Synthesis and evaluation of 2-phenylamino-1,4-naphthoquinones derivatives as potential hypoglycaemic agents

Abstract Due to the severe side effects revealed by most of the currently used antidiabetic medicines, search for finding new and safe drugs to manage diabetes is continued. Naphthoquinones possessing strong antioxidant properties have been employed as candidates for diabetes therapy. Present study is aimed at finding the antioxidant and hypoglycaemic potential of some novel derivatives of 2-phenylamino-1,4-naphthoquinones (PAN) including chloro, nitro, methyl and bromo (5a-d) derivatives synthesized by single pot experiment. Product crystals were purified by TLC and characterized by FT-IR. The antioxidant potential of the compounds was assayed through DPPH radical scavenging and reducing power activities noted as UV-vis. absorbance. The DPPH assay has showed the powerful antioxidant activity of nitro and bromo derivatives, while the nitro derivative showed the significant reduction potential towards FRAP assay. Hypoglycaemic potential of the compounds was studied in rat animal model. All synthesized compounds revealed better hypoglycaemic activity; however, the chloro-derivative exhibited the more potent hypoglycaemic activity showing about 43% reduction in the mean blood glucose levels of the treated animals. As the bioreduction of naphthoquinones may be influenced by changing its redox properties, it has been noticed that the e-donating resonance effect (+R) of chloro group has shown the significant effects on biological activity through stabalization of its imine form which limits the potential of generation of free radicals during bioreduction of quinones and thus has been proposed as the reason of its hypoglycaemic activity. Future studies employing the properties of e-donating groups of PAN may optimize the drug-receptor interaction for better drug designing and drug development strategies against diabetes and also for the clinical trials.

Resumo Em razão dos graves efeitos colaterais causados pela maioria dos medicamentos antidiabéticos atualmente utilizados, continua a busca por novos medicamentos seguros para o controle do diabetes. As naftoquinonas, que possuem fortes propriedades antioxidantes, têm sido empregadas como candidatas à terapia do diabetes. O presente estudo visa encontrar o potencial antioxidante e hipoglicemiante de alguns novos derivados de 2-fenilamino-1,4-naftoquinonas (PAN), incluindo derivados de cloro, nitro, metil e bromo (5a-d) sintetizados por experimento em pote único. Os cristais do produto foram purificados por TLC e caracterizados por FT-IR. O potencial antioxidante dos compostos foi testado por meio de atividades de sequestro de radicais DPPH e redução de energia observada como absorção no UV-vis. O ensaio DPPH mostrou a poderosa atividade antioxidante dos derivados nitro e bromo, enquanto o derivado nitro mostrou o potencial de redução significativo para o ensaio FRAP. O potencial hipoglicêmico dos compostos foi estudado em modelo animal de rato. Todos os compostos sintetizados revelaram melhor atividade hipoglicemiante; no entanto, o derivado cloro apresentou atividade hipoglicêmica mais potente, com redução de 43% nos níveis médios de glicose no sangue dos animais tratados. Como a biorredução de naftoquinonas pode ser influenciada pela alteração de suas propriedades redox, notou-se que o efeito da doação eletrônica por ressonância (+R) do grupo cloro tem sido significativo na atividade biológica por meio da estabilização de sua forma imina, que limita o potencial de geração de radicais livres durante a biorredução de quinonas, e, portanto, tem sido proposto como a razão de sua atividade hipoglicemiante. Estudos futuros empregando as propriedades de grupos de doação eletrônica de PAN podem otimizar a interação droga-receptor para melhor planejamento de medicamentos e estratégias de desenvolvimento de medicamentos contra o diabetes e também para os ensaios clínicos.

