Title of Invention

"A HEXAHYDRO-2, 2-DIMETHYL-5-(2-NITROETHYL)- [1,3]DIOXOLO[4,5-B]PYRAZINE COMPOUND"

Abstract A compound hexahydro-2,2-dimethyl-5-(2-nitroethyl)- [1,3]dioxolo[4,5-b]pyrazine  obtained  from  Channa  striatus,   the compound represented asthe said compound being useful as an anti-neoplastic agent.
Full Text FIELD OF THE INVENTION
The present invention relates to a hexahydro-2, 2~dimethyl-5-(2-nitroethyl)- [1,3]dioxolo[4,5-b] pyrazine compound
The present invention also relates to a process for the isolation of a novel potent antineoplastic agent useful in pharmacological-therapeutic areas from the skin extract of the snake head fish Channa striatus, locally known as "Shol" fish. Background of the invention
Cancer is a major growing cause of mortality worldwide. Cancer affects men and women of all age groups and a wide variety of organs of the body. The incidence and mortality increase with age. Cancer is characterized by uncontrolled growth of the malignant cells which in turn invades the surrounding tissue as well as spreads to distant organ systems by the process called metastasis. Physical, chemical, immunobiological stimuli induces this mitotic abnormality as well as various phases of cell growth and multiplication.
The development of effective cancer therapy is a major focus of biomedical research. As a result of fundamental and applied research, certain previously lethal malignancies have become curable. Cancer therapy is distributed among three interacting subspecialties. The role of surgeon, the radiation oncologist, and the medical oncologist will continue to evolve as new agents became clinically available and a multimodality approach to cancer becomes the rule. The discovery of biologic agents of potential clinical importance continues at a rapid pace Tumor vaccine and gene transfer strategies are emerging from the laboratory and preclinical testing. New chemothera;peutic drugs, many with novel mechanisms of action, have entered clinical trials. Immunotherapy or cancer vaccine is effective in the tr5eatment of a malignant disease has recently been approved for clinical use. Manipulation of the host immune response to cancer is a major goal of biologic/immunotherapy. Animal studies have conclusively shown that
the immune system can recognize and eliminate malignant tumors in vivo Cancer immunotherapy seeks to evoke effective immune response to human tumors. Approaches have included administration of monoclonal antibodies, immunomodulatory cytokines, autologous and allogeneic immuno-competent cells, and tumor vaccines, Christopher and Donald (1998) (Christopher A.S. and Donal W K. (1998) Principles of cancer therapy In Harrison's
Chemother Pharmacol, 47(1), 34-40). Patients with cancer and chronic inflammatory disorders have used shark cartilage preparations for many years throughout the world, Miller et. Al. (1998) (Miller D R, Anderson G.T, Start J J, Granick J L and Richardson D (1998) Phase l/ll trial of the safety and efficacy of shark cartilage in the treatment of advanced
,L
cancer J. Clin Oncol, 16(11), 3649 - 3655). Shark liver oil is a natural source of alkylglycerols has been used for over 40 years as both a therapeutic and preventive agent in the treatment of neoplastic disorders and as an immune booster in infectious diseases, Pugiiese et. Al. (1998) (Pugiiese P T, Jordan K, Cederberg H and Brohult J (1998) Some biological actions of alkylglycerols from shark liver oil, J Altern Complement Med., 4(1), 87 - 99). So, fish is closely related with human society for its nutritional and therapeutic values.
In the present laboratory, we have for the first time isolated and purified a novel bioactive molecule from the skin extract of common murrel Channa striatus, which possesses pptent antineoplastic property. This novel compound, is alkaloid in nature and showed max 209.6 nm, containing -NH, -N02 as functional groups and the molecular weight was found to be 413 dalton. The tentative structure of the novel compound has been worked out. This novel compound showed potent
antineoplastic activity in both animal (in vivo) and human cell line

