2-amino benzoic acid (anthranilic acid).

                    IMG_20190823_112750.jpg
                      Chemical reaction involving synthesis of 2-(2-nitroethylidene amino) benzoic acid from 2-amino benzoic acid (anthranilic acid)
                      Calculations for the synthesis of 2-(2-nitroethylidene amino) benzoic acid from 2-amino benzoic acid (anthranilic acid)
                      S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole Equivalent Density
                      1. NaOH pellets 9.6 g 40.00 gmol-1 240 mm 3.29 -
                      2. Nitromethane 4.32g + 4.32g 61.04 gmol-1 80.47 mm + 80.47 mm 1.1023 + 1.1023 1.14 g/ml
                      3. Anthranilic acid 10 g 137.14 gmol-1 73 mm 1 -
                      4. Conc. HCl 25 ml + 3.3 ml - - - -
                      5. Distilled H2O 75 ml + 10 ml - - - -

                      PROCEDURE:

                      A saturated solution of 9.6g NaOH was prepared in 10ml H2O. 4.32ml nitromethane was added dropwise to this solution at 0oC with constant stirring until the white paste obtained. Then the above solution was allowed to attain 40oC temperature (either by shaking or by heating). Then 4.32ml nitromethane was again added slowly at 40-45oC (either by slow rotation of round bottom flask in heating mentle at that temperature or shaking) to obtain a clear solution. Reaction mixture was heated to 55oC for 10 minutes and kept aside until the RT attained. Poured the solution into crushed ice and acidified with conc. HCl (approx. 12-15ml) so that the pH becomes 3-4, to get methazoic acid. Then, a solution of 10g anthranilic acid was prepared in 3.3ml conc. HCl and 75ml distilled water and resultant solution of methazoic acid was added immediately to it. Mixture was allowed to stand at room temperature for 12 hours. The solution was filtered and the obtained residue was washed with water and dried to compute yield.

                      IMG_20190823_112251.jpg
                        Mechanism showing synthesis of 2-(2-nitroethylidene amino) benzoic acid from 2-amino benzoic acid (anthranilic acid) 

                         STEP 3: AIM: To synthesize 3-nitroquinolin-4-ol from 2-(2-nitroethylidene amino) benzoic acid.

                        IMG_20190823_112733.jpg
                          Chemical reaction involving synthesis of 3-nitroquinolin-4-ol from 2-(2-nitroethylidene amino) benzoic acid
                          Calculations for the synthesis of 3-nitroquinolin-4-ol from 2-(2-nitroethylidene amino) benzoic acid
                          S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole equivalent Density
                          1. 2-(2-nitroethylidene amino) benzoic acid 10 g 208.13 gmol-1 48.04 mm 1 -
                          2. Potassium acetate 4.80 g 98 gmol-1 49 mm 1.019 -
                          3. Acetic anhydride 20 ml - - - -

                          PROCEDURE:

                          A solution of 10g of 2-(2-nitroethylidene amino) benzoic acid in 20ml acetic anhydride was stirred and heated to 105oC until a clear solution obtained. The mixture was allowed to cool at room temperature and 4.80g potassium acetate was added to it. It was refluxed at 100oC for 15 minutes with vigorous stirring until the solid started precipitating. Then cooled down to RT. The residue was filtered and washed with glacial acetic acid until it became colourless. Then it was washed with water and dried in oven.

                          IMG_20190823_073340.jpg
                            Mechanism showing synthesis of 3-nitroquinolin-4-ol from 2-(2-nitroethylidene amino) benzoic acid

                            STEP 4: AIM: To synthesize 4-chloro-3-nitroquinoline from 3-nitroquinolin-4-ol.

                            IMG_20190823_112650_1.jpg
                              Chemical reaction involving synthesis of 4-chloro-3-nitroquinoline from 3-nitroquinolin-4-ol
                              Calculations for the synthesis of 4-chloro-3-nitroquinoline from 3-nitroquinolin-4-ol
                              S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole equivalent Density
                              1. 3-nitroquinolin-4-ol 1.1 g 190.03 gmol-1 5.8 mm 1 -
                              2. Phosphoryl chloride 3.3 ml 153.33 gmol-1 35.4 mm 6.10 1.645 g/ml

                              PROCEDURE:

                              1.1g 3-nitroquinolin-4-ol was taken in a round-bottomed flask and 3.3ml phosphoryl chloride was added to it. The mixture was refluxed for one hour at 110oC.

                              WORK-UP:

                              After refluxing for an hour, the contents were transferred in a beaker containing ice, in slots with stirring. After cooling, transfer it into the separating funnel and shaken. Brine solution was added to it and shaken continuously. It was then filtered by applying cotton on the funnel and ethyl acetate was evaporated.

                              STEP 5: AIM: To synthesize 4-isobutylamino-3-nitroquinoline from 4-chloro-3-nitroquinoline.

