Description:FIELD OF THE INVENTION
The present invention relates to compositions of 5-hydroxy-5-aryl-pyrrol-2-ones and their use in the treatment of various diseases.

BACKGROUND OF THE INVENTION
A number of adverse physiological conditions are associated with the immune system for e.g. cancer. The immune system comprises of the innate and the adaptive immune system. Presently, all known drugs influence the innate and the adaptive immune system in the same way, i.e., either up regulating both or down regulating both. Thus, the drugs either act as immune system simulator or an immune suppressant.

Standard immune therapeutics are mostly re-proposed anticancer agents, immune suppressants, such as alkylating agents and natural products, as cyclosporin. Then, there is the large class of anti-inflammatory steroids, such as dexamethasone and all impair the body’s immune response. A small time window allows treatment and while their use in Covid-19 SARS-2 was recommended, in SARS 1 it was contraindicated. On the other end immune stimulators, such as interferons in general and IFN-gamma in particular are useful, but come with very serious life threatening adverse reactions. Thus, simulation of the immune system can be problematic.

Although, immune therapy is emerging now in form of monoclonal antibody therapies, but costs are generally very high. In contrast, modulating the immune system into the healthy range would allow an effective treatment of all immune related diseases without the serious life-threatening effects involved with immune stimulators, or compromising on one part of the body’s immune system e.g. in case of steroids or the significant costs associated with monoclonal antibody therapies. Stimulation of an immune system is problematic, as seen for interferons. Thus, there is a need for an approach to restore the immune system into the healthy range.

5-hydroxy-5-aryl-pyrrol-2-ones class of compounds have been reported to act as Cholecystokinin (CCK) receptor ligand. The Indian Patent no. 405278 describes compounds having a 5-hydroxy-5-arylpyrrol-2-one core structure that are disclosed therein as having CCK binding activity. It also discloses methods for preparation of fifteen 5-hydroxy-5-aryl-pyrrol-2-ones, and their binding with the receptor. However, their immunology of any of the disclosed compounds or their compositions has not been reported.

Thus, there is an immense and unmet need for an immune regulator drug and compositions therefor which act as effective treatments against immune mediated physiological adverse conditions and have a beneficial effect over a wide range of diseases and their associated symptoms.

In selected figures all applications to immunology, where the immune system is up or down regulated, are disclosed in detail.

OBJECTS OF THE INVENTION
It is an object of the present invention to provide 5-hydroxy-5-aryl-pyrrol-2-ones derivatives and compositions thereof useful as immune system regulators.
It is another object of the present invention to provide 5-hydroxy-5-aryl-pyrrol-2-ones derivatives and compositions thereof as agents act as immune system regulators by modulating both the adaptive and innate parts of the immune system to bring them within healthy range.
It is another object of the present invention to provide 5-hydroxy-5-aryl-pyrrol-2-ones derivatives and compositions thereof useful as immune regulators capable of acting as anti-inflammatory agents, anti-viral agents, anti-cancer agent, modulators/regulators of innate and adaptive immune system, particularly cytotoxic T-cells & helper cells.
It is another object of the present invention to provide 5-hydroxy-5-aryl-pyrrol-2-ones derivatives and compositions thereof useful in the repair of organs, as seen for the lung, gut and pancreas.

SUMMARY OF THE INVENTION
The current disclosure provides that the 5-hydroxy-5-aryl-pyrrol-2-ones class of compounds disclosed in Indian Patent no. 405278 and their compositions have additional unanticipated clinical benefits as immune regulators i.e. by modulating both the adaptive and the innate parts of the immune system resulting in the values to come in the healthy range. This allows the 5-hydroxy-5-aryl-pyrrol-2-ones to be effective in the treatment of multitude of physiological adverse conditions including inflammation, viral infections, cancer, blood sugar etc. The unanticipated broad clinical application extends to almost the entire range immune mediated inflammatory diseases. For e.g. they are found to be useful as anti-inflammatory agents, anti-viral agents, anti-cancer agents and modulators of innate / adaptive immune system. They are also found to act as modulators of lymphocytes, particularly cytotoxic T-cells, as well as helper T-cells. These compounds and compositions thereof are also useful in organ repair, as seen for the lung, gut and pancreas. These additional unanticipated clinical benefits of the 5-hydroxy-5-aryl-pyrrol-2-ones also significantly improve patient outcomes.

One embodiment of the present invention is to provide a pharmaceutical composition comprising a 5-hydroxy-5-aryl-pyrrol-2-one compound of formula (I):

(I)
wherein
X is halogen, alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy or alkenylcarbonyl,

Y is hydroxy or alkoxy group.

R is selected from phenyl, cyanophenyl, or halogenated phenyl.

R1 is selected from is alkyl, branched alkyl, alkenyl, alkynyl, cycloalkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyloxy, alkenylcarbonyl, phenyl, phenyl alkyl, dialkyl phenyl, benzyl, or benzyl alkyl; and
pharmaceutically acceptable excipients and/or carriers

Another embodiment of the present invention provides a pharmaceutical composition, wherein X is selected from H, F, Br, Cl, I and methyl.

Another embodiment of the present invention provides a pharmaceutical composition having the one of following formulae.

Another embodiment of the present invention provides a pharmaceutical composition, wherein the therapeutically effective amount compound of formula I is in the range of 100ng/kg to 20mg/kg.

Another embodiment of the present invention provides 5-hydroxy-5-aryl-pyrrol-2-ones of formula I and compositions thereof, wherein the use of a compound in the preparation of a medicament, for the treatment or prophylaxis of inflammation or a related condition.

Another embodiment of the present invention provides 5-hydroxy-5-aryl-pyrrol-2-ones of formula I and compositions thereof, wherein the use of a compound in the preparation of a medicament, for the treatment of pain, immune mediated physiological condition including inflammation or a related condition, pain, fever, pneumonia, viral infection and in particular for inflammation of the gut, pancreas or lung.

Another embodiment of the present invention provides 5-hydroxy-5-aryl-pyrrol-2-ones of formula I and compositions thereof, wherein the use of a compound in the preparation of a medicament, for the symptomatic treatment of pneumonia associated with micro-organisms (bacteria, in particular gram negative microorganisms, fungus) and viral infections, such as Dengue fever or Corona-19 virus infection, hepatitis and multiple sclerosis, associated / not associated with the Epstein bar virus.

Another embodiment of the present invention provides 5-hydroxy-5-aryl-pyrrol-2-ones of formula I and compositions thereof, wherein the use of a compound in the preparation of a medicament, for the treatment or prophylaxis of inflammation, which is mediated by cytokines, such as IL-6 or related conditions, often termed cytokine storm, including sepsis.

Another embodiment of the present invention provides 5-hydroxy-5-aryl-pyrrol-2-ones of formula I and compositions thereof, wherein the compound used to treat viral diseases including Covid-19, Dengue fever, HIV, hepatitis, multiple sclerosis, lime disease and shingles, including pain associated with shingles.

Another embodiment of the present invention provides 5-hydroxy-5-aryl-pyrrol-2-ones of formula I and compositions thereof, as highly effective anti-inflammatory agent, even in the presence of potent steroids.