studies (in vitro).
Objects of the invention:
The main objective of the present invention is to provide a novel antineoplastic compound useful for pharmacological purposes and obtained from the skin extract of the Indian snake head fish Channa striatus, locally known as "Shol" fish.
Another object of the present invention is to provide a process for the isolation of a novel antineoplastic agent useful for therapeutic application in neoplasia and as a biomedical research probe/tool
Yet another object of the present invention is to provide a process for the isolation of a novel antineoplastic agent from easily available resources such as the common edible snake head fish Channa striatus, a natural product of Indian origin
Detailed Description of the Invention:
Accordingly, the present invention provides a novel anti-neoplastic compound and a process for its isolation, the compound being useful for various pharmacological purposes.
The said compound has been named as hexahydro-2,2-dimethyl-5-(2-nitroethyl)-[1,3]dioxolo[4,5-b]pyrazine and is represented as under:
(Formula Removed)
The said novel antineoplastic compound has been purified from the skin of a common edible jish skin Channa striatus, available in India. The compound has been punfied by simple and economic chromatographic methods
Further, the invention provides a pharmaceutical composition comprising the said novel antineoplastic compound and a therapeutically acceptable carrier The amount of the compound in the composition may vary from 50 to 60 µg. The composition may be prepared using known pharmacological techniques
In an embodiment, the compound is obtained by fractionating and purifying (isolation and purification) the skin extract and this step may be effected using known chromatographic methods such as thin layer chromatography on silica gel followed by two steps silica gel column chromatography to its crystalline form.
By the process of the present invention a novel and potent antineoplastic compound has been purified from the skin extract of Indian common murrel Channa striatus, locally known as "Shol" fish by thin layer chromatography followed by two steps silica gel column chromatography. The novel antineoplastic compound is devoid of haemorrhegic, heamolytic and defibnnogenating activity. This novel antineoplastic compound had no toxic effects on liver, kidney tissues.
This antineoplastic compound possesses haematinic effects and showed potent antineoplastic effect in animal (in vivo) and human carcinoma cell line studies (in vitro).
Accordingly, the present invention relates to a compound hexahydro-2, 2-dimethyl-5-(2-nitroethyl)- [1,3]dioxolo[4,5-b] pyrazine obtained from Channa striatus, the compound represented as
(Formula Removed)
(Fig 1) the said compound being useful as an anti-neoplastic agent
Detailed description of the accompanying drawings
Figure 1 shows UV spectrum of CSS-ANF Figure 2 shows IR spectrum of CSS-ANF Figure 3 shows 1H NMR spectrum of CSS-ANF Figure 4 shows 13C NMR spectrum of CSS-ANF Figure 5 shows FAB-MASS spectrum of CSS-ANF Figure 6 shows tentative structure of CSS-ANF
Example 1 Purification of the novel antineoplastic compound
To isolate antineoplastic compound, thin layer chromatography of
Channa striatus methanolic skin extract was done in preactivated glass
plates (20 x 10 cm) coaled with silica gel G 60, using solvent system
isopropanol • 0.1 (N) HCI (7:3 v/v) Zones were visualized in (1) UV (254
nm) chamber, (2) iodine vapour and Ri valve calculated.
i) A singlet at 5 1 26
ii) Two protons at 8 0 87 as double doublet.
The results are shown in figure 3. The 13C NMR spectrum of compound was studied in CDCI3 at FX300 MHZ spectrometer, Signals were obtained at 5 99 65, 76 59, 65.87, 31 92, 29.70, 22.70, 15.27 and 14 11. Fast atom bombardment mass spectra (FAB-MS) of compound was done with a Jeol FX500 spectrometer. The molecular weight of the compound was found to be 413.
Example 2 UV-spectrum
The ultraviolet spectrum of the compound recorded in a Perkin Elmer Lamda 15 UV/VIS spectrophotometer in spectral ethanol showed max 209 6 nm (Fig 1)
Example 3 IR-spectrum
The infrared spectrum of the compound in KBr pellets was done in Perkin Elmer IR spectrophotometer showed presence of — NH and -N02 functional groups (Fig 2)
Example 4 1H-NMR spectrum
The 1H-NMR spectrum of the compound was measured in CDCI3 at FX 300 MHz spectrometer (Fig. 3). The spectrum revealed the following characteristic features.
i) One proton singlet at 55.3
n) One proton triplet at 82.35.
in) One proton singlet at 52.10
iv) A broad singlet at 51 79.
v) A singlet at 51.26.
vi) Two protons at 50.87 as double doublet
Example 5 13C-NMR spectrum
The 13C-NMR spectrum of compound was studied in CDCI3 at FX300 MHz spectrometer (Fig. 4). Signals were obtained at 5 99.65, 76.59, 65 87, 31.92,29.70,22.70, 15 27 and 14.11.
Example 6
FAB-MS spectrum
Fast atom bombardment mass spectra (FAB-MS) of compound was done with a Jeol FX500 spectrometer (Fig. 5). The molecular weight of the compound was found to be 413 dalton
Structure elucidation
The novel antineoplastic compound isolated and purified from the skin extract of Indian common murrel Channa striatus, locally called "Shol" fish to jts_vellowish needle shaped crystalline forms gave positive test for alkaloid (Dragendorffs positive) The UV spectrum showed max 209 6 proving it to be a 5 membered cyclic compound The IR spectrum shows the presence of —NH and -N02 group The mass 413 (odd) further proves that it is a nitrogeneous compound mainly alkaloid in nature