                              IMG_20190823_112627.jpg
                                Chemical reaction involving synthesis of 4-isobutylamino-3-nitroquinoline from 4-chloro-3-nitroquinoline
                                Calculations for the synthesis of 4-isobutylamino-3-nitroquinoline from 4-chloro-3-nitroquinoline
                                S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole equivalent Density
                                1. 4-chloro-3-nitroquinoline 1g 208 gmol-1 4.807 mm 1 -
                                2. Isobutylamine 0.621 ml or 0.4571 g 73.13 gmol-1 6.25 mm 1.3 0.736 g/ml
                                3. Triethylamine 1.0128 ml or 0.7348 g 101.9 gmol-1 7.211 mm 1.5 0.726 g/ml
                                4. DCM 20 ml   - - -

                                PROCEDURE:

                                To a solution of 1 g of 4-chloro-3-nitroquinoline in 20 ml DCM, 0.621 ml isobutylamine and 1.012 ml triethylamine was added to it and reaction was refluxed at 45oC for two hours with stirring. After two hours of refluxing, round-bottomed flask was removed from the condenser and DCM was evaporated on rotary evaporator. Water was added to it for the precipitate formation and wait for 4 hours. Filtered the compound and dried.

                                STEP 6: AIM: To synthesize 3-amino-4-isobutylaminoquinoline from 4-isobutylamino-3-nitroquinoline.

                                IMG_20190823_112558.jpg
                                  Chemical reaction involving synthesis of 3-amino-4-isobutylaminoquinoline from 4-isobutylamino-3-nitroquinoline
                                  Calculations for the synthesis of 3-amino-4-isobutylaminoquinoline from 4-isobutylamino-3-nitroquinoline
                                  S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole equivalent Density
                                  1. 4-isobutylamino-3 nitroquinoline 100 mg 245 gmol-1 - - -
                                  2. Ethyl acetate 10 ml 88.11 gmol-1 - - 0.902 g/ml
                                  3. Pd/C 10 mg - - - -
                                  4. Sodium sulphate 50 mg 142 gmol-1 - - -

                                  PROCEDURE:

                                  100 mg of 4-isobutylamino-3-nitroquinoline was taken in a 100 ml round-bottomed flask and 10 ml ethyl acetate was added to it. 10 mg palladium on activated charcoal was added to it and 50 mg sodium sulphate was also added. A balloon filled with hydrogen gas was fitted to the flask and putted on stirrer for 2 hours. TLC was taken at regular intervals to check the completion of reaction and after completion, the product was filtered on celite band and dried.

                                  STEP 7: AIM: To synthesize 1-isobutyl-1H-imidazo[4,5-c]quinoline from 3-amino-4-isobutylaminoquinoline.

                                  IMG_20190823_112024.jpg
                                    Chemical reaction involving synthesis of 1-isobutyl-1H-imidazo[4,5-c]quinoline from 3-amino-4-isobutylaminoquinoline
                                    Calculations for the synthesis of 1-isobutyl-1H-imidazo[4,5-c]quinoline from 3-amino-4-isobutylaminoquinoline
                                    S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole equivalent Density
                                    1. 3-amino-4-isobutylaminoquinoline 1.075g 215 gmol-1 5.00 mm 1 -
                                    2. Triethyl orthoformate 1.5 ml or 1.48 g 148 gmol-1 10.00 mm 2 0.891g/ml
                                    3. Toluene 20 ml 92 gmol-1 - - -

                                    PROCEDURE:

                                    1.075 g of 3-amino-4-isobutylaminoquinoline was taken in a round-bottomed flask and 1.5 ml triethyl orthoformate and 20 ml dry toluene was added to it and was refluxed at 100-110oC for 3-4 hours. After reaction completion, toluene was evaporated on rotary evaporator and compound was dried. Column chromatography was used for purification.

                                    STEP 8: AIM: To synthesize 1-isobutyl-1H-imidazo[4,5-c]quinoline 5-oxide from 1-isobutyl-1H-imidazo[4,5-c]quinoline.

                                    IMG_20190823_113725.jpg
                                      Chemical reaction involving synthesis of 1-isobutyl-1H-imidazo[4,5-c]quinoline 5-oxide from 1-isobutyl-1H-imidazo[4,5-c]quinoline
                                      Calculations for the synthesis of 1-isobutyl-1H-imidazo[4,5-c]quinoline 5-oxide from 1-isobutyl-1H-imidazo[4,5-c]quinoline
                                      S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole equivalent Density
                                      1. 1-isobutyl-1H-imidazo[4,5-c]quinoline 400 mg 225 gmol-1 1.777 mm 1 -
                                      2. 3-chloroperoxy benzoic acid 766 mg 172.56 gmol-1 4.44 mm 2.5 -
                                      3. MeOH:DCM:CHCl3 0.1 : 1 : 1 30 ml - - - -

                                      PROCEDURE:

                                      400 mg of 1-isobutyl-1H-imidazo[4,5-c]quinoline was taken in a round-bottomed flask. 766 mg mcpba was added to it. 30 ml of solvent system MeOH:DCM:CHCl3 in the ratio 0.1:1:1 was added and mixture was refluxed for 4 hours and the product was purified using column chromatography.