BRIEF DESCRIPTION OF THE DRAWINGS
The novel features and characteristics of the disclosure are set forth in the description. The disclosure itself, however, as well as a preferred mode of use, further objectives and advantages thereof, will best be understood by reference to the following description of an illustrative embodiment when read in conjunction with the accompanying drawings. One or more embodiments are now described, by way of example only, with reference to the accompanying drawings wherein like reference numerals represent like elements and in which:
Figure 1 illustrates inflammatory pathways from pain via pyrexia towards immune mediated diseases under involvement of cytokines.
Figure 2 illustrates a graphical representation to determine the potentiation of tramadol anti-pain effect by compounds.
Figure 3 illustrates a graphical representation to determine anxiolytic property of Pyrrol 8 compared to standard diazepam.
Figure 4 illustrates a graphical representation to determine the anti-depressant property of Pyrrol 8 compared to desimipramine.
Figure 5 illustrates a graphical representation to determine the antipyretic assay in rats compared to Aspirin.
Figure 6 illustrates a graphical representation to determine anti-inflammatory efficacy in the rat paw oedema assay.
Figure 7 illustrates a graphical representation of survival with respect to analysis of death rate in a viral disease example Dengue fever for control with no treatment and treated by Pyrrol 5.
Figure 8 illustrates role of Pyrrol 5 in inflammation and stimulation of production of lymphocytes.
Figure 9 illustrates CRP levels before and after Pyrrol 5 +BC and BC treatments in moderate Covid – 19 patients.
Figure 10 illustrates a graphical representation of levels of IL6 cytokines before and after Pyrrol 5+BC and BC treatments in moderate Covid – 19 patients.
Figure 11 illustrates a graphical representation to determine percentage of lymphocytes before and after Pyrrol 5+BC and BC treatments in moderate Covid- 19 patients.
Figure 12 illustrates the percentage of the neutrophils before and after Pyrrol 5+BC and BC treatments in moderate Covid – 19 patients.
Figure 13 illustrates and represents the ratio of neutrophils to lymphocytes (NLR) before and after Pyrrol 5+BC and BC treatments in moderate Covid – 19 patients.
Figure 14 illustrates the effect of treatments on glucose levels in moderate Covid-19 patients and the restoration of pancreatic tissue with steroids alone and adjunctive Pyrrol 5treatment.
Figure 15 illustrates the proposed pathways of Pyrrol 5 interaction on the adaptive immune system (T-cells) and innate immune system (neutrophils) in the fight against intruders in the form of micro-organisms (bacteria, fungi) and viruses. The same MOA may apply to cancer.
Figure 16 illustrates the increase of CD3 T –cells in human subjects.
Figure 17 illustrates the increase of CD4 T -cells, Helper T cells in human subjects.
Figure 18 illustrates the increase of CD8 T-cells, cytotoxic T-cells in human subjects.
Figure 19 illustrates the packed cell volume in LPS induced inflammation in mice.
Figure 20 illustrates the change of polymorphs/neutrophils in the LPS model in mice.
Figure 21 illustrates the percentage of lymphocytes in the LPS model for a dose range and in conjunction to dexamethasone.
Figure 22 illustrates the immune based anticancer activity in Lung cancer cells in mice for untreated control group compared to the treatment group.

The figures 1-22 depict embodiments of the disclosure for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the assemblies, structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.
 
DETAILED DESCRIPTION OF THE INVENTION

According to the present invention there is provided pharmaceutical compositions and uses of 5-hydroxy-5-aryl-pyrrol-2-one compound of formula (I):

(I)
wherein
X is halogen, alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy or alkenylcarbonyl;

Y is hydroxy or alkoxy group;

R is phenyl, cyanophenyl, or halogenated phenyl;
R1 is alkyl, branched alkyl, alkenyl, alkynyl, cycloalkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyloxy, alkenylcarbonyl, phenyl, phenyl alkyl, dialkyl phenyl, benzyl, or benzyl alkyl; and pharmaceutically acceptable excipients and/or carriers.

Preferably said alkyl-containing moieties are C1-C18, preferably C1-C12. Preferably said alkenyl- and said alkynyl-containing moieties are C2-C18, preferably C2-C12..Preferred substituents for R1 are C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, benzyl, cyclohexyl, phenyl, benzyl (C2-4) alkyl, or phenyl (C2-4) alkyl. Preferably, X is F, Br, Cl, I.

It will be understood that formula (I) is intended to embrace all possible isomers, including optical isomers and mixtures thereof, including racemates. It will also be understood that formula (I) is intended to embrace all possible polymorphs, crystal, impurity, N-oxide, ester, hydrate or any combination thereof. In addition, the present invention includes within its scope prodrugs of the compounds of formula (I). In general, such prodrugs will be functional derivatives of the compounds of formula (I) which are readily convertible in vivo into the required compound of formula (I). Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed H. Bungaard, Elsevier, 1985.

The scope of the invention also extends to salts, particularly physiologically acceptable salts and hydrates of the compounds of formula (I).

The pharmaceutically acceptable salts of the compounds of formula (I) include the conventional non-toxic salts or the quarternary ammonium salts of the compounds of formula (I) formed, eg, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of formula (I) also include those formed from a base, such as an alkali or alkaline earth metal hydroxide, or an organic base, such as an amine or a quarternary ammonium hydroxide. Some of the preferred chemical compounds of formula (I) are given below.

Pyrrol 5

Pyrrol 8

Pyrrol 22
Pyrrol 23

The present invention also resides in a pharmaceutical composition for the treatment of a disease, said composition comprising a therapeutically effective amount of a compound of formula (I):

(I)
wherein
X is halogen, alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy or alkenylcarbonyl;

Y is hydroxy or alkoxy group;

R is phenyl, cyanophenyl, or halogenated phenyl;

R1 is alkyl, branched alkyl, alkenyl, alkynyl, cycloalkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyloxy, alkenylcarbonyl, phenyl, phenyl alkyl, dialkyl phenyl, benzyl, or benzyl alkyl; and

pharmaceutically acceptable excipients and/or carriers.

Preferably said alkyl-containing moieties are C1-C18, preferably C1-C12.Preferably alkenyl- and said alkynyl-containing moieties are C2-C18, preferably C2-C12. Preferably R1 is C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, benzyl, cyclohexyl, phenyl, benzyl (C2-4) alkyl, or phenyl (C2-4) alkyl. Preferably X is selected from F, Br, Cl, and I.

The present invention also resides in pharmaceutical composition comprising a therapeutically effective amount of a compound having one of the following formulae:

According to another aspect of the invention the therapeutically effective amount compound of formula I is in the range of 100ng/kg to 20mg/kg.

The compounds and compositions according to the present invention may be administered via oral, topical, intraperitoneal or intra-venous route.

In another embodiment, the compounds of formula I and pharmaceutical compositions thereof according to the present invention are useful in the preparation of a medicament for the treatment or prophylaxis of an immune mediated physiological adverse condition, including inflammation or a related condition, pain, fever, cancer, pneumonia, viral disease, blood glucose.

Another embodiment of the present invention provides compounds of formula I and pharmaceutical compositions thereof, wherein the use of a compound in the preparation of a medicament, for the treatment or prophylaxis of inflammation or a related condition.

Another embodiment of the present invention provides compounds of formula I and pharmaceutical compositions thereof, wherein the use of a compound in the preparation of a medicament, for the treatment of pain, immune mediated diseases and in particular for inflammation of the gut, pancreas or lung.