The 1H NMR spectrum shows that it is not an aromatic compound and shows protons corresponding to -NH, -CH2, -CH3 and -CH 13C NMR spectrum shows 9 carbon atoms The complete structure of the novel antineoplastic compound is pending. The antineoplastic compound isolated and purified from the skin extract of Indian snake headed fish Channa striatus, locally known as "Shol" fish as mentioned in example 1 was obtained in pure crystalline state is alkaloid in nature, (Dragendorffs positive) with molecular weight of 413 Daltons.
In the following examples the biological activities of the novel antineoplastic compound were determined.
Example 7
EAC-tumor cell growth inhibition {in vivo)
The effect of the novel antineoplastic compound on EAC-tumor cell growth inhibition in mice (in vivo) was assayed according to Sur ef. at. (2001) (Sur P, Das M, Gomes A, Vedasiromoni J R, Sahu N P, Banerjee Sr Sharma R M and Ganguly D K (2001) Trigonella foenum graecum (Fenugreek) seed extract as an antineoplastic agent. Phytother Res, 15, 257-259). The novel antineoplastic compound treatment (50 p.g/20 g, i.p./day for 10 days) significantly inhibited Ehrlich ascites carcinoma cell (EAC-cell) growth in male albino mice as compared with the control EAC mice. The EAC-cell count in control mice was 680.83 ± 10.60 where as novel compound treated mice showed EAC cell count 187.16 ± 7.97*. about 72.50 ± 1.07% inhibition of cell growth (n = 6, *P Example 8
Bioassay of EAC — cell survivability [in vivo)
The bioassay of the novel antineoplastic compound on EAC-tumor cell viability was done by the modified method of Fernandes and Klubes (1979) (Fernandes D L and Klubes P (1979) A biochemical and pharmacological study of therapeutic synergism with 5-Fluorouracil plus cyclophosphamide in murine L1210 Leukemia. Cancer Res, 39, 1396 - 1404). The hovel antineoplastic compound treatment (50 ng/20 g, i.pVday for 1 day) significantly decreased EAC - cell viability. In control EAC-mice, the cell count was 802.66 ± 5.73 where as antineoplastic compound treated mice showed cell count 321.16 ± 7.27*. about 39.98 ± 0.87% decrease (n = 6, *P Example 9
Determination of survival time {in vivo)
The antineoplastic efficacy of the novel compound was done through the determination of median survival time (MST) with reference to tumor bearing control and 5-Fluorouracil (5-FU) treated mice according to the method of Sur and Ganguly (1994) (Sur P and Ganguly D K (1994) Tea plant root extract (TRE) as an antineoplastic agent Planta Med, 60, 106-109). According to the method, the animals surviving more than 60 days were considered to be cured. It was observed that, novel antineoplastic compound treatment (50 p.g/20 g, i pVday for 20 days) increased the survivability of EAC bearing mice as compared with control and 5-FU (200 |j.g/20g, i pVday for 20 days) treated standard group. Median survival time (MST) for the control group was found to be 36 days, while it was 52 days or 144% increase of life span for 5-FU treated standard group. MST for novel antineoplastic compound treated group was found >60 days indicating full protection (n = 6) or >166% increase of life span.
Example 10
EAC - cell growth inhibition {in vivo) (oral route)
In example 2, it was observed that the novel compared treatment (intraperitoneal route) significantly decreased the EAC - cell growth in mice as compared to control EAC mice. It was also observed that the novel compound treatment (oral route) (50 ng/20 g, p.o./day for 10 days) significantly inhibited Ehrlich ascites carcinoma cell growth in male albino mice as compared with the control EAC bearing mice. In control mice, the cell count was 740.55 ± 12.85, where as the novel compound treated mice showed cell count 310.25 ± 8.75* about 58.10 ± 2.40% inhibition of EAC cell growth (n=6, *P Example n
Determination of survival time {in vivo) (oral route)
In example 4, it was observed that the novel antineoplastic compound (intraperitoneal) treatment increased the survival time of EAC-bearing mice In oral route treatment of the novel antineoplastic compound (50 µg/20 g, po/day for 20 days) markedly increase the survivability of EAC bearing mice as compared with control group. Median survival time (MST) for the control group was found to be 28 days while it was found 50 days for the novel antineoplastic compound treated group indicating >214% increase of life span as compared to control EAC bearing mice survival time.
Example ' 12
Effect on human myeloid leukemic U-937 cell growth assay {in vitro)
In vitro human carcinoma cell growth inhibition assay was done according to the method of Sur et a/. (1995) (Sur P, Chatterjee S P, Roy P and Sur B (1995) 5-Nitrofuran derivative of fatty acid hydrazides induce differentiation in human myeloid leukemic cell lines, Cancer Letter, 94, 27-32). It was observed that the novel antineoplstic compound produced dose dependent inhibition of U-937 cell growth as compared to control untreated cells. In control the cell count was 70.83 ± 3.57 and 87.66 ± 4.06 in 24 hours and 48 hours respectively. The novel antineoplastic compound at dose of 10 n.g/ml showed a marked reduction in cell count (24 hours — 53.66 ± 2.77, 48 hours - 69.36 ± 6.26) about 24.23 ± 3.28% and 20.86 ± 2.54% inhibition in cell growth. Similarly, the novel compound at a dose of 50 µg/ml produced marked reduction in cell count (24 hours - 38.50 ± 3.41, 48 hours - 56.18 ± 2.48) about 45.65 ± 2.55% and 35.92 ± 2.35% inhibition in U-937 cell growth as compared to control U-937 growth.