Synthesis and evaluation of 2-phenylamino-1, 4-naphthoquinones derivatives as potential hypoglycaemic agents / Síntese e avaliação de derivados de 2-fenilamino-1, 4-naftoquinonas como potenciais agentes hipoglicemiantes

Due to the severe side effects revealed by most of the currently used antidiabetic medicines, search for finding new and safe drugs to manage diabetes is continued. Naphthoquinones possessing strong antioxidant properties have been employed as candidates for diabetes therapy. Present study is aimed at finding the antioxidant and hypoglycaemic potential of some novel derivatives of 2-phenylamino-1,4-naphthoquinones (PAN) including chloro, nitro, methyl and bromo (5a-d) derivatives synthesized by single pot experiment. Product crystals were purified by TLC and characterized by FT-IR. The antioxidant potential of the compounds was assayed through DPPH radical scavenging and reducing power activities noted as UV-vis. absorbance. The DPPH assay has showed the powerful antioxidant activity of nitro and bromo derivatives, while the nitro derivative showed the significant reduction potential towards FRAP assay. Hypoglycaemic potential of the compounds was studied in rat animal model. All synthesized compounds revealed better hypoglycaemic activity; however, the chloro-derivative exhibited the more potent hypoglycaemic activity showing about 43% reduction in the mean blood glucose levels of the treated animals. As the bioreduction of naphthoquinones may be influenced by changing its redox properties, it has been noticed that the e-donating resonance effect (+R) of 'chloro' group has shown the significant effects on biological activity through stabalization of its imine form which limits the potential of generation of free radicals during bioreduction of quinones and thus has been proposed as the reason of its hypoglycaemic activity. Future studies employing the properties of e-donating groups of PAN may optimize the drug-receptor interaction for better drug designing and drug development strategies against diabetes and also for the clinical trials.

Em razão dos graves efeitos colaterais causados pela maioria dos medicamentos antidiabéticos atualmente utilizados, continua a busca por novos medicamentos seguros para o controle do diabetes. As naftoquinonas, que possuem fortes propriedades antioxidantes, têm sido empregadas como candidatas à terapia do diabetes. O presente estudo visa encontrar o potencial antioxidante e hipoglicemiante de alguns novos derivados de 2-fenilamino-1,4-naftoquinonas (PAN), incluindo derivados de cloro, nitro, metil e bromo (5a-d) sintetizados por experimento em pote único. Os cristais do produto foram purificados por TLC e caracterizados por FT-IR. O potencial antioxidante dos compostos foi testado por meio de atividades de sequestro de radicais DPPH e redução de energia observada como absorção no UV-vis. O ensaio DPPH mostrou a poderosa atividade antioxidante dos derivados nitro e bromo, enquanto o derivado nitro mostrou o potencial de redução significativo para o ensaio FRAP. O potencial hipoglicêmico dos compostos foi estudado em modelo animal de rato. Todos os compostos sintetizados revelaram melhor atividade hipoglicemiante; no entanto, o derivado cloro apresentou atividade hipoglicêmica mais potente, com redução de 43% nos níveis médios de glicose no sangue dos animais tratados. Como a biorredução de naftoquinonas pode ser influenciada pela alteração de suas propriedades redox, notou-se que o efeito da doação eletrônica por ressonância (+R) do grupo "cloro" tem sido significativo na atividade biológica por meio da estabilização de sua forma imina, que limita o potencial de geração de radicais livres durante a biorredução de quinonas, e, portanto, tem sido proposto como a razão de sua atividade hipoglicemiante. Estudos futuros empregando as propriedades de grupos de doação eletrônica de PAN podem otimizar a interação droga-receptor para melhor planejamento de medicamentos e estratégias de desenvolvimento de medicamentos contra o diabetes e também para os ensaios clínicos.

Serum lipoprotein (a) levels in liver diseases caused by hepatitis.

Lipoprotein (a) [LP(a)] is a genetic variant of low density lipoprotein (LDL) and is mainly synthesized in liver. We conducted a study to evaluate the association of serum [Lp(a)] level with hepatitis viral infections. A total of 130 patients including 50 patients with acute viral hepatitis (AVH), 30 with chronic active hepatitis (CAH), 30 with cirrhosis of liver and 20 patients with fulminant hepatic failure (FHF) were analysed for different hepatitis viral markers and Lp(a) level in their serum samples. For comparison, 50 healthy persons were also tested for Lp(a) level. Serum Lp(a) level in patients in all the disease groups was significantly reduced compared to that observed in controls. Lp(a) level could not be detected in 40 per cent cases with AVH, 46.6 per cent with CAH, 70 per cent with cirrhosis and 80 per cent of FHF patients. On correlating Lp(a) level to viral etiology in these patients, it was found that the extent of diminution in Lp(a) level did not follow any trend with some particular viral infection and was recorded nearly same in all the infections. The findings of this study suggested that serum Lp(a) level was significantly (P<0.001) reduced in patients with liver diseases irrespective of the viral etiology.