Another embodiment of the present invention provides compounds of formula I and pharmaceutical compositions thereof, wherein the use of a compound in the preparation of a medicament, for the symptomatic treatment of pneumonia associated with micro-organisms (bacteria, in particular gram negative microorganisms, fungus) and viral infections, such as Dengue fever or Corona-19 virus infection, hepatitis and multiple sclerosis, associated / not associated with the Epstein bar virus.

Another embodiment of the present invention provides compounds of formula I and pharmaceutical compositions thereof, wherein the use of a compound in the preparation of a medicament, for the treatment or prophylaxis of inflammation, which is mediated by cytokines, such as IL-6 or related conditions, often termed cytokine storm, including sepsis.

Another embodiment of the present invention provides compounds of formula I and pharmaceutical compositions thereof, wherein the compound used to treat viral diseases including Covid-19, Dengue fever, HIV, hepatitis, multiple sclerosis, lime disease and shingles, including pain associated with shingles.

Another embodiment of the present invention provides compounds of formula I and pharmaceutical compositions thereof, wherein the compound is a highly effective anti-inflammatory agent, even in the presence of potent steroids.

In another embodiment, the compounds of formula I and pharmaceutical compositions according to the present invention are useful in the preparation of a medicament, for the treatment or prophylaxis of inflammation which is mediated by cytokines, such as IL-6 or related conditions.

In another embodiment, the compounds of formula I and pharmaceutical compositions according to the present invention are useful for modulating immune functions, glucose levels or both.

In another aspect, the compounds of formula I and pharmaceutical compositions thereof according to the present invention is useful for modulating lymphocytes, particularly cytotoxic T-cells.

In another aspect, compounds of formula I and pharmaceutical compositions thereof according to the present invention are useful for modulating neutrophils and bring them into a medically acceptable range.

In another aspect, the compounds of formula I and pharmaceutical compositions thereof according to the present invention is useful for inducing an increase in T-cells, particularly CD4 cells or CD8 cells or both.

In another aspect, the present invention also relates to a method of treatment of a mammal afflicted with a disease or prophylaxis in a mammal at risk of a disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or compositions thereof.

In another aspect the present invention provides a method of treatment of a mammal afflicted with a disease or prophylaxis in a mammal at risk of a disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound having one of the following formulae as disclosed above.

The administration of the experimental agents can done via oral, topical, intraperitoneal or intra-venous route.

According to an aspect of the present invention, the disease is inflammation or a related condition or immune mediated disease such as cancer.

According to another aspect of the present invention the disease is a viral disease and/or symptoms associated with a viral disease such as pneumonia.

According to another aspect of the present invention the viral disease is Dengue fever, COVID-19 virus infection, HIV, hepatitis or multiple sclerosis.

According to another aspect of the present invention the inflammation is mediated by cytokines, such as IL-6 or related conditions.

According to another aspect of the present invention, the method of useful for inducing an increase in T-cells, particularly CD4 cells or CD8 cells or both.

Another aspect of the present invention provides use of the compound of formula (I) wherein the treatment or prophylaxis of inflammation which is mediated by cytokines, such as IL-6 or related conditions.

Another aspect of the present invention provides use of the compound of formula (I) wherein the treatment or prophylaxis comprises administering therapeutically effective amount of compound of formula (I) in the range of 100ng/kg body weight of the patient and 20mg/kg body weight of the patient. The administration may be done via oral, intraperitoneal or intra-venous route.

Another aspect of the present invention provides use of the compound of formula (I) or compositions thereof wherein the compound is capable of modulating immune response or blood glucose or both.

Another aspect of the present invention provides use of the compound of formula (I) or compositions thereof, wherein the compound is capable of modulating lymphocytes including an increase total number of lymphocytes, particularly cytotoxic T-cells.

Another aspect of the present invention provides use of the compound of formula (I) or compositions thereof wherein the compound is capable of modulating neutrophils and they restore homeostasis into a medically acceptable range.

Another aspect of the present invention provides use of the compound of formula (I) or compositions thereof wherein the compound is capable of increasing T-cells, particularly CD4 or CD8 or both.

Another aspect of the present invention provides use of a compound of formula (I) or compositions thereof to potentiate the analgesic effects of SE/ NE reuptake inhibitors.

Another aspect of the present invention provides the use of these compounds of formula (I) or compositions thereof to potentiate the analgesic effect of opiates.

Another aspect of the present invention provides the use of these compounds of formula (I) or compositions thereof to modulate immune functions.

Figure 1 shows the anti-inflammatory analgesic at the interface of the therapeutic areas pain and inflammation. The efficacy is broadly confirmed in arrays using mice and rats and over a wide array of animal models from hotplate / tail flick via antipyretic assays towards formalin tests, finally targeting immune inflammation. We propose a possible understanding according to cholinergic pathways. Here, acetylcholine is playing a key role and reports show ACh is involved in chronic pain, in addition to immune stimulation of T cells. T cells itself produce Ach, thus in an autocrine manner, directly leading to cytokine reduction. In the spleen ACH by macrophages and T cells is inhibiting cytokines (IL-1, 2, 6, 14, 17) on the nicotinic acetyl choline receptor. The alpha 7 subunit of the nicotinic acetyl choline receptor is a highly rated molecular pain target. Indirectly ACH is released from the vagus nerve down shifting the pain threshold, in particular in the spinal cord. Interestingly, the dose range we found to possess anti-inflammatory in the formalin test as well as analgesic activity in the tail flick / hotplate assay and for immune modulation was found identical.

Figure 2 shows the CCK B antagonist (Pyrrol 8) did not show a significant tramadol potentiation. The phenylethyl-derivative (Pyrrol 5) had a 4-fold potentiation of the analgesic effect of tramadol. A similar, but smaller activity was observed for the cyclopentyl-derivative (Pyrrol 7). Tramadol is acting via NA/SE pathways and therefore in addition to opiate interactions these cross-talks are of particular therapeutic interest in pain, anxiety and depression.

Figure 3 shows the isobutyl derivative 8, at a 0.1mg/kg dose, showed a significant anxiolytic effect, which is at a 0.5 mg/kg dose similar to the standard anxiolytic diazepam at a 1 mg/kg dose. These CNS effects are unexpected, novel and useful to create a therapeutic profiling of the molecules.

Figure 4 shows the isobutyl derivative 8, showed good antidepressant properties compared to the standard antidepressant desimipramine at a 10mg/kg dose. The 0.5 mg dose of the molecule from the disclosed generic scope, has a greater magnitude of action than the standard desimipramine in the forced swimming test.

Figure 5 shows Pyrrol 5 data with respect to pyrexia are depicted. Compared to aspirin (200mg/kg) the potency of Pyrrol 5 was 20 times higher with a 10 mg /kg oral dose. The magnitude of the antipyretic effect was significantly greater than the standard and in line with the expected anti-inflammatory activity at a nano-molar range. The pyrexia is a direct effect of cytokine release, as TNF-alpha and interleukin-6 and these actions are inhibited in a dose dependent manner at low drug molecule concentrations over a prolonged time period.