Example - 13
MTT assay with U-937 cell line (in vitro)
The chemosensitivity of U-937 cell line was determined by the conventional MTT assay according to the method of Kawada et al. (1995) (Kawada M, Amemiya M, Ishijuka M and Takeuchi T (1995) Different induction of apoptisis in B-16 melanoma and EL-4 lymphoma cells by cytostain and bactobolin Jpn J. Can. Res, 90, 219-225) It was observed that the novel antineoplastic compound (10 ^ig and 50 ng/ml) showed a marked reduction (22.39% and 43.84% respectively) in U-937 cell viability as compared with untreated control U-937 cells indicating the cytotoxic nature of the novel antineoplastic compound.
Example 14
H - Thymidine incorporation assay (in vitro)
The effect of novel compound on DNA synthesis in U-937 cell line was assayed by incorporation of 3H-thymidine according to the method of Sur ef. a/. (1995) (Sur P, Chatterjee S P, Roy P and Sur B (1995) 5-Nitrofuran derivatives of fatty acid hydrazides induce differentiation in human myeloid leukemic cell lines. Cancer Letter, 94, 27-32). The U-937 cell line was treated with the novel antineoplastic compound at a dose of 10 and 50 p.g/ml for 24 hours. The novel compound produced a dose dependent inhibition (15.31 ± 0.68% and 34.99 ± 1.20% respectively) of 3H-thymidine incorporation to treated U-937 cell line as compared to untreated control U-937 cell line.
Example 15
Effect on human myeloid leukemic HL-60 celt growth assay (in vitro)
The effect of novel antineoplastic compound on HL-60 cell line was assayed according to Sur et. al. (1995) (Reference was mentioned in Example 7). HL-60 cells were treated with the novel antineoplastic compound at a dose of 10, 20, 50 and 100 jig/ml which produced a concentration and time dependent inhibition of HL-60 cell
grqwth as compared with untreated control HL-60 cells The control cell count at 12 hours, 24 hours and 48 hours' was 2.74 ± 0.40, 3.83 ± 0.40 and 4.56 ± 0.38 The novel compound at 10 ^g/ml produced 26.26 ± 0.16%, 21.40 ± 0.24% and 37.48 ± 0.21% inhibition at 12, 24 and 48 hours of observation. The novel compound at 20 µg/ml produced 32.83 ± 0.07, 31.07 ± 0.21 and 42 75 + 0.68% inhibition whereas 50 µg/ml produced 47.06 ± 0 35%, 50.90 ± 0.63% and 51.08 ± 0.14% inhibition of HL-60 cell growth at 12, 24 and 48 hours as compared to control untreated HL-60 cell growth. Similarly, the novel compound at a dose level of 100 µg/ml produced 55.44 ± 0.18%, 58.74 ± 0.37% and 64.23 ± 0.46% inhibition of HL-60 cells at 12, 24 and 48 hours of observation as compared to control untreated HL-60 cells.
Example 16
MTT assay with HL-60 cell line {in vitro)
The viability of HL-60 treated with the novel antineoplastic compound was determined by the MTT assay according to the method of Kawada ef. al. (1995) (Reference was mentioned in example 8). Treatment of HL-60 cells with the novel antineoplastic compound (10, 20, 50 and 100 µg/ml) showed dose dependent inhibition of HL-60 cells as compared to control untreated HL-60 cells. The novel antineoplastic compound (10, 20, 50 and 100 µg/ml) showed 20.06%, 28.42%, 43.47% and 55.85% cell inhibition (cytotoxicity) as compared to control untreated HL-60 cells.
Example 17
3H -Thymidine incorporation assay (in vitro)
3H-thymidine incorporation assay in novel compound treated HL- cells was done according to the method of Sur ef. al. (1995) (Reference was mentioned in example 9). HL-60 cell line was treated with the novel antineoplastic compound at a doses of 10 and 50 u.g/mt for 24 hour which produced a dose dependent (21.07 +: 0.48% and 49.82 ± 0.90% inhibition respectively) inhibition of 3H-thymidine incorporation to treated HL-60 cell line when compared to untreated HL-60 control cell line.
Example 18
Effect on hematological parameters / haemogram {in vivo)
To assess the novel antineoplastic compound on haemogram, male albino mice (n=6) was treated with the compound at a dose of 50 µg/20 g i p /day for 10 days, showed a haematinic effect on mice haemogram. The novel antineoplastic compound significantly increased the total RBC count (Control - 8.71 ± 0.12, treated - 9.31 ± 0.09**, **P Example . 19
Effect on hepato-renal system {in vivo)
To detect, the novel antineoplastic compound effect on metabolism and organ toxicity (liver, kidney etc.), male albino mice was treated with the novel antineoplastic compound at a dose of 50 µg/20 g, i.p./day for 10 days. After treatment scheduler blood was drawn from the 0 9% saline treated control group and from the compound treated experiment group (n=6) and biochemical parameter was estimated. It was observed that the novel antineoplastic compound had no significant effect on sugar (Control - 88.07 ± 2.41, treated - 83.22 ± 1.82), cholesterol (Control - 78.00 ± 2.22, treated - 73.40 ± 2.83), SGOT (Control - 103.25 ± 4.15%, treated - 94.86 ± 3.07), SGPT (Control - 36.25 ± 2.56, treated - 28.65 ± 1.89), alkaline phosphatase (Control - 72.70 ± 3.20, treated - 65.57 ± 2.86), urea (Control - 45.25 ± 2.53, treated - 43.25 ± 1.41), creatinine (Control - 0.54 ± 0.06, treated - 0.52 ± 0.08) indicating the novel antineoplastic compound did not interfere with metabolism and organ toxicity.
Example ' 20
Hemorrhagic activity {in vivo)
Cutaneous haemorrhagic activity of the novel antineoplastic compound was assayed in vivo in male albino Swiss mice (20g) after the method of Kondo et al.