Oxidant-antioxidant system: role and significance in human body.

Present article gives a holistic view of the causes, role and conrol of oxidative stress in the development and progression of various human diseases. Several types of reactive species are generated in the body as a result of metabolic reactions in the form of free radicals or non-radicals. These species may be either oxygen derived or nitrogen derived and called prooxidants. They attack macromolecules including protein, DNA and lipid etc. causing cellular/tissue damage. To counter their effect, the body is endowed with another category of compounds called antioxidants. These antioxidants are produced either endogenously or received from exogenous sources and include enzymes like superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, minerals like Se, Mn, Cu and Zn, and vitamins like vitamin A, C and E. Other compounds with antioxidant activity include glutathione, flavonoids, bilirubin and uric acid etc.. In a healthy body, prooxidants and antioxidants maintain a ratio and a shift in this ratio towards prooxidants gives rise to oxidative stress. This oxidative stress may be either mild or severe depending on the extent of shift and remains the cause of several diseases such as cardiovascular diseases, neurological diseases, malignancies, renal diseases, diabetes, inflammatory problems, skin diseases, aging, respiratory diseases, liver diseases and different types of viral infections. As more and more reports are pouring in, a lot of information is being unfolded about oxidative stress in relation to several other diseases.

Sero-epidemiology of hepatitis E virus (HEV) in urban and rural children of North India.

OBJECTIVE: To estimate the prevalence of anti-HEV IgG and IgM antibodies to ORF3 peptide of Hepatitis E virus genome in an age stratified urban and rural population of children. DESIGN: Cross sectional survey. SETTING: Pediatric out-patient clinics in a tertiary hospital and a rural dispensary. METHODS: Study subjects between 6 months and 10 years with minor, non-hepatic illnesses were recruited for the study from March to December 1996. Baseline demographic details, drinking water source, sewage disposal methods, reasons for attending the hospital, histories of parenteral exposure in the past 12 months and acute hepatitis in the subjects and the family in the previous six months were obtained. Serum anti-HEV IgG antibodies were screened in all subjects, and in those who were positive, anti-HEV IgM antibodies were assayed as an indicator of recent infection. Serum aminotransferase (ALT) was estimated in those who were anti-HEV IgM antibody positive. RESULT: Out of 2160 subjects recruited, 2070 samples could be screened for anti-HEV IgG antibodies. In the urban population (n = 1065) anti-HEV IgG antibodies were detected in 306 subjects (28.7%; 95% CI 26.0-31.6) and of these 131 (42.8%; 95%CI 37.2-48.6) were anti-HEV IgM antibody positive. Amongst 1005 rural children, anti-HEV IgG antibodies were present in 239 (23.8%; 95% CI 21.1-26.4) and IgM antibodies in 113 (47.3%; 95% CI 40.9-53.7) children. The antibodies were present since the first year of age till 10 years of age and, increased with advancing age. Serum transaminases were raised in 7.5% (9/120) and 5.5% (5/88) of subjects with anti-HEV IgM antibodies in urban and rural centers respectively. Overall the seroprevalence of IgG antibodies against HEV were significantly more in urban as compared to that in rural subjects (p = 0.011). However, proportion of children with anti-HEV IgG carrying IgM antibodies was similar in the two study groups (p = 0.298). A model for estimating expected prevalence of anti-HEV IgG antibodies was developed. The observed antibody prevalence in both urban and rural subjects at each age interval after 48 months was less as compared to the expected levels and this gap increased with advancing age categories. It appeared that there was a decay of HEV antibodies with time. CONCLUSIONS: Children are susceptible to HEV infection since early infancy. The probability of exposure to HEV during childhood was higher in urban than rural population. Seropositivity to HEV antibodies increased by over 2 times beyond 4 years of age as compared to younger age. Anti-HEV IgG antibodies appear to wean off with increasing age.

Impact of hepatitis C virus infection on renal transplant outcome in India--a single centre study.

BACKGROUND: Hepatitis C virus (HCV) infection is currently the main cause of hepatotropic viral infection in renal transplant (RT) recipient throughout the world. Contrary reports are available as regard graft and patient survival and liver disease outcome in these patients. From India, outcome of HCV positive patients following RT has not been documented. Herewith, we present results of RT in HCV positive patients at our centre. METHODS: Study design was prospective case control with primary end point being graft and patient survival and the exposure being HCV infection. Between June 1995 till February 1998, 128 patients had RT at our hospital, of which, 37 (28.9%) were anti-HCV positive at the time of RT. All the patients were on triple immunosuppressive therapy. As a policy of unit, none of the donor had HBV and/or HCV infection. Anti-HCV positive patients formed the subjects (Gr. I), while anti-HCV negative patients severed as control (Gr. II). Anti HCV was done using 3rd generation ELISA tests kit. HCV-RNA could not be done due to non-availability. None of the positive patient was treated with anti-viral therapy. Acute rejection, serious infections, patient and graft survival and outcome of liver disease was compared in these patients. RESULTS: Mean age of the patients, number of males, number of pre-RT haemodialysis and blood transfusion, donor age and HLA-mismatch were comparable in both the groups. Mean follow-up in Gr. I was 28 +/- 9.4 months and in Gr. II 31.4 +/- 7.6 months. At the end of this follow-up, acute rejection was seen in 43% and 33.3% patient in Gr. I and II respectively. In Gr. I, serious infections were seen in 30% while the same in Gr. II was 11.8% (p < 0.01). There was no difference in graft survival in Gr. I and II (72% and 66%) and the patient survival were also similar (72% and 66%). Of the deaths in Gr. I, 80% died of sepsis and 20% died of liver cell failure related to one each of hepatitis B and hepatitis E. Of the deaths in Gr. II, 65% died of sepsis and 17% died of hepatic cell failure. But, there was no difference in causes of deaths in these two groups. In both the groups, none of liver related death was due to isolated HCV infection. There was no effect of donor age, HLA mismatch, number of haemodialysis and pre-RT blood transfusion on the survival of graft as well as patient. CONCLUSION: In conclusion, HCV infection is major problem in RT with us. In a short follow-up of nearly 30 months, graft and patient survival is same in HCV positive and negative patients. However, serious infections are significantly more common in HCV positive patients.

Hepatitis C virus infection during haemodialysis in India.

BACKGROUND: Hepatitis C viral (HCV) infection is a major cause of hepatitis during haemodialysis after the control of hepatitis B following vaccination and isolation measures. Magnitude of this problem in India has not been studied and there are only few reports of HCV infection during haemodialysis from this country. This study was conducted to find out the incidence of HCV infection in patients of end stage renal disease (ESRD) and haemodialysis (HD), along with its source and to find out the clinical course of HCV positive patients. METHODS: HCV infection was diagnosed by detecting anti-HCV antibodies using the IIIrd generation ELISA kits. RESULTS: Of the 208 consecutive patients of ESRD accepted for the study, HCV prevalence was 4.3% at the time of start of study. Of the all risk factors studied, past history of jaundice and number of blood transfusion (BT) were significantly higher in HCV positive patients as compared to HCV negative patients. Of the 208 patients, 20 (9.6%) died, 119 (57.2%) lost follow-up and 69 (33.2%) got renal transplant (RT). Incidence of HCV in patients who died, lost follow-up and got RT was 10%, 3.4% and 36.2% while prevalence was 15%, 4.2% and 42% respectively. In these groups, duration of HD was 8.4, 5.2 and 22.7 weeks respectively while the mean blood transfusion (BT) was 1.2, 0.8 and 5.4 in number respectively. Mean age of patients in these groups was 37.85, 37.9 and 32.53 years and percentage of males were 65%, 75% and 89.9% respectively. At no stage of follow-up, patients with HCV infection had any symptoms or high serum bilirubin. Major abnormality was fluctuating ALT in these patients. HCV in 512 units of blood transfusions given to these patients and healthy volunteers was 1.17% and 0.66% respectively. Marked increase of HCV infection while patients were on HD is likely to be due to nosocomial spread. Blood transfusion was not found to be important source of HCV infection. Longer the patients remain on HD; more will be chance of HCV infection. CONCLUSION: It is concluded that HCV is a major cause of concern in haemodialysis patients in India and the predominant source of spread of infection is nosocomial. In our set-up, blood transfusion is not an important source of infection. Majority of these patients remains asymptomatic at least for the short terms follow-up.

Comparison of two schedules of hepatitis B vaccination in patients with mild, moderate and severe renal failure.

Seroconversion rate following hepatitis B vaccination in patients of chronic renal failure (CRF) has been in the range of 10%-82% in various studies. Different approaches have been tried to improve seroconversion rate. We studied two schedule of hepatitis B vaccination, 0,1,2 (Group A) and 0,1,2,6 (Group B) in mild (creatinine 1.5 to 3.0 mg%), moderate (creatinine 3.0 to 6.0 mg%) and severe CRF (creatinine > 6.0 mg%). Between Oct. 93 to Oct. 95, 117 patients with CRF who were negative for HBsAg and anti-HBs were included in the study. Forty micrograms of recombinant vaccine "ENGIREX" (20 micrograms in each deltoid region) was given in both the groups. Number of cases of mild, moderate and severe CRF were 18, 15 and 42 in group A and 12, 13 and 17 in group B, respectively. One month after the last dose of vaccination, anti-HBs was measured using ELISA kit (Abbot Laboratories, India). Anti-HBs titres of > 10 IU/L were taken as criteria of positive seroconversion. In group A seroconversion rate was 87.5%, 66.6% and 35.7% in mild, moderate and severe CRF respectively while same results in group B were 100%, 77% and 36.36%, respectively. We conclude that patients of chronic renal failure should be vaccinated at very early stage of the disease using 40 micrograms of vaccine. Four doses schedule of 0,1,2,6 give better results than three doses schedule in early CRF.

Hepatitis E virus: a global view of its seroepidemiology and transmission pattern.

Hepatitis E virus (HEV) infection causes epidemic outbreaks as well as sporadic disease in many parts of the world. It has been detected in travellers from endemic regions and also in native citizens of developed countries. In contrast to epidemics where predominantly adults are infected, HEV is found to be a common cause of acute sporadic hepatitis in children as well. A high incidence of HEV infection has been noted in pregnant ladies. Further, HEV has an association with other hepatotropic viruses and induces fulminant hepatic failure both with and without the simultaneous presence of other viruses. Transmission of HEV occurs predominantly by the faeco-oral route. However, the parenteral route has also been implicated. There is evidence to suggest vertical transmission of HEV via the intrauterine and perinatal routes. However, a number of questions remain unanswered. The available data do not explain the occurrence of HEV infection predominantly in adults during epidemics, possibility of contact transmission and means of protection against this infection. More detailed studies are needed to provide the actual status of HEV epidemiology in different parts of the world.

Prevalence of hepatitis C virus antibodies in the general population & in selected groups of patients in Delhi.

We report on the prevalence of anti-HCV antibodies in healthy individuals and patient groups with different liver diseases. The healthy population comprising 234 voluntary blood donors and 65 pregnant women with no history of liver diseases, had a per cent positivity of 1.5 anti-HCV in each group. The patients groups comprising 32 with acute viral hepatitis, 110 with fulminant hepatic failure, 65 with subacute hepatic failure, 33 with chronic active hepatitis, 45 with cirrhosis and 10 with hepatocellular carcinoma, had anti-HCV per cent positivity of 12.5, 43.6, 41.5, 48.5, 8.8 and 0 respectively. Anti-HCV was also tested in sera from 9 patients who had developed post-transfusion hepatitis and was recorded in 2 (22.2%) within one year of transfusion.

Plasma lipid profile in gallstone patients from North India.

One hundred and thirty five patients with gallstones along with eighty nine matched controls were studied ultrasonographically to look for any association with hyperlipidemias. Plasma cholesterol and triglycerides were estimated by colorimetric methods and lipoproteins were classified according to Beaumont's classification. Male to female ratio in gallstone patients was 1:3. Mean plasma cholesterol and triglyceride values were higher in male gallstones patients as compared to controls (166.40 +/- 54.21 vs 40.26 +/- 32.80 mg/dl, p <0.01 and 182.65 +/- 84.49 vs 133.18 +/- 52.37 mg/dl, p <0.01 respectively). In female gallstone patients, on the other hand, only plasma triglyceride levels were raised as compared to control (182.65 +/- 84.49 vs 133.18 +/- 52.32 mg/dl, p <0.01). Prevalence of type IIb and type IV was 24.32% and 29.72% in male gallstone patients and 13.2 and 39.70% respectively in female gallstone patients. Thus, more than half of our gallstone patients had hyperlipidemia, the commonest types amongst them being type IIb and type IV.