Figure 6 shows the paw oedema formation was gradually increased within the 1st hour for the control group; and the maximum effect was reached over the period of 3 hours. Animals treated with 200 mg/kg aspirin showed fewer swollen paws and the paw volumes were significantly less than the control group after 2 and 3 hours. The anti-inflammatory effect of API example 5 was superior at 10 mg/kg to the group receiving 200 mg/kg aspirin and significantly larger in magnitude than the activity of Aspirin. API Pyrrol 5 at a dose of 10mg/kg resulted in an anti-inflammatory effect of even a higher magnitude with a 20 times higher potency compared to aspirin. In this rat paw oedema assay (induced by carrageenan) the addition of esterase inhibitors enhanced the anti-inflammatory action of test molecules and this may support our proposed cholinergic pathway involvement further.

Figure 7 shows mice were injected with unadapted Dengue virus, resulting in a dose-dependent transient viremia lasting several days and peaking on day 3 after infection. Increased levels of pro-inflammatory cytokine IL-6 were measured to provide experimental evidence of the key involvement of inflammation. Oral administration of API example 5 led to a reduction of splenomegaly and pro-inflammatory cytokine levels and most importantly death. The results in mice with respect to a reduced death rate validate the experimental agent to be studied clinically in viral diseases, in which death is triggered by inflammation. This finding is seen in this selected model virus and other viral infections such as COVID-19 and other viruses.

Figure 8 shows the role of Pyrrol 5 in inflammation and stimulation of production of lymphocytes. The molecules within the generic scope trigger via an increase of calcium ion concentrations, the T cell activation; and indirectly via cytokine release the proliferation of lymphocytes. Clinically the lymphocytes were modulated into the middle of the healthy reference range in inflamed human subjects in-line with the postulated action.

Figure 9 shows higher concentrations of CRP on day 1 in both the Pyrrol 5+BC and BC arms. A significant decline is noticed on day 15 after treatments, more importantly in patients administered with Pyrrol 5+BC, the CRP levels dropped by >90%, proving the anti- inflammatory properties of the compound in human subjects. CRP is a best biomarker to monitor inflammation. Interestingly even in presence of potent anti-inflammatory steroids, such as dexamethasone, a stronger reduction of marker CRP was found in humans.

Figure 10 shows the change in the levels of IL6 cytokines as per the severity of inflammation. Immune response requires presence of cytokines. Excess cytokines cause organ damage within body as observed in Covid-19 infections. Reduction in cytokines is observed during high inflammation. Very high IL-6 levels are found in sepsis, and the higher the value the stronger the reduction of IL-6 was found in humans. Low IL-6 levels in presence of API example stimulate immune system to initiate lymphocyte proliferation. Thus, for the first time an immune regulator was disclosed. The immune regulator may find more therapeutic applications in immune mediated inflammatory diseases in the future.

Figure 11 shows an increase of lymphocytes from 14% to 27% after treatment with Pyrrol 5+BC, whereas the rise noted in BC is only 14 % to 19%. The lymphocytes are the immune cells required to fight against the Covid-19 infection. This doubling in lymphocytes is providing more helper ad more killer T cells. In HIV-AIDS in the end of life the number and percentage of helper T cells tend to go towards zero. Here and for hepatitis, the increase in helper T cells is therapeutically proven. In cancer the cytotoxic T cells kill distorted cancer tissue and this cancer approach via a small molecule is a first in class. It is a smart therapeutic approach for biologics, such as monoclonal antibodies, and these molecules may replace modern antibody based cancer therapies.

Figure 12 illustrates the levels of neutrophils before and after Pyrrol 5+BC and BC treatments. Higher levels of neutrophils cause NETosis. Pyrrol 5 may reduce the number of neutrophils by cholinergic inflammatory pathway. The percentage of neutrophils is reduced from 81% to 65 % on Pyrrol 5+BC treatment and increased from 72% to 76% in BC treatment highlighting the effect of Pyrrol 5 on reduction of neutrophils. Neutophils and macrophages are part of the innate immune system and here reduction is only in the middle of the healthy range. There is no reduction towards zero, as seen for IL-6 antibody therapies. Reduction of NEtosis is important in general and in particular of key importance, when inflammation goes towards sepsis (and death). Hyper inflammation is effectively inhibited by the disclosed API molecule.

Figure 13 represents the NLR ratios of Pyrrol 5+BC and BC before and after treatments. NLR is a significant indicator of infection or inflammation. The NLR ratio reduced from 8.5 to 2.8 in Pyrrol 5+BC arm and from 11.1 to 5.9 in BC arm asserting better recovery with Pyrrol 5. The NLR is a convenient cellular biomarker to monitor inflammation. The reduction is highly significant and the molecule is a best in class in reduction of this neutrophil to lymphocyte ratio. This biomarker is featured in numerous review articles and NLR may be considered a gold standard inflammation biomarker for a range of diseases from cancer, IBD (inflammatory bowel disease), psoriasis, RA (rheumatoid arthritis ) to even inflammation of the pancreas termed pancreatitis.

Figure 14 shows decline in glucose levels of Pyrrol 5+BC treated patients, which is of great importance to pre diabetic and diabetic patients. It may be concluded that the agent repaired the pancreatic organ, as seen in mice for the gut mice and the lung in humans. Diabetes type 1 may be linked to viral infection (and thereafter trigger an immune response ) which irreversibly destroyed the B cells of the pancreas organ. Here, in Covid 19 patients glucose levels were fully restored and this applied to diabetic patients, who were diabetic prior to the Covid 19 infection as well as patients, who became diabetic after covid-19 infection. This repair of the organ was also seen in the gut and the lung of Covid-19 patients, who needed less oxygen and were discharged earlier from hospital.

Figure 15 shows the concept of efficacy of the API in a Covid–19 infection. Pyrrol 5 was efficient in its use in Covid-19 patients as example for a viral infection. The immune modulator stimulated lymphocytes and reduced neutrophils. Reduction of NETosis stopped hyper-inflammation and prevented its progression of an inflammatory disease via sepsis to death. For the first time a decoupling of immune and inflammation was achieved. For the first time the innate immune system, containing at a cellular level macrophages and neutrophils was down regulated and the adaptive immune system, containing helper and cytotoxic T cells, was up-regulated.

Polymorphs are neutrophils and in man a significant reduction of neutrophils was observed when treated with Pyrrol 5. Lymphocytes are subdivided into B cells and T cells also termed B and T lymphocytes. T lymphocytes play a key role in fighting infection and T cells contain killer T cells and Helper T cells as part of an orchestrated immune response.

Figure 16 is the first in detailed analysis of the subtype of lymphocyte with respect to the cluster of differentiation (CD). In Figure 16 the increase in CD3 is shown. Lymphocytes contain B and T cells and in particular the T cells subpopulations were analysed. CD3 cells are T cells with the CD3 marker attached and serve various applications, such as immune regulations. Regulatory T cells were decreased in response to Covid-19 infection. The adjunct treatment increased the numbers into the healthy range.

Helper T-cells: Helper T cells are targeted by HIV AIDS infection and the CD4 were analysed in a clinical laboratory (Figure 17). (CD4 cells = Helper T-cells) CD4 cells are essential in the orchestrated immune response and were analysed. As a result of the Covid-19 infection on day 1 the CD4 numbers were low in BC and adjunct group (Figure 17). The increase in the adjunct group is higher on day 15 for the adjunct arm compared with BC alone. The figure 17 shows the change of helper T cells in humans.