(1969) (Kondo S, Ikezawa H, Murata R and Ohsaka A (1969) Studies on the quantitative method of determination of haemorrhagic activity of Habu snake venom Jap J. Med. Sci. Biol, 13, 43-51). Minimum haemorrhagic dose (MHD) was defined as the amount to test substance when injected intradermally, produced a haemorrhagic spot of 10 mm diameter within 24 hours of observation. Saline (0.9%) was used as negative control, Russel's viper venom (5 µg/20g, i.d ) was used as positive control. The novel antineoplastic compound (50, 100, 200 µg/20g, i.d ) did not produce any haemorrhagic spot observed within 24 hours as compared with positive control (10 mm diameter) Russel's viper venom. Thus the novel antineoplastic compound was found to be non-haemorrhagic in nature.
Example 21
Defibrinogenating activity (in vivo)
Defibrinogenating activity of the novel antineoplastic compound was assayed in male albino mice according to the method of Theakston and Reid (1983) (Theakston R D G and Reid H A (1983) Development of simple standard assay procedure for the characterization of snake venom. Bull WHO, 61, 949-956). The novel antineoplastic compound 50 ng, 100 ng and 200 µg in 0.9% satine was injected intravenously through the caudal vein in male albino mice (20g). After 1 hr. blood was collected from retro-orbital plexus and defibrinogenating activity was recorded. The blood of the compound treated animals coagulated within the same time as that of the control animal (0.9% saline injected, i.v.) within 2.00 ± 0.20 minute, indicating absence of defibrinogenating activity.
Example . 22
Haemolytic activity {in vitro)
Haemolytic activity of the novel antineoplastic compound (50, 100 and 200 u.g) was assayed in vitro by incubating with 1 ml 2% suspension of rat, guineapig, sheep, mice and human RBC at 37°C for 30 minute. The RBC suspension was centrifuged at 2000 r.p.m. for 15 minutes and degree of lysis was measured photometrically at 540 nm against negative control (0.9% saline) and positive control (100% distilled water).
The 'novel antineoplastic compound did not haemolyse the RBC indicating non-aemolytic nature of the novel antineoplastic compound.
Example 23
Detormination of LD50 {in vivo)
The LD50 of the novel antineoplastic compound was determined after Litchfield and Wilcoxon (1949) (Litchfield J T and Wilcoxon F (1949) A simplified method of evaluating dose effects experiments, J. Pharmacol. Exp. Ther. 26, 99-102). The different doses of the novel antineoplastic compound was administered into the male albino mice (20g) intravenously and mortality was recorded upto 24 hours. The LD50 of the novel antineoplastic compound was found to be 12.5 mg/kg (i.v.) in male albino mice.
Example 24
Isolated guineapig heart (in vitro)
Isolated guineapig heart was prepared after Langendroff (1895) (Langendroff O (1895) Untersuchungen am Uberlebenden Sangetierheazen, Plfugers. Arch. Ges. Physiol, 61, 291-293). On isolated guineapig heart, the novel antineoplastic compound (50 µg) did not produce significant change in heart rate and amplitude of contraction but at dose of 200 µg produced significant decrease in frequency of heart rate and amplitude of contraction leading to complete cardiac arrest within 60.50 ± 5.45 minute (n=6) indicating cardiotoxic nature at higher dose level.
Example 25
Isolated rat phrenic nerve diaphragm preparation {in vitro)
To assess the novel antineoplastic compounds effect on nerve muscle preparation, isolated rat phrenic nerve diaphragm was prepared after Bulbring (1946) (Bulbring E (1946) Observation on the isolated pohrenic nerve diaphragm preparation of the rat. Br. J. Pharmacol, 1, 38-42). The preparation was suspended in Tyrode solution in a 8 ml bath and gassed with 95% 02 and 5% C02. It was observed the
noyel antineoplastic compound 50 µg/ml had no significant effect on the nerve muscle preparation but at a dose level of 200 |ig/ml produced irreversible blockade of electrically induced twitch response within 48.33 ± 3.20 mm (n=6) indicating neurotoxic nature of the compound at higher dose level.
From the overall examples it may be concluded that, a novel antineoplastic compound, provisionally designated as CSS-ANF (where, CSS = Channa striatus skin, ANF = Antineoplastic factor) has been isolated and purified from the skin extract of the Indian snake head fish Channa striatus, locally known as "Shol" fish The compound is an alkaloid with molecular weight of 413 daltons. The structure of the novel antineoplastic compound has been worked out. This novel antineoplastic compound showed potent antineoplastic activity in several in vivo and in vitro experimental models where it inhibits EAC-cell growth, viability of EAC-cell, increased survival time of EAC bearing mice in both intraperitoneal and oral route treatment of the novel compound. The novel antineoplastic compound inhibit two human myeloid leukemic cell lines (U-937 and HL-60) growth, showed cytotoxicity by MTT assay. The antineoplastic compound action may be mediated through DNA level as indicated by 3H-thymidine incorporation experiments. The novel antineoplastic compound possesses haematinic effect, and had no interference with metabolism and organ toxic effects. The novel antineoplastic compound is devoid of defibrinogenating, haemorrhagic and haemolytic activity. The novel antineoplastic compound at higher doses possesses lethality, cardiotoxicity and neurotoxicity in experimental models.
The main advantages of the present invention are :
1. The novel antineoplastic compound (CSS-ANF) has been purified from the skin extract of Indian snake head fish Channa striatus, locally known as "Shol" fish, has the widest natured distribution in the freshwaters of India, Sri Lanka, Pakistan, Bangladesh, Burma, Malaya, Thailand, North Borneo, Vietnam, Cambodia, Philippines, Indonesia, Siam, Shanghai, Korea, Kampuchia etc. Chondar (1999) (Chondar S L (1999) In Biology of Fin fish and Shell fish, 1st edition, SCSC Publishers, Howrah, India, 435-444).