                                      STEP 9: AIM: To synthesize N-(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)benzamide from 1-isobutyl-1H-imidazo[4,5-c]quinoline 5-oxide.

                                      IMG_20190823_113706.jpg
                                        Chemical reaction involving synthesis of  N-(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)benzamide from 1-isobutyl-1H-imidazo[4,5-c]quinoline 5-oxide
                                        Calculations for the synthesis of N-(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)benzamide from 1-isobutyl-1H-imidazo[4,5-c]quinoline 5-oxide
                                        S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole equivalent Density
                                        1. 1-isobutyl-1H-imidazo[4,5-c]quinoline 5-oxide 255 mg 241 gmol-1 1.058 mm 1 -
                                        2. Benzoyl isocyanate 233 mg 147.13 gmol-1 1.587 mm 1.5 -
                                        3. Anhydrous DCM 20 ml 85 gmol-1 - - -

                                        PROCEDURE:

                                        255 mg of 1-isobutyl-1H-imidazo[4,5-c]quinoline 5-oxide and 233 mg of benzoyl isocyanate was taken in a round-bottomed flask and 20 ml anhydrous DCM was added to it and mixture was refluxed at 45oC for 30 minutes. The product was filtered and dried.

                                        STEP 10: AIM: To synthesize 4-Amino-1-isobutyl-1H-imidazo(4,5-c)quinoline from N-(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)benzamide.

                                        IMG_20190823_113642.jpg
                                          Chemical reaction involving synthesis of 4-Amino-1-isobutyl-1H-imidazo(4,5-c)quinoline from N-(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)benzamide
                                          Calculations for the synthesis of 4-Amino-1-isobutyl-1H-imidazo(4,5-c)quinoline from N-(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)benzamide
                                          S. No. Chemicals Weight or Volume Molecular weight Millimoles Mole equivalent Density
                                          1. N-(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)benzamide 200 mg 344 gmol-1 - - -
                                          2. Anhydrous MeOH 20 ml 32 gmol-1 - - -
                                          3. Sodium methoxide Excess 54 gmol-1 - - -

                                          PROCEDURE:

                                          200 mg of N-(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)benzamide was taken in a round-bottomed flask and anhydrous MeOH was added to dissolve it. Excess of sodium methoxide was added and was refluxed for 1 hour. The product was filtered and dried and purified with the help of column chromatography.

                                          RESULTS AND DISCUSSION

                                          Synthesis of desired imidazoquinoline i.e. imiquimod was achieved from the commercially available pthalamide using the above mentioned ten steps synthesis. Quantitative yield was achieved for the chlorination reaction using phosphorus oxychloride (POCl3) instead of relatively expensive phenyl dichlorophosphate. The crude product obtained was used as it is for the regioselective displacement of chloro functionality at C4 position using isobutylamine in the presence of triethylamine to afford 4-isobutylamino-3-nitroquinoline which was purified using silica gel column chromatography. The subsequent nitro reduction was carried out using Pd/C catalyst under hydrogen atmosphere at 55 psi and the residue obtained was refluxed with triethylorthoformate in the presence of toluene to obtain 1-isobutyl-1H-imidazo[4,5-c]quinoline. This derivative was converted into an oxide which on treatment with benzoyl isocyanate gives amide derivative and further refluxing with sodium methoxide resulted in a mixture which on column chromatographic purification afforded desired Imiquimod. The overall yield for this process was found to be quite high and was analysed with the 1H NMR spectroscopy.

                                          CONCLUSION

                                          The desired imidazoquinoline i.e. Imiquimod was synthesized on a large scale and its 1H NMR data is given below.

                                          IMG_20190825_114851.jpg
                                            1H NMR spectrum of Imiquimod
                                            IMG_20190825_114801.jpg
                                              Elaborated spectra region from 6.9 ppm to 8.4 ppm
                                              IMG_20190825_114733_1.jpg
                                                Elaborated spectra region from 1.0-6.0 ppm

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                                                ACKNOWLEDGEMENTS

                                                I would like to give my special thanks to Dr. Deepak B. Salunke, Assistant Professor, Department of Chemistry, Punjab University, without his assistance and dedicated involvement in every step throughout the process, this project would have never been accomplished.

                                                I would also express my thanks to Indian Academy of Sciences for providing an opportunity under the aegis of SRFP for providing necessary infrastructure and facilities at Punjab University.

                                                At last, I gave my sincere thanks to Mr. Deepender Kaushik with whom I worked with during the course of this research training and with his constant support and dedication towards my work always paves the way to make this training accomplished.

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