Cytotoxic T-cells: Cytotoxic or killer T cells are critical in fighting a viral infection. In addition in Figure 18, CD 8 T cells are shown before and after treatment. Data are plotted for the CD8 T-cells in figure 18. In conclusion the increase in lymphocytes was most importantly seen in an increase in helper and killer T cells. On days 1 for all hospitalised patients the numbers were at the low reference range and by day 15 CD8 numbers were increased and the increase is higher in the adjunct group compared to BC.
In addition to viral uses, the application was extended from viral to bacterial applications, using the LPS model in mice. The PLS model in mice was used and here (LPS) lipopolysaccharide induced lung inflammation. LPS is part of the cell wall of gram negative micro-organisms. Lung injury by LPS is increasing the packed cell volume, which is reduced by positive control dexamethasone (figure 19). Efficacy of Pyrrol 5 is fully confirmed using this parameter and the activity is lost when dexamethasone and Pyrrol 5 is combined. Here, the neutrophils are increased from 3 to 13% on LPS treatment and reduced for positive control dexamethasone and for PNB-001/ Pyrrol 5 in a dose dependent matter (figure 20). The effect on neutrophils is totally lost when Pyrrol 5 is combined with dexamethasone. Again, for this second parameter adjunct treatment resulted in a loss of activity (figure 20).
On LPS treatment lymphocytes are increased and on positive control dexamethasone they were reduced (Figure 21). For treatment with Pyrrol 5 as single agent lymphocytes are increased and the error is minimal. The same effect is observed in mice for the entire group uniformly as in the MAD phase 1 study in humans. Here, the organism was not healthy, but infected with the experimental agent LPS. The effect is smaller on adjunct treatment dexamethasone and Pyrrol 5 and then again lost at the high dose.

In addition to viral- and bacterial- therapeutic application, in this invention the use of hydroxypyrrolones in cancer immune therapy is disclosed. Previously the use of hydroxypyrrolones as CCK antagonists were published to block CCK related cancers by pathway inhibition. This known effect was associated with the inhibition of the CCK pathways, which are an established growth factor for only CCK related cancers. (This is not working on non-CCK related cancers). Here, the new invention is a new mode of action for the generic scope of molecules, which act indirectly against cancer cells via the immune system by killing cancer cells/tissue with T cells. Thus, all cancers are removed from the organism, including non-CCK related cancers by immune therapy. Opposite to a normal approach to block pathways, with the activation of killer cells, such as cytotoxic T cells (Figure 22) cancer tissue is eaten by specialised lymphocytes. The tumour is killed by the hosts own immune response in form of cytotoxic killer cells. This effect is achieved by a small molecule, which is easily produced and not by a complex, expensive and unstable biomolecule (antibody).
For treating of any immune regulated physiological conditions, the effective dosage range may be preferably about 100ng/kg to about 1.0 mg/kg of CCK antagonist may be administered orally (p.o.), in single or divided doses per day (b.i.d.). Other routes of administration are also suitable.

In the effective treatment of a disclosed therapeutic application, preferably about 0.05 mg/kg to about 10.0 mg/kg of agent may be administered orally (p.o.), in single or divided doses per day (b.i.d.). Other routes of administration are also suitable.
For directly introducing therapeutic effects, the effective dosage range is preferably from about 100 ng/kg to about 10 mg/kg by intraperitoneal administration. Oral administration is an alternative route, as well as others.

For directly treating viral infections such as Dengue fever and SARS, the effective dosage range is preferably from about 100 ng/kg to about 1 mg/kg by intraperitoneal-, oral or IV administration.

For immune activity cancer, as seen for lung cancer, is responding to a low dose as in Covid 19 patients. The active dose range is stimulation the cytotoxic T cells, which kill the cancer and viral infected cells. This is studied in mice for gram negative bacteria, but is universal for all types of infection.
While it is possible for an active ingredient to be administered alone as the raw chemical, it is preferable to present it as a pharmaceutical formulation. The formulations, both for veterinary and for human medical use, of the present invention comprise an active ingredient in association with a pharmaceutically acceptable carrier therefor and optionally other therapeutic ingredient(s). The carrier(s) must be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The invention will now be further described by way of examples.

The compounds of formula (I) can be prepared by reaction of appropriately-substituted furan-2(5H)-ones with the corresponding amine, as described in Indian Patent no. 405278

Examples of pyrolones 1-24 are prepared from intermediates and the experimental details are included in the selected examples.

Example 1: Preparation of 5-hydroxy-5-aryl-pyrrol-2-ones
Preparation of 4-Chloro-5-hydroxy-5-aryl-1,5-dihydro-pyrrol-2-ones 1-24.
The compounds were prepared according to the methods as described in Indian Patent no. 405278 briefly outlined below.

General Method: The relevant amine (3 times excess) was added to a solution of building block (0.7 mol) in ether (10 ml) and it was stirred on ice for 30 minutes. It was allowing to warm up to RT over the time. The resultant mixture was poured into 5 ml water and separated by a separating funnel. The mixture was washed with water three times. The organic layer was dried over magnesium sulphate and the solvent was removed under vacuum. All compounds gave an oily solid which were passed through a column (80% ether, 20% petrol ether). The resulting fractions were dried from excess solvent under vacuum to yield crystals.

The following active molecules (Pyrrol 1-24) were prepared:

Pyrrol 1 4-Chloro-1-cyclopropyl-5-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 2 4-Chloro-1-(3,4-dimethyl-phenyl)-5-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 3 4-Chloro-5-hydroxy-1-isopropyl-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 4 4-Chloro-5-hydroxy-1-methyl-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 5 4-Chloro-5-hydroxy-1-phenethyl-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 6 4-Chloro-1-hexyl-5-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 7 4-Chloro-1-cyclopentyl-5-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 8 4-Chloro-5-hydroxy-1-isobutyl-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 9 1-Benzyl-4-chloro-5-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 10 4-Chloro-5-hydroxy-5-phenyl-1-((S)-(-)-1-phenyl-ethyl)-1,5-dihydro-pyrrol-2-one

Pyrrol 11 4-Chloro-5-hydroxy-5-phenyl-1-((S)-(-)-1-phenyl-ethyl)-1,5-dihydro-pyrrol-2-one

Pyrrol 12 4-Chloro-1-cyclohexyl-5-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 13 4-Chloro-5-(4-chloro-phenyl)-1-cyclopropyl-5-hydroxy-1,5-dihydro-pyrrol -2-one

Pyrrol 14 4-Chloro-5-(4-chloro-phenyl)-5-hydroxy-1-isopropyl-1,5-dihydro-pyrrol-2-one

Pyrrol 15 4-Chloro-5-(4-chloro-phenyl)-5-hydroxy-1-methyl-1,5-dihydro-pyrrol-2-one

Pyrrol 16 4-Chloro-5-(4-chloro-phenyl)-5-hydroxy-1-phenethyl-1,5-dihydro-pyrrol-2-one

Pyrrol 17 4-Chloro-5-(4-chloro-phenyl)-1-hexyl-5-hydroxy-1,5-dihydro-pyrrol-2-one

Pyrrol 18 4-Chloro-5-(4-chloro-phenyl)-1-cyclopentyl-5-hydroxy-1,5-dihydro-pyrrol-2-one