The novel antineoplastic compound (CSS-ANF) has been purified using easy and cheaper conventional biochemical methods.
The novel antineoplastic compound (CSS-ANF) significantly inhibited EAC-tumor cell growth in mice {in vivo), decreased EAC cell viability in mice (in vivo), increased survival time of EAC bearing mice {in vivo) in both oral and peritoneal route treatments, showed cytotoxicity by MTT assay on EAC tumor cell {in vitro).
The novel antineoplastic compound (CSS-ANF) significantly inhibited human carcinoma cell line U-937. HL-60 growth {in vitro), showed cytotoxicity by MTT assay {in vitro), reduced 3H-thymidine incorporation to treated cancer cells {in vitro) indicating DNA mediated mechanism of action of the compound.
The novel antineoplastic compound (CSS-ANF) had haematinic effect, had no toxic effects on liver, kidney tissues.
The novel antineoplastic compound (CSS-ANF) is devoid of haemorrhagic, haemolytic and defibrinogenating activity.
The novel antineoplastic compound (CSS-ANF) showed lethality, cardiotoxicity and neurotoxicity at higher doses but possesses potent antineoplastic activity in lower doses and at this dose level the compound (CSS-ANF) had no lethality, cardiotoxicity and neurotoxicity but has beneficial effects as revealed from the previous examples.
The yield of the novel antineoplastic compound (CSS-ANF) was obtained about 0.02%.





