Pyrimethamine and Our research Question

[KieranCo]

Hi everyone. We are seven students from Sydney Grammar School (Harry, James, Oliver, Alex , Hugo, Duncan and Kieran) and we are hoping to contribute to the Open Source Malaria Project. We'd like to do this by continuing on from the work of last years students by investigating the activity of pyrimethamine analogues.

The first stage of our project is to do a literature search to identify pyrimethamine analoues that have already been synthesized and to see if their activity against T. gondii and P. falciparum has been investigated. At this point it looks like a lot of analogues have been synthesized and we are still sorting through which could be useful.

We are very open to any suggestions from the community about our research methods!

Below is a background that we have prepared about our project.

Pyrimethamine

Background

Gertrude Elion (1988 Nobel laureate) developed pyrimethamine (1) in 1952 in GlaxoSmithKline (GSK) laboratories, and it became available on the market in 1953.

1

GSK sold pyrimethamine, under the trade name daraprim, for 57 years after its initial introduction. It was shown to be active against the Plasmodium falciparum parasite, the most common malaria parasite and responsible for the most deaths world-wide,1 as well as the less common Plasmodium malariae and Plasmodium vivax.2 Initially, pyrimethamine was used for malaria in combination with drugs such as sulfadoxine, but due to its heavy use, resistance to it appeared within two years in countries several African countries.3,4 This pattern continued wherever pyrimethamine was used, and by 1967 resistance has spread to Asia.5 Today it is no longer the standard treatment for malaria due to this widespread resistance.

Currently, pyrimethamine is used in conjunction with other drugs such as sulphonamide to treat other parasitic infections such as toxoplasmosis (caused by Toxoplasma gondii)6 and isosporiasis (caused by Isospora belli).7 It can also be used to treat infections such as neosporosis in dogs.8

Rights to daraprim were bought by CorePharma in 2010, and then again by Impax Laboratories in 2015. The rights were then bought by Turing Pharmaceuticals in the same year, and a 5000% price hike of the drug occurred in the US later in the same year.9,10 This caused a huge public uproar, but even after the massive media pressure, the price has not decreased for patients.

Mechanism of action

Pyrimethamine acts by inhibiting the dihydrofolate reductase in the protists responsible for these diseases. This blocks the synthesis of tetrahydrofolic acid, a key precursor in DNA and RNA synthesis.11 Consequently, pyrimethamine leads to a failure of nuclear division, acting against the infection.

Why is research into pyrimethamine and its analogues important?

In 2015, there were an estimated 214 million cases of malaria and 438 000 deaths.12 Most of these deaths occur in children under 5 years old, and typically occur in developing countries in Africa and South-East Asia.12 The most common treatment for malaria currently is artemisinin used in combination with other drugs, but parasite resistance to artemisinin has been detected in Asia.13,14 In some areas along the Cambodia-Thailand border P. falciparum has become resistant to most antimalarial treatments, including artemisinin.14,15

T. gondii is one of the most widely distributed and successful parasites known.16 The impact of the protozoan on most healthy adult humans is minimal but if the immune system of the host is compromised by HIV infection or immunosuppressive drugs the infection can become acute and, if untreated, ultimately fatal.16 As the parasite can also move across the placenta, infection in pregnancy is associated with severe foetal damage and spontaneous miscarriage.17 Although further work need to be done, growing evidence has shown that T. gondii is developing significant resistance to a number of drugs, including pyrimethamine.18,19

Consequently it is essential that new, effective antiparasitic medicines are developed. Pyrimethamine analogues are an attractive target for our research because a viable synthetic route has been developed that can be performed in school laboratory conditions by high school students.

Our research question

As a team, we intend to research which analogues of pyrimethamine have been made previously and what has been found about their biological activity. Specifically we would like to find analogues that have not been tested against P. Falciparum, or even develop entirely new analogues. We would then synthesise these and test their activity against malaria.

References

1. Rich, S. M.; Leendertz, F. H.; Xu, G.; Lebreton, M.; Djoko, C. F.; Aminake, M. N.; Takang, E. E.; Diffo, J. L. D.; Pike, B. L.; Rosenthal, B. M.; Formenty, P.; Boesch, C.; Ayala, F. J.; Wolfe, N. D. The origin of malignant malaria. Proc. Natl. Acad. Sci. 2009, 106,14902–14907.

2. Shute, P.G.; Maryon, M. The effect of pyrimethamine (daraprim) on the gametocytes and oocysts of Plasmodium falciparum and Plasmodium vivax. Trans. R. Soc. Trop. Med. Hyg. 1954, 48, 50-63.

3. Burgess, R. W.; Young, M. D. The development of pyrimethamine resistance by Plasmodium falciparum. Bull World Health Organ. 1959, 20, 37–46.

4. Jones S. A. Resistance of P. falciparum and P. malariae to pyrimethamine (daraprim) following mass treatment with this drug; a preliminary note. East Afr. Med. J. 1954, 31, 47-9.

5. Wongsrichanalai, C.; Pickard, A. L.; Wernsdorfer, W. H.; Meshnick, S. R. Epidemiology of drug-resistant malaria. Lancet Infect. Dis. 2002, 2, 209-218.

6. Chio, L.C.; Queener, S.F. Identification of highly potent and selective inhibitors of Toxoplasma gondii dihydrofolate reductase. Antimicrob. Agents Chemother. 1993, 37, 1914-1923.

7. Ebrahimzadeh, A.; Bottone, E.J. Presistent diarrhea caused by Isospora belli: therapeutic response to pyrimethamine and sulfadiazine. Diagn. Microbiol. Infect. Dis. 1996, 26, 87-89.

8. Barber, J. S.; Trees, A. J. Clinical aspects of 27 cases of neosporosis in dogs. Vet. Rec. 1996, 139, 439-443.

9. McCarthy, M. Drug's 5000% price rise puts spotlight on soaring US drug costs. BMJ (Online), 2015, 351, h5114.

10. Bloomberg. Drug Goes From $13.50 to $750 Overnight. http://www.bloomberg.com/news/videos/2015-09-21/why-turing-increased-price-of-daraprim-over-500- Sept 22, 2015 (accessed Jun 20, 2017].

11. McCutchan, T. F.; Welsh, J. A.; Dame, J. B.; Quakyi, I. A.; Graves, P. M.; Drake, J. C.; Allegra, C. J. Mechanism of pyrimethamine resistance in recent isolates of Plasmodium falciparum. Antimicrob. Agents Chemother. 1984, 26, 656-659.

12. World Health Organization. World malaria report 2015. 2015. Available http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/

13. Tilley, L.; Straimer, J.; Gnädig, N. F.; Ralph, S. A.; Fidock, D. A. Artemisinin action and resistance in Plasmodium falciparum. Trends Parasitol. 2016, 32, 682-696.

14. Dondorp, A. M.; Fairhurst, R. M.; Slutsker, L.; MacArthur, J. R.; Guerin, P. J.; Wellems, T. E.; Ringwald, P.; Newman, R. D.; Plowe, C. V. The threat of artemisinin-resistant malaria. N. Engl. J. Med. 2011, 365, 1073-1075.

15. Dondorp, A. M.; Yeung, S.; White, L.; Nguon, C.; Day, N. P.; Socheat, D.; von Seidlein, L. Artemisinin resistance: Current status and scenarios for containment. Nature Rev. Microbiol. 2010, 8, 272-280.

16. Sims, P. F. G. Drug Resistance in Toxoplasma gondii. In Antimicrobial drug resistance: Clinical and epidemiological aspects. Mayers, D. L. Ed.; Humana Press: Totowa, NJ, 2009, pp 1121-1126.

17. Thulliez, P. Maternal and foetal infection. In Toxoplasmosis: A comprehensive clinical guide. Joynson, D. H. M., Wreghitt, T. G., Eds.; Cambridge University Press: Cambridge, UK, 2001, pp 193-213.

18. Thaithong, S.; Chan, S.-W.; Songsomboon, S.; Wilairat, P.; Seesod, N.; Sueblinwong, T.; Goman, M.; Ridley, R.; Beale, G. Pyrimethamine resistant mutations in Plasmodium falciparum. Mol. Biochem. Parasitol. 1992, 52, 149-157.

19. Tanaka, M.; Gu, H.-M.; Bzik, D. J.; Li, W.-B.; Inselburg, J. W. Dihydrofolate reductase mutations and chromosomal changes associated with pyrimethamine resistance of Plasmodium falciparum. Mol. Biochem. Parasitol. 199, 39, 127-134.

[drc007]

ChEMBL contains the structures and activity versus dihydrofolate reductase

https://www.ebi.ac.uk/chembl/target/inspect/CHEMBL1939

[mattodd]

Hi @KieranCo . Thanks for this. A few questions. 1) You're the Sydney Grammar School team doing lit background on Daraprim, right? @alintheopen ? 2) If so, could you edit the post above with a little paragraph of intro so that people know this? It helps for people to know who they're dealing with so they can pitch their help in the most useful way. 3) At the top or bottom of your post can you clarify what you're looking for from us, the community? You need help with something specific? 4) If we are treating the above entry as a bit like a wiki, to keep track of your progress, are you happy for other people to edit it? Or are you keeping an active document (like a living report) somewhere else? 5) When's your deadline? i.e. when are you finishing up? Welcome aboard, by the way!

[AJIC]

Hi @mattodd,

Thanks for being so welcoming! Sorry for the slow reply, we've been on school holidays for the past few weeks, and we're just getting back into it. In answer to your questions:

4. We're happy to treat the entry as a wiki, so people can edit it. We've got a spreadsheet with the results of our ongoing lit search.

5. Our deadline is really the end of the year. However, this term we have our major exams so we may not be as active as we will be next term.

[TrentWallis]

Whatever you think about Sci-Hub, some of the papers you're after might be more easily obtained there. To use Panisko, D.M. (1990) from the above list as an example, sure you could pay €40, or see if it's online somewhere, or if someone at a university has access to it, or investigate an inter-library loan, or deliver the original manuscript by carrier pigeon... or you could just find the URL or doi and paste it into http://sci-hub.ac/...

[mattodd]

Well, we at USyd, and others at other Unis, will have access to all these papers and are well within our rights to share with scientific collaborators behind a paywall (e.g. a shared Dropbox account, or something like that). If this is ever something that's needed, then please just say. There may even be solutions that allow for guest access to the lit via the Library for a discrete period - I can ask.

[cdsouthan]

@AJIC You might (as might OSM also) consider joining/exploiting http://www.citeulike.org/

You can create fully open, tagged and annotated literature lists. Its totally flexible e.g. you can paste key sections of full text (from Sci-Hub or wherever) into the public notes pages.

We (http://dev.guidetopharmacology.org/) use this for open literature curation eg http://www.citeulike.org/user/cdsouthan/tag/immpharm

[juliasmith2]

We're Julia and Loops from the Broadrup Lab at Haverford College. We have synthesized pyrimethamine and two analogues. Our notebook can be found at: http://malaria.ourexperiment.org/synthesis_of_darapr

Our first step uses acid chlorides, which provide access to many different analogues. The second step uses TMS diazomethane. The third step is the traditional ring closing with guanidine. We are interested in next steps, and we would be willing to commit several hours a week to this in the fall.

[MFernflower]

@juliasmith2 there are a few interesting daraprim like molecules: http://www.rcsb.org/pdb/explore/explore.do?structureId=3FRA This is pretty cool because you can see how the molecule has a total death grip on the bacterial DHFR protein https://en.wikipedia.org/wiki/Trimetrexate This is said to work on malaria but I cannot seem to find a lot on it

[mattodd]

Wow @AJIC @KieranCo - your lit search spreadsheet is growing! Need any help, e.g. in how to triage compounds? Have you found any compounds that have not yet been evaluated? Or any obvious analogs that have not been made? Are there any papers you've not yet been able to access?

[mattodd]

Just found this site which seems to have a daraprim synthesis available for download, though I've not taken a look. Found via this article which, well, I'm not 100% sure about.

[MFernflower]

^ Neat concept - but that website has no data available for download - I will be keeping an eye on it however!!!!

[mcoster]

Just found this site which seems to have a daraprim synthesis available for download, though I've not taken a look. Found via this article which, well, I'm not 100% sure about.

Oh my... Anyone contemplating making their own pharmaceuticals should reflect on the MPTP story and seriously consider how they will do their own QC. Last time I looked, NMR spectrometers were a tad expensive for home use.

[MFernflower]

@mcoster It's one thing to make a dye or non edible chemical at home (I've made a few copper pigments) but it seems really dangerous to try to make things you will be administering to yourself. Also did you have a look at what they want to be able to make later on - Sofosbuvir!

[mcoster]

In a world where some people are willing to turn themselves into Smurfs, I have stopped being surprised at what people are willing to put into their bodies..

[MFernflower]

@HThawley http://www.sciencedirect.com/science/article/pii/S0006295216303082 section 13 details DHFR inhibitors

[HThawley]

We think we’re almost done with the literature search, just a few last things to clear up. The next thing we have to do is organise the information on the analogues, so we would like some advice on how best to do this. We figure we’ll need to collate all the analogues that have been made, organise them by functional group location on daraprim, we’ll need to give them some sort of code (for example BGSGS001 for daraprim) , and include computer strings. We’ll also include information about past testing against toxoplasmosis, neosparosis, or P. Falciparum (Pretty much everything has been tested against toxoplasmosis, but it looks like some haven’t yet been tested against malaria). The main problem we have, is that we don’t have very many computers with chemdraw, so getting the strings will be a long and tedious process.

The main thing we would like help with is the best way to organise the analogue data, and if anyone knows of any reliable freeware programs that can get computer strings for the compounds.

[rbroadrup-HC]

Do you have experience using Chemdraw for Excel? It allows you to make a spreadsheet where you draw the editable molecule in 1 cell.

You can then generate a variety of outputs usings special functions. You can even convert to .sd files. I've used this method to create databases of 1000s of molecules that can then be used with other valuable programs.

On Aug 10, 2017 6:22 PM, "HThawley" notifications@github.com wrote:

We think we’re almost done with the literature search, just a few last things to clear up. The next thing we have to do is organise the information on the analogues, so we would like some advice on how best to do this. We figure we’ll need to collate all the analogues that have been made, organise them by functional group location on daraprim, we’ll need to give them some sort of code (for example BGSGS001 for daraprim) , and include computer strings. We’ll also include information about past testing against toxoplasmosis, neosparosis, or P. Falciparum (Pretty much everything has been tested against toxoplasmosis, but it looks like some haven’t yet been tested against malaria). The main problem we have, is that we don’t have very many computers with chemdraw, so getting the strings will be a long and tedious process.

The main thing we would like help with is the best way to organise the analogue data, and if anyone knows of any reliable freeware programs that can get computer strings for the compounds.

— You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub https://github.com/OpenSourceMalaria/OSM_To_Do_List/issues/509#issuecomment-321689480, or mute the thread https://github.com/notifications/unsubscribe-auth/AS409niJIXz0uQ51yFtlgj1qmIr90tbOks5sW4KRgaJpZM4OJC07 .

[TrentWallis]

If you don't have success with the above, ACD Chemsketch is free and can generate SMILES/InChI from structures.

[mattodd]

Sounds awesome @HThawley . Can't wait to see this diagram, and if you've identified a way forward synthetically. The human-readable summary is almost as important as the detailed search you've done.

Compound numbering helps a lot in discussion, yes. @alintheopen and I briefly mentioned this when we spoke yesterday. OSM-SGS-XXX. Or yes, BG-SGS-XXX (BG = "Breaking Good") would be great given that malaria is perhaps not the principal focus of these compounds. I would advise having the codes and strings in a spreadsheet (correlating to the lit source where you found the compounds) as well as having a human-readable diagram summarising your findings.

For drawing, head over to the discussion at #531 . Chemaxon have something called Marvin that you should check out. I can discuss with them soon about whether we could obtain a shared licence for BG or OSM users. There's a demo online you can try, but I suspect we will need a licence to use properly.

[MFernflower]

@mattodd I use the free version of Marvin sketch - I have not fully checked the EULA but I assume it's fine as OSM is a charity organization - it would be very nice if they would be able to give us all a shared osm license however!

[ErinSheridan]

@rbroadrup-HC I've just had a look at Chemdraw for Excel and it's ideal for what we want to do, but we're still hampered by only having Chemdraw on one computer accessible to everyone so I'd rather not rely on Chemdraw.

But it looks like we have some good options for drawing structures and generating strings now, thanks @TrentWallis, @mattodd and @MFernflower for your suggestions!

[rbroadrup-HC]

Sounds good, and please let me know if you later go with the Chemdraw option and I can be helpful. Years ago, if memory serves, they used to be easier on the wallet for individual academic purchases.

On Aug 11, 2017 12:36 AM, "ErinSheridan" notifications@github.com wrote:

@rbroadrup-HC https://github.com/rbroadrup-hc I've just had a look at Chemdraw for Excel and it's ideal for what we want to do, but we're still hampered by only having Chemdraw on one computer accessible to everyone so I'd rather not rely on Chemdraw.

But it looks like we have some good options for drawing structures and generating strings now, thanks @TrentWallis https://github.com/trentwallis, @mattodd https://github.com/mattodd and @MFernflower https://github.com/mfernflower for your suggestions!

— You are receiving this because you were mentioned.

Reply to this email directly, view it on GitHub https://github.com/OpenSourceMalaria/OSM_To_Do_List/issues/509#issuecomment-321730496, or mute the thread https://github.com/notifications/unsubscribe-auth/AS409n9h_J12c6E-SHIyS2Hd6mTFsKIRks5sW9pOgaJpZM4OJC07 .

[drc007]

@ErinSheridan If the molecules are in the literature you should search ChemSpider for example daraprim http://www.chemspider.com/Chemical-Structure.4819.html?rid=991e3363-d082-4a5a-96a6-0ad426588730 If you click on more details, you can just copy the image of the structure and the SMILES, InChI etc. So no need to redraw.

[ErinSheridan]

@ceyoung123, here are a few links to get you started.

Daraprim - The story so far (not recently updated). Summary of 2016 daraprim synthesis Our literature research so far, mostly complete just needs some tidying Collating information about pyrimethamine analogues from the literature search - very much a work in progress!

We used Google Scholar to search for the keywords, and I have a USyd library unikey that we used to locate the papers.

[ErinSheridan]

At the end of last term we had to make some decisions about what analogues we would like to synthesise before the end of the year since we needed to order the chemicals. We wanted to use the same route as last year as far as possible. Obviously we know that it works, but also all of the variation can be introduced in the two starting materials. Since we were running up against a deadline, based on the (then-incomplete) collation of the compounds that have been made and the phenylacetonitriles available to purchase, we bought the chemicals to make the following pyrimethamine analogues (we're still waiting on 3,5-bis(trifluoromethyl)phenylacetonitrile for 7-9).

With the now almost complete collation of the compounds that have been previously made, it turns out that compounds 7-9 would be novel.

Compounds 1-6 have been made, but it's not entirely clear what parasites they have been tested against. They have been definitely been tested against Plasmodium falciparum (Barlin et al, 1996), but we haven't found anything that suggests they've been tested against Toxoplasmosis gondii.

It is obviously impossible for us to synthesise all 9 compounds before the end of the year. We've started with 1 since we're received 3-(trifluoromethyl)phenylacetonitrile and it's otherwise the same as pyrimethamine. We'll chose another one to work on depending on when we get 3,5-bis(trifluoromethyl)phenylacetonitrile since ideally we'd like to try to make 7 at least.

In addition we do also have 3,4-(methylenedioxy)phenylacetonitrile, but the trifluoromethyls looked like a nice series to start with.

So far we've tried Step 1 to produce compound 1, SGS17-1-1, and although further analysis is required it looks positive so far. Next we're going to try and do Step 2 on the crude mixture which has been successful in the past.

Does anyone have any suggestions about how we can check if 1-6 have been tested against T. gondii (or even Isospora belli or Neospora caninum)?

Any other thoughts about the planned syntheses are welcome!

InChI for compounds 1-9 1 InChI=1S/C13H13F3N4/c1-2-9-10(11(17)20-12(18)19-9)7-4-3-5-8(6-7)13(14,15)16/h3-6H,2H2,1H3,(H4,17,18,19,20) 2 InChI=1S/C12H11F3N4/c1-6-9(10(16)19-11(17)18-6)7-3-2-4-8(5-7)12(13,14)15/h2-5H,1H3,(H4,16,17,18,19) 3 InChI=1S/C11H9F3N4/c12-11(13,14)7-3-1-2-6(4-7)8-5-17-10(16)18-9(8)15/h1-5H,(H4,15,16,17,18) 4 InChI=1S/C13H13F3N4/c1-2-9-10(11(17)20-12(18)19-9)7-3-5-8(6-4-7)13(14,15)16/h3-6H,2H2,1H3,(H4,17,18,19,20) 5 InChI=1S/C12H11F3N4/c1-6-9(10(16)19-11(17)18-6)7-2-4-8(5-3-7)12(13,14)15/h2-5H,1H3,(H4,16,17,18,19) 6 InChI=1S/C11H9F3N4/c12-11(13,14)7-3-1-6(2-4-7)8-5-17-10(16)18-9(8)15/h1-5H,(H4,15,16,17,18) 7 InChI=1S/C14H12F6N4/c1-2-9-10(11(21)24-12(22)23-9)6-3-7(13(15,16)17)5-8(4-6)14(18,19)20/h3-5H,2H2,1H3,(H4,21,22,23,24) 8 InChI=1S/C13H10F6N4/c1-5-9(10(20)23-11(21)22-5)6-2-7(12(14,15)16)4-8(3-6)13(17,18)19/h2-4H,1H3,(H4,20,21,22,23) 9 InChI=1S/C12H8F6N4/c13-11(14,15)6-1-5(2-7(3-6)12(16,17)18)8-4-21-10(20)22-9(8)19/h1-4H,(H4,19,20,21,22)