Pyrrol 19 4-Chloro-5-(4-chloro-phenyl)-5-hydroxy-1-isobutyl-1,5-dihydro-pyrrol-2-one

Pyrrol 20 1-Benzyl-4-chloro-5-(4-chloro-phenyl)-5-hydroxy-1,5-dihydro-pyrrol-2-one

Pyrrol 21 4-Chloro-5-hydroxy-1-phenethyl-5-phenyl-1,5-dihydro-pyrrol-2-one

Pyrrol 22 1-Benzyl-4-chloro-5-(4-fluoro-phenyl)-5-hydroxy-1,5-dihydro-pyrrol-2-one

Pyrrol 23 4-Chloro-5-(4-fluoro-phenyl)-5-hydroxy-1-isobutyl-1,5-dihydro-pyrrol-2-one

Pyrrol 24 N-Fluorobenzyl-4-chloro-5-(4-fluoro-phenyl)-5-hydroxy-1,5-dihydro-pyrrol-2-one

Example 2: Mortality after viral Dengue infection for 4-Chloro-5-hydroxy-1-phenethyl-5-phenyl-1,5-dihydro-pyrrol-2-one (5)
Dengue fever is an infectious disease caused by the dengue virus (DENV), and it is the most prevalent mosquito-borne viral disease in humans. Dengue fever occurs in tropical and subtropical regions and threatens an estimated 2.5 billion people. Growing urbanization, failure to control the mosquito vector, and increase in long-distance travel have contributed to the continuing spread and increase of the disease.

Although in most cases fever and other symptoms abate after 3–5 days, some patients deteriorate and progress to life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), which can reach a fatality rate of >20%. No vaccine or antiviral treatment for dengue infection exists.
IN VIVO

Dose groups Vehicle/Standard/Test Route of administration Dose Mortality
G1 Control - - N=8

G3 Pyrrol 5 PO 10 mg/kg N=2

Table 1. Individual animal mortality and clinical signs.

After only 1 week the mortality in the control group was 80% compared to 20% in the treatment group, giving a reduction of mortality by more than 50%, which was considered efficient (Table 1).

Inclusion criteria: T, rectal body temperature, T>38ºC; the mortality in dengue fever in man is about 20 % and here the virus load was increased in a dose range finding study prior to the main experiment. Organs were obtained from the study using mice and no immunosuppression was applied in the experiment (Table 2). The death rate was set to about 80 % using a high dose of the 2017 dengue strain (Table 1). The results were featured in drawings under figure 7.

Example 3: Bioactivity linked to viral inflammation
In the following sections the bioactivity linked to inflammation is studied. Body temperature served as inclusion criteria into the study and pre-clinical endpoints are level of inflammation marker IL-6 and size of the spleen.

Ex vivo – isolated tissue preparation
Inflammatory immune response during dengue infection
To investigate the effect of Dengue infection on mice, we studied the extent of the inflammatory immune response, which had been linked to the severity of symptoms in patients with dengue fever.

A clearly visible marker for the virus infection-induced inflammatory response is the enlargement of the spleen, as this is also observed in patients with dengue fever.

Mass of the spleen in mice
Uninfected Day 7
Control 0.06±0.02 g 0.16±0.05 g
Pyrrol 5 0.07± 0.01 g 0.56±0.1 g
Table 2: Mass of the spleen in mice

The size of the spleen was increased by 35 times with 1 week and this is a good marker to monitor infection as a result to a virus in general (Table 2).

Pyrrol molecule 5 reduced the swelling significantly and the reduction of the smelling of the spleen is in high correlation with a reduced death rate.

In vitro- Inflammation marker IL-6: The extent of the inflammatory immune response in Dengue infected mice was further explored by analyzing the levels of pro-inflammatory cytokines.

Interleukin (IL)-6 is an inflammation marker, which have been reported to be higher in patients with acute dengue infection. After infection, the levels of pro-inflammatory cytokines is increased until day 3 and IL-6 was measured at the end of the experiment on day 7. Levels returned to normal on week 2 after infection, when the virus infection was overcome by the host organism. These results correlate in man and mice with dengue infection and confirmed a causal link between the inflammatory response and the Dengue-infection in mice.

Inflammatory cytokine level IL-6 after infection in pg/ml
Day 0 Day 3 Day 7
Control 11±2 19±3 135±19
Pyrrol 5 (10 mg/kg) 10±2 19±4 29±5
Table 3. Analysis of inflammation marker IL-6

Initially inflammation marker IL-6 is low even in presence of fever, then from day 3 onwards inflammation is increasing, peaking at day 7, when the high inflammation correlates with a high constant decline of survival (Table 3). The pyrrol resulted in a reduction of inflammation marker IL-6 by 80%, in line with smaller spleen inflammation and a reduced death rate.

The 10 mg/kg dose was efficient in three analgesia-, four inflammation models and for the antipyretic effect. The 10 mg /kg dose of Pyrrol 5 would translate into a 70 mg dose in man and this dose is within the extremely save tested range in the G1-G4 MAD study.

Example 3: Clinical evaluation of Pyrrol-5 in moderate Covid 19 patients
RNA viruses also represent an enormous public health problem worldwide. Well-known RNA viruses include influenza virus (including the avian and swine isolates), hepatitis C virus (HCV), West Nile virus, SARS-coronavirus, COVID-19, respiratory syncytial virus (RSV), and human immunodeficiency virus (HIV). Vaccines exist only for influenza virus.

Accordingly, drug therapy is essential to mitigate the significant morbidity and mortality associated with these viruses. Unfortunately, the number of antiviral drugs is limited, many are poorly effective.

Coronavirus disease 2019 (COVID-19) has widely spread over the globe since its first case in December 2019 in Wuhan, China. COVID19 is spread via droplets or direct contact and infects the respiratory tract resulting in pneumonia. Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering, that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, it was shown that NETs is highly relevant in COVID-19 pathophysiology. This example is concerned with a clinical example.

NLR is a predictive biomarker of turning the infection into serious and Pyrrol 5 is required to stimulate the production of lymphocytes (Figure 8).

Method: The study was conducted at two sites in India (Study Registration NO. CTRI/2020/10/028423) and the 8 score ordinal scale was applied to evaluate efficacy.

Ordinal scale for clinical improvement eligibility

The inclusion criteria of the study were laboratory confirmed SARS-CoV-2 infection as determined by PCR within 2 days of randomization; patients having pneumonia with no signs of severe disease with SpO2 ≤94% (range 90- 94%) on room air; patients with any two of the following signs or symptoms suggestive of COVID-19: fever, cough, dyspnoea, or hypoxia; respiratory rate more or equal to 24 per minute; radiographic infiltrates as confirmed by imaging (chest x-ray).
The patients were randomized into two parallel groups in 1:1 ratio, one group to receive Pyrrol 5 along with best clinical care (Pyrrol 5+ BC) and the second group to receive only best clinical care (BC).

Primary efficacy endpoints - A significant improvement from baseline from Day 5 onwards till end of the study in Pyrrol 5+BC arm, while patients of BC arm showed improvement from Day 8. The mean ordinal scale for Pyrrol 5+BC arm was 0.22, and 1.12 in BC arm and the mean change in ordinal scale from baseline to Day 15 was statistically significant (P=0.042).