WE CLAIM:
1. A compound hexahydr o-2, 2-dimethyl-5-(2-nitroethyl)- [1,3]dioxolo[4,5-b] pyrazine obtained from Channa striatus, the compound represented as


(Formula Removed)
(Fig 1) the said compound being useful as an anti-neoplastic agent.
2. A pharmaceutical composition as and when prepared by using the compound of claim 1 together with pharmaceutically acceptable carrier.
3. A process for isolation of a novel anti-neoplastic compound as claimed in claim 1, comprising the steps of:
a. preparing an extract of the skin of Channa striatus,
b. subjecting the extract of thin layer chromatography, and
c. separating the fraction that shows anti-neoplastic activity and has Rf
0.35 on UV and iodine chamber.
4. A compound substantially as hereindescribed and illustrated with
reference to the examples.

Documents:

832-del-2003-abstract.pdf

832-del-2003-claims.pdf

832-del-2003-complete specification(granted).pdf

832-DEL-2003-Correspondence Others-(06-03-2012).pdf

832-del-2003-Correspondence Others-(11-04-2012).pdf

832-del-2003-correspondence-others.pdf

832-del-2003-correspondence-po.pdf

832-del-2003-description (complete).pdf

832-del-2003-drawings.pdf

832-del-2003-form-1.pdf

832-del-2003-form-18.pdf

832-del-2003-form-2.pdf

832-del-2003-form-26.pdf

832-del-2003-form-3.pdf

832-del-2003-form-4.pdf

832-del-2003-form-5.pdf

832-del-2003-GPA-(11-04-2012).pdf


Patent Number 235763
Indian Patent Application Number 832/DEL/2003
PG Journal Number 36/2009
Publication Date 04-Sep-2009
Grant Date 24-Aug-2009
Date of Filing 17-Aug-2003
Name of Patentee INDIAN COUNCIL OF MEDICAL RESEARCH
Applicant Address V. RAMALINGASWAMI BHAWAN, ANSARI NAGAR, POST BOX 4911, NEW DELHI 110 029, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 ANTONY GOMES DEPARTMENT OF PHYSIOLOGY, UNIVERSITY COLLEGE OF SCIENCE & TECHNOLOGY, UNIVERSITY OF KOLKATA, 92, ACHARYA PRAFULLA CHANDRA BOSE ROAD, KOLKATA 700 009
PCT International Classification Number A61P 35/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA