1. 6-Amino-2-thio-, 6-aminouracils as Precursors for the Synthesis of antiviral and antimicrobial methylene bis(2-thiouracil) and tricyclic pyrimidines. Shaker Youssif*1 and Sahera F. Mohamed2 1 Medical Chemistry Division, Faculty of Medicine, University of Jazan, P.O.Box:114 Jazan Saudi Arabia 2 Department of Chemistry, Faculty of Education for Girls, Jazan, Saudi Arabia Summary. A several compounds of 5-arylmethylene bis(1-methyl-6- amino-2-thiouracils) 2-7 and 5-aryldipyrimidopyridines 9-14 were prepared by stirring of 6-amino-1-methyl-2-thiouracil (1) and 6- amino-1-benzyluracil (8) with ethanol in the presence of HCl or refluxing with AcOH. The structures of the novel compounds were characterized by elemental analysis and spectroscopic methods. The effect of both synthesized groups of the novel compounds on both HAV and HSV type 1 were investigated.

2. There are two compounds, (4) and (13), were effective inhibitors of virus replication, as determined by a virus yield inhibition assay. Compound (4) showed 54 % and 63 % virucidal effect on HSV type 1 with concentration of 10 μg/ml and 20 μg/ml respectively. Whereas 95.7 % and 100 % inhibitory concentrations against HSV type 1 were 10 μg/ml and 20 μg/ml for compound (13). On the other hand, some compounds showed inhibitory effect on both gram +ve, gram –ve bacteria as well as yeast and fungi

3. Results and Discussion Chemistry The stirring of 1-methyl-6-amino-2-thiouracil (1) in absolute ethanol in the presence of hydrochloric acid at room temperature or refluxing with glacial acetic acid afforded 5- arylmethylene bis(6-amino-1-methyl-2-thiouracil) (2-7) in a good yield. While, at the same conditions, 6-amino-1-benzyluracil (8) afforded the cyclic pyrido[2,3-d][6,5-d] dipyrimidines (9-14) in a good yield. 1H-nmr for compounds 2-7 showed the absence of d characterised for CH-5 of uracil and presence of d 7.56-7.59 characterised for 2 NH2 at C-6,6`. While, 1H-nmr for compounds 9- 14 showed the absence of d 7.56-7.59 characterised for NH2-6 and presence of d 11.23-11.98

4. R2R2 R1R1 RCompd. no H F H OH Cl H OCH 3 H OCH 3 H Cl 234567234567 Reagents: a) Abs. ethanol/HCl, r.t.; b) CH3COOH, reflux

5. R2R2 R1R1 RCompd. no H F H OH Cl H OCH 3 H OCH 3 H Cl 9 10 11 12 13 14

6. Pharmacology: The newly synthesized compounds have been tested for their antimicrobial activity against some microorganisms. The following microorganisms have been selected. 1-Viruses, a) HSV-1 b) HAV-27 2-Bacteria, a) Gram +ve bacteria; Bacillus subtilis, b) Gram - ve bacteria; Escherichia coli 3- Yeast; Candida tropicalis (NRC) 4- Fungi; Aspergillus niger (NRC) All bacterial, yeast and fungal strains were obtained from Microbial Biotechnology department (NRC); Egypt.

7. Hepatitis AHepatitis A virus (HAV) is classified with the enterovirus group of the Picornaviridae family. HAV has a single molecule of RNA surrounded by a small (27 nm diameter) protein capsid and a buoyant density in CsCl of 1.33 g/ml. Many other picornaviruses cause human disease, including polioviruses, coxsackieviruses, echoviruses, and rhinoviruses (cold viruses). The term hepatitis A (HA) or type A viral hepatitis has replaced all previous designations: infectious hepatitis, epidemic hepatitis, epidemic jaundice, catarrhal jaundice, infectious icterus, Botkins disease, and MS-1 hepatitis. Hepatitis A is usually a mild illness characterized by sudden onset of fever, malaise, nausea, anorexia, and abdominal discomfort, followed in several days by jaundice. The infectious dose is unknown but presumably is 10-100 virus particles. Hepatitis A is diagnosed by finding IgM-class anti- HAV in serum collected during the acute or early convalescent phase of disease. Commercial kits are available.HAV is excreted in feces of infected people and can produce clinical disease when susceptible individuals consume contaminated water or foods. Cold cuts and sandwiches, fruits and fruit juices, milk and milk products, vegetables, salads, shellfish, and iced drinks are commonly implicated in outbreaks. Water, shellfish, and salads are the most frequent sources. Contamination of foods by infected workers in food processing plants and restaurants is common. Picornaviridae

8. HSV Type-1 and Type-2 Herpes Simplex Virus SITE and TYPE There are actually two types of herpes simplex virus, HSV-1 and HSV-2. These are very similar in many ways, and both can cause either oral herpes or genital herpes. They do, however, prefer to live in different areas, and they follow different patterns of reactivation. For this reason, it's useful to find out which type you have, by asking your health care provider to request this information from the lab test that is done to diagnose your herpes. Let's talk about HSV-1 and HSV-2.Let's talk about HSV-1 and HSV-2 GENITAL HSV-2: HSV-2 accounts for about 2/3 of new genital infection, but is responsible for 90-95% of recurrences. About 90% of those who have HSV-2 infection do not know that they are infected (Fleming, 1997). GENITAL HSV-1: This infection is often transmitted from the mouth of one person to the genital of another, through giving and receiving oral sex, HSV-1 causes about 1/3 of new genital infections (about 75% of new genital infections in college students), but only recurs about once every other year, after the first year of being infected.

9. ("cold sores" or "fever blisters"). About 60% of the US population over the age of 12 is infected with HSV-1 virus. However, only about one third of people who are infected recall ever having any symptoms. ORAL HSV-2: It's rare to find someone who has oral HSV-2, but it can happen. After recovery from a possible first episode, such an infection is of little consequence in most cases, since oral HSV-2 is not likely to reactivate and cause signs or symptoms. Many thanks to Terri Warren, RN, ANP, MS, Med who is the owner of Westover Heights Clinic in Portland, Oregon, a private clinic specializing in the diagnosis and treatment of sexually transmitted diseases. Ms. Warren has been in practice for 22 years, and during that time has had a special interest in the area of genital herpes. Terri Warren is also author of The Updated Herpes Handbook.The Updated Herpes Handbook

10. ("cold sores" or "fever blisters"). About 60% of the US population over the age of 12 is infected with HSV-1 virus. However, only about one third of people who are infected recall ever having any symptoms. ORAL HSV-2: It's rare to find someone who has oral HSV-2, but it can happen. After recovery from a possible first episode, such an infection is of little consequence in most cases, since oral HSV-2 is not likely to reactivate and cause signs or symptoms. Many thanks to Terri Warren, RN, ANP, MS, Med who is the owner of Westover Heights Clinic in Portland, Oregon, a private clinic specializing in the diagnosis and treatment of sexually transmitted diseases. Ms. Warren has been in practice for 22 years, and during that time has had a special interest in the area of genital herpes. Terri Warren is also author of The Updated Herpes Handbook.The Updated Herpes Handbook

11. ORAL HSV-1: HSV-1 causes the vast majority of oral herpes ("cold sores" or "fever blisters"). About 60% of the US population over the age of 12 is infected with HSV-1 virus. However, only about one third of people who are infected recall ever having any symptoms. ORAL HSV-2: It's rare to find someone who has oral HSV-2, but it can happen. After recovery from a possible first episode, such an infection is of little consequence in most cases, since oral HSV-2 is not likely to reactivate and cause signs or symptoms. Many thanks to Terri Warren, RN, ANP, MS, Med who is the owner of Westover Heights Clinic in Portland, Oregon, a private clinic specializing in the diagnosis and treatment of sexually transmitted diseases. Ms. Warren has been in practice for 22 years, and during that time has had a special interest in the area of genital herpes. Terri Warren is also author of The Updated Herpes Handbook.The Updated Herpes Handbook

12. Microscopy image of a herpes simplex virus.

13. Latency and Recurrent disease HSV1 and HSV2 can establish a latent infection in the ganglia of the nerves that supply the site of the primary infection Genital area - sacral ganglia Oro-facial - trigeminal ganglion Following primary oro-facial infection, the virus enters sensory nerve endings and travels up the axon and establishes a latent infection in the trigeminal ganglion. The viral genome persists in an episomal form (plasmid) in the nucleus of the neurone. No viral genes are expressed. This state of latency may persist for many years. In a percentage of people, the virus will reactivate: - A cycle of viral replication occurs in the neurone and virus particles travel down the axon to reinfect the skin or mucous membrane in the area supplied by the nerve. Reactivation may be provoked by a number of stimuli: including sunlight, stress, febrile illnesses, menstruation or immunosuppression

15. HSV-1HAV-27 50 % inhibitory concentration % of virucidal effect 50 % inhibitory concentration % of virucidal effectAr Compd No. 20 μg/ml 10 μg/ml 20 μg/ml10 μg/ml >20 9.25 22.22 >20 20 - <5 - 45 32 63 45 27 35.5 44.5 31 50 0 100 0 ND٭ ND 54 31 ND 0 27 0 95.7 0 >20 - >20 23.25 >20 - >20 - 26 6 23 0 36 43 40 0 33 10 36 0 6 36 23 0 23 0 36 -C 6 H 5 -4.F.C 6 H 4 -2.CH 3 O.C 6 H 4 -3.CH 3 O.C 6 H 4 -4.HO.C 6 H 4 -2,4.Cl.C 6 H 3 -C 6 H 5 -4.F.C 6 H 4 -2.CH 3 O.C 6 H 4 -3.CH 3 O.C 6 H 4 -4.HO.C 6 H 4 -2,4.Cl.C 6 H 3 2 3 4 5 6 7 9 10 11 12 13 14 Table (1) Antiviral activities of the newly synthesized 5-arylmethylene bis(6- amino- 1-methyl -2-thiouracils) 2-7 and 5-aryldipyrimidopyridines 9-14. ND, Not determined. Each value is the mean of triplicate assays.

16. Table( 2 ) Antimicrobial activity of the newly synthesized compounds Diameter of the inhibition zone (mm)Compound No. A.nigerC. tropicalisE. coliB. subtilis 10.0 8.0 10.0 0.0 10.0 12.0 8.0 0.0 8.0 0.0 11.0 0.0 8.0 0.0 11.0 12.0 10.0 14.0 13.0 0.0 8.0 0.0 2 3 4 5 6 7 9 10 11 12 13 14