Patients receiving Pyrrol 5+ BC were hospitalized for lesser duration compared to BC (Mean 9.45 vs. 9.80 days). 95% of patients who received Pyrrol 5+BC were discharged from the hospital by the end of Day 15 and, 75% of patients were discharged from BC arm.
The mean duration of supplemental oxygen requirement for patients in Pyrrol 5+BC arm was 5.45 days and BC arm patients required supplemental oxygen for 7.10 days which is relatively higher. 17 patients from Pyrrol 5+BC arm were off oxygen support on day 14, while 13 patients were off oxygen support from BC arm (P=0.14).

CRP and ESR were analysed as inflammation biomarkers at molecular level and cellular level respectively. Greater than 90% of CRP reduced in Pyrrol 5+BC group; less than 80% of CRP reduced in BC group (Figure 9). The current finding is larger than any known anti-inflammatory agent.

The cytokine IL-6 was analysed as an inflammation or immune marker. A reduction of cytokines is observed during high inflammation and low levels of IL6 resulted in increase of cytokines leading to stimulation of immune system (Figure 10) by initiating lymphocyte proliferation.

The number of lymphocytes increased from 14% to 27% which is the optimal reference range, in Pyrrol 5+BC arm after 14 days of treatment. BC arm showed only 14% to 19% increase. The change between the arms on day 15 for Adjunct with Pyrrol 5 versus BC is significant with a P=0.032 (Figure 11). Neutrophil to lymphocyte ratio (NLR) is the most relevant Covid 19 biomarker. The patients had elevated NLR ratio on day 1 of hospitalization as a result of Covid-19 infection. The ratio dropped from 8.5 to 2.8 for Pyrrol 5+BC treatment and from 11.1 to 5.9 for BC treatment (Figure 13). The difference between the two arms are significant with P=0.010. Pyrrol 5+BC treatment showed reduction in glucose blood concentration (Figure 14).

Example 4: Evaluation of cluster of differentiation CD of Pyrrol 5 on lymphocytes
Lymphocytes contain B and T cells and in particular the T cells subpopulations were analysed. CD3 cells are T cells with the CD3 marker attached and serve various applications such as immune regulations. Regulatory T cells were decreased in response to Covid infection. The adjunct treatment increased the numbers into the healthy range. (Figure 16).

Helper T cells are targeted by HIV AIDS infection and the CD4 were analysed in a clinical laboratory.

CD4 cells / Helper T-cells: CD4 cells are essential in the orchestrated immune response and were analysed. As a result of the Covid-19 infection on day 1 the CD4 numbers were low in BC and adjunct group. The increase in the adjunct group is higher on day 15 for the adjunct arm compared with BC alone. (Figure 17)

Cytotoxic or killer T cells are critical in fighting a viral infection. In addition, the before and after values were plotted for the CD8 T-cells. (Figure 18). On days 1 for all hospitalised patients the numbers were at the low reference range and by day 15 CD8 numbers were increased and the increase is higher in the adjunct group compared to BC.

In conclusion the increase in lymphocytes was most importantly seen in an increase in helper and killer T cells.

Example 5: Bacterial activityby LPS model / bacterial inflammation/infection in mice
LPS from gram negative organism is applied to the lung and the inflammation cascade is triggered. The body was fighting back by increasing the number of lymphocytes (figure 21). Remarkable and for the first time inflammation and immune response were separated.

LPS instillation and sampling procedure: Mice, under ketamine–xylazine (50 mg/kg and 10 mg/kg; i.p;) anesthaesia, were instilled intratracheally with 50 ul saline solution or saline containing LPS (10 ug per mouse), after blunt dissection of the neck soft tissues to expose the trachea. At different times (4, 6, 12 and 24 h) after endotoxin injection, the animals killed by ether hyper anaesthesia; the BALF was obtained by gently washing the lung cavities with repeated 600 ul saline lavages up to a total volume of - 3 ml and was put into an ice-cold conical plastic tube. The lung was then removed to test the MPO activity. Betamethasone, or vehicle and Pyrrol 5 10, 30 and 100 mg/kg were administered orally 1 h before and 6 h after LPS- instillation; After 1 h the last dose of standard and test compounds the animals killed by ether hyperanaesthesia; the BALF was- obtained by gently washing the lung cavities with repeated 600 ul saline lavages up to a total volume of 3 ml and was put into an ice-cold conical plastic tube. The lung was then removed to test the following activity.

Here, under LPS immune stimulation the neutrophils, as well as macrophages were reduced (Figure 20). This is a serious issue in sepsis when fighting against the organism is required, but cytokine levels must be controlled and neutrophils are part of a toxic storm of neutrophils, caused by cytokines and termed cytokine storm.

Here, molecules of the generic scope reduced inflammation /cytokine storm under fully maintained immune response of the adaptive immune system.

Example 6. Cancer activity via killer T-cells
A human tumor was transplanted into a mouse model and proliferation was studied in form of tumor volume. The full experimental details were previously and repeatedly published by Lattmann et al and were summarized briefly.

Human xenograft study: In vivo experiments in mice - assessment of anti-tumour inhibition
Pure strain NMRI mice aged between 6 and 8 weeks from our inbred colony were used for transplanting SCLC and gastric tumours. Animals were fed on RM3E diet (Lillco-England) and water ad libitum. Approximately 2 mm cubes containing 2x 105 cells of tumour fragments were transplanted subcutaneously in the inguinal region via a trocar in a volume of 0.2 ml. Tumour bearing mice were randomised in groups of 6 animals per group and the treatment was started approximately 10 days after transplantation when the tumour was fully established.

The test compounds were administered in appropriate formulations. The effect of chemotherapy was assessed weekly after transplantation by measuring the tumour volume. Mice were killed after drug treatment or when weight loss reached 20% of body weight and the effects were measured by the differences in tumour volume as expressed:
% [inhibition] = Treated volume / control volume x 100

Here, the untreated tumor has grown exponentially and in the treatment group up to 90% inhibition of tumor growth was found (figure 22).

Typically, the inhibitors of cancer pathways are studied and here the body’s own defense system, and not a toxic chemical or biologic agent, killed the dysregulated cells. Thus, cancer tissue was removed from the host organism. The effect is not requiring the blockage of pathway with usually very high level of inhibition, leading to drug related toxicity. Other doses (figure 22) were tested and the same curve was obtained for the large dose range from 1, 3, 10, 30 to 100 mg/kg.
, Claims:We claim:
1. A pharmaceutical composition for the treatment of a disease, said composition comprising a therapeutically effective amount of a compound of formula (I):

(I)
wherein
X is halogen, alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy or alkenylcarbonyl;

Y is hydroxy or alkoxy group;

R is phenyl, cyanophenyl, or halogenated phenyl;

R1 is alkyl, branched alkyl, alkenyl, alkynyl, cycloalkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyloxy, alkenylcarbonyl, phenyl, phenyl alkyl, dialkyl phenyl, benzyl, or benzyl alkyl; and

pharmaceutically acceptable excipients and/or carriers.

2. The pharmaceutical composition as claimed in claim 1, wherein said alkyl-containing moieties are C1-C18, preferably C1-C12.

3. The pharmaceutical composition as claimed in claim 1, wherein said alkenyl- and said alkynyl-containing moieties are C2-C18, preferably C2-C12.

4. The pharmaceutical composition as claimed in claim 1, wherein R1 is C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, benzyl, cyclohexyl, phenyl, benzyl (C2-4) alkyl, or phenyl (C2-4) alkyl.

5. The pharmaceutical composition as claimed in claim 1, wherein X is selected from F, Br, Cl, and I.

6. A pharmaceutical composition comprising a therapeutically effective amount of a compound having one of the following formulae:

7. The pharmaceutical composition as claimed in claims 1 or 6, wherein the therapeutically effective amount compound of formula I is in the range of 100ng/kg to 20mg/kg.

8. The pharmaceutical composition as claimed in any of claims 1-7, wherein the composition is administered via oral, topical, intraperitoneal or intra-venous route.

9. The pharmaceutical composition as claimed in claims 1 or 6, for use in the preparation of a medicament for the treatment or prophylaxis of inflammation or a related condition.

10. The pharmaceutical composition as claimed in claims 1 or 6, for use in the preparation of a medicament, for the treatment of pain, pyrexia, inflammation of the gut or lung, or immune mediated diseases such as cancer.

11. The pharmaceutical composition as claimed in claims 1 or 6, for use in the preparation of a medicament, for the treatment of a viral disease and/or symptomatic treatment of pneumonia associated with a viral disease.
12. The pharmaceutical composition as claimed in claim 11, wherein the viral disease is Dengue fever, COVID-19 virus infection, HIV,hepatitis or multiple sclerosis.

13. The pharmaceutical composition as claimed in claims 1 or 6, for use of a compound in the preparation of a medicament, for the treatment or prophylaxis of inflammation which is mediated by cytokines, such as IL-6 or related conditions.

14. The pharmaceutical composition as claimed in claims 1 or 6, wherein the composition is useful for modulating immune functions, glucose levels or both.

15. The pharmaceutical composition as claimed in claims 1 or 6, wherein the composition is useful for modulating lymphocytes, particularly cytotoxic T-cells.

16. The pharmaceutical composition as claimed in claims 1 to 6, wherein the composition is useful for modulating neutrophils and bring them into a medically acceptable range.

17. The pharmaceutical composition as claimed in claims 1 to 6, wherein the composition is useful for inducing an increase in T-cells, particularly CD4 cells or CD8 cells or both.

18. A method of treatment of a mammal afflicted with a disease or prophylaxis in a mammal at risk of a disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I):

(I)
wherein

X is halogen, alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy or alkenylcarbonyl;

Y is hydroxy group or alkoxy group;

R is phenyl, cyanophenyl, or halogenated phenyl;

R1 is alkyl, branched alkyl, alkenyl, alkynyl, cycloalkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyloxy, alkenylcarbonyl, phenyl, phenyl alkyl, dialkyl phenyl, benzyl, or benzyl alkyl.

19. The method as claimed in claim 18, wherein said alkyl-containing moieties are C1-C18, preferably C1-C12.

20. The method as claimed in claim 18, wherein said alkenyl- and said alkynyl-containing moieties are C2-C18, preferably C2-C12.

21. The method as claimed in claim 18, wherein R1 is C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, benzyl, cyclohexyl, phenyl, benzyl (C2-4) alkyl, or phenyl (C2-4) alkyl.
22. The method as claimed in claim 18, wherein X is selected from F, Br, Cl and I.

23. A method of treatment of a mammal afflicted with a disease or prophylaxis in a mammal at risk of a disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound having one of the following formulae:

;

24. The method as claimed in claims 18 or 23, wherein the administration is done via oral, topical, intraperitoneal or intra-venous route.

25. The method as claimed in claims 28 or 23, wherein the disease is inflammation or a related condition.
26. The method as claimed in claims 18 or 23, wherein the disease pain, pyrexia, inflammation of the gut or lung, or immune mediated disease such as cancer.

27. The method as claimed in claims 18 or 23, wherein the disease is a viral disease and/or symptoms associated with a viral disease such as pneumonia.

28. The method as claimed in claim 27, wherein the viral disease is Dengue fever, COVID-19 virus infection, HIV, hepatitis or multiple sclerosis.

29. The method as claimed in claim 25, wherein the inflammation is mediated by cytokines, such as IL-6 or related conditions.

30. The method as claimed in claims 18 or 23, wherein method is useful inducing an increase in T-cells, particularly CD4 cells or CD8 cells or both

31. The method as claimed in claim 18 or 23, wherein the therapeutically effective amount of compound of formula (I) is in the range of 100ng/kg body weight of the patient and 20mg/kg body weight of the patient.

32. The method as claimed in claim 31, wherein the therapeutically effective amount compound of formula I administered in single dose or divided doses.

33. A use of a compound of formula (I) for the treatment of a disease by administering to a patient in need thereof, a therapeutically effective amount of a compound of formula (I):

(I)
wherein
X is halogen, alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy or alkenylcarbonyl;

Y is hydroxy group or alkoxy group;

R is phenyl, cyanophenyl, or halogenated phenyl;

R1 is alkyl, branched alkyl, alkenyl, alkynyl, cycloalkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyloxy, alkenylcarbonyl, phenyl, phenyl alkyl, dialkyl phenyl, benzyl, or benzyl alkyl.

34. The use as claimed in claim 33, wherein said alkyl-containing moieties are C1-C18, preferably C1-C12.

35. The use as claimed in claim 33, wherein said alkenyl- and said alkynyl-containing moieties are C2-C18, preferably C2-C12.

36. The use as claimed in claim 33, wherein R1 is C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, benzyl, cyclohexyl, phenyl, benzyl (C2-4) alkyl, or phenyl (C2-4) alkyl.

37. The use as claimed in claim 33, wherein X is F, Br, Cl and I.

38. A use of a compound having one of the following formulae for the treatment of a disease:

;

39. The use as claimed in claim 33 or 38, wherein the disease is inflammation or a related condition.

40. The use as claimed in claim 33 or 38, wherein the disease pain, pyrexia, inflammation of the gut or lung, or immune mediated diseases such as cancer.

41. The use as claimed in claim 33 or 38, wherein the disease is a viral disease and/or symptom associated with a viral disease such as pneumonia.

42. The use as claimed in claim 41, wherein the viral disease is Dengue fever, COVID-19 virus infection, HIV, hepatitis or multiple sclerosis.

43. The use as claimed in claim 38, wherein the treatment or prophylaxis of inflammation which is mediated by cytokines, such as IL-6 or related conditions.

44. The use as claimed in claim 33 or 38, wherein the therapeutically effective amount of compound of formula (I) is in the range of 100ng/kg body weight of the patient and 20mg/kg body weight of the patient.

45. The use as claimed in claim 33 or 38, wherein the administration is done via oral, intraperitoneal or intra-venous route.

46. The use as claimed in claims any of claims 33 to 45, wherein the compound is capable of modulating immune response or blood glucose or both.

47. The use as claimed in any of claims 33 to 45, wherein the compound is capable of modulating lymphocytes including an increase total number of lymphocytes, particularly cytotoxic T-cells.

48. The use as claimed in any of claims 33 to 45, wherein the compound is capable of modulating neutrophils and bring them into a medically acceptable range.

49. The use as claimed in any of claims 33 to 45, wherein the compound is capable of increasing T-cells, particularly CD4 or CD8 or both.