Prevalence of hepatitis B virus infection in healthy persons in North India.

BACKGROUND. There is scant information on the main methods through which hepatitis B virus infection is transmitted in India. We, therefore, studied the prevalence of hepatitis B surface antigen and antibody to hepatitis B surface antigen in voluntary blood donors as well as in those healthy groups who have a high risk of contracting this infection. METHODS. The groups at risk studied included commercial sex workers (635), eunuchs (28), truck drivers (217), professional blood donors (1117) and health care workers (1313). In addition, 20,435 voluntary blood donors were also studied. RESULTS. Hepatitis B surface antigen (and its antibody) was positive in 2.6% (14%) of voluntary blood donors, 3.6% (19%) of commercial sex workers, 5% (16%) of truck drivers, 12% (9%) of professional donors, 1.4% (19%) of health care workers and none (18%) of the eunuchs. Except professional donors and truck drivers, none of these groups had a higher positivity than the normal population (2.6%). CONCLUSIONS. Our results indicate that in India the so-called high risk groups, other than truck drivers and professional blood donors, are unlikely to represent major sources of infection.

Chronic hepatitis in a large Indian hospital.

BACKGROUND. In developed countries as well as in Southeast Asia, the hepatitis B and C viruses are the main causes of chronic hepatitis. In India, however, there have been no major investigations on the aetiology of chronic hepatitis. (The hepatitis E virus which is responsible for half the sporadic and most of the epidemic cases of acute viral hepatitis in India does not cause chronic disease.) We, therefore, studied the profile of chronic hepatitis in India. METHODS. The clinical presentation, aetiology, serology and histological changes were studied prospectively in 48 patients with chronic hepatitis admitted to the All India Institute of Medical Sciences, New Delhi. Of these, 44 (92%) had chronic active hepatitis, 3 (6.3%) had chronic persistent hepatitis and 1 (2%) had chronic lobular hepatitis. RESULTS. The hepatitis B virus was the aetiological agent in 24 (50%) of these patients, the hepatitis D virus in association with hepatitis B virus in 10 (21%), the hepatitis C virus in 7 (15%) and the non-A, non-B viruses other than the hepatitis C virus in 6 (13%). One patient (2.0%) had autoimmune chronic active hepatitis. Jaundice at presentation was seen in 33 (69%) patients and more than half had hypoalbuminaemia (< 3 g/dl) with a prolonged prothrombin time. Alanine aminotransferase levels were less than 5 times above normal in over two-thirds of the patients. The highest alanine aminotransferase values were observed in patients with hepatitis D virus infection whereas the lowest were seen in patients with non-A, non-B related chronic active hepatitis. Histological examination revealed bridging necrosis in 40 (91%) patients with chronic active hepatitis indicating a severe form of disease. Replication of the hepatitis B virus was seen in 13 patients with chronic hepatitis, 5 of whom had hepatitis D virus-induced chronic hepatitis. Patients with hepatitis B virus replication had higher alanine aminotransferase values and more severe bridging necrosis than patients who did not have replicating viruses. Higher alanine aminotransferase values, ascites and oesophageal varices were encountered more frequently in patients with hepatitis B and D virus than in those with non-A, non-B related chronic hepatitis. CONCLUSION. Chronic hepatitis is not uncommon in India. It presents with evidence of severe disease and, as elsewhere, is most frequently caused by the hepatitis B virus.

HBV--status in professional blood donors in north India.

Present study demonstrates the efficacy and significance of routine screening assays used for HBsAg testing in donor blood in different blood banks of Delhi city. Blood from professional donors already screened in blood banks were cross checked using micro-ELISA technique developed at All India Institute of Medical Sciences and the results were compared. HBsAg carrier rate in these professional donors was found to be 11.7% by micro ELISA as against only 6% reported in blood banks using RPHA and latex agglutination assays. Thus, assays used in blood banks were found to be missing nearly 50% HBsAg positive cases as compared to micro-ELISA. A small group of professional donors was also screened for anti-HBs and results explained in comparison of normal values.