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28th June 2016 @ 02:15

On Friday 3rd June 2016, first year students from the Special Studies Program (SSP) at The University of Sydney presented the research performed in their weekly laboratory class. The students held a fantastic film festival, where they premiered shorts (aimed at a HSC audience) about nanoscience. Additionally, students presented research posters based on their synthesis of four brand new antimalarial compounds. We didn't quite get the biological data (i.e. how well the compounds could kill the malaria parasite) in time for the final presentation...but we have them now, so read on!

It's my second year working on a completely open research-focused laboratory course, and once again I've been throughly impressed by our student's research efforts. More coming on the nanoscience project elsewhere, but this blog entry will discuss the SSP lab's contribution to the Open Source Malaria (OSM) consortium and attempt to place their results within the context of the OSM project.

Malaria

Malaria is one of the world’s oldest diseases and remains one of the most deadly. In 2015 there were 213 million recorded cases of malaria and 306 000 deaths, the vast majority being children under the age of five. The current frontline medicines are artemisinin combination therapies (ACTs) that combine a potent antimalarial, artemisinin with other drugs (Figure 1). Artemisinin is isolated from the sweet wormwood plant and was employed in traditional Chinese medicine long before it was marketed as an antimalarial.

Figure 1: Structure of artemisinin, a potent antimalarial

Malaria is a mosquito-borne disease carried by parasites of the genus Plasmodium. There are five strains, of which the most lethal is Plasmodium falciparum.[ii] The parasite is transmitted following a bite from a female anopheles mosquito whilst feeding on blood to nourish her eggs and simultaneously injecting malaria sporozites. The sporozites then travel to the victim’s liver, leading to the development of flu-like symptoms such as high fever and chills.

Although significant progress has been made in the treatment malaria, the emergence of artemisinin resistant strains means that there is an urgent need for a new drug to treat the disease. As malaria mainly affects people in less economically developed countries, there are additional complications associated with disease detection and treatment. Criteria for a new malaria medicine require a single dose cure costing no more than 1 USD. Not only are these criteria difficult to achieve, they also mean that there is a reduced market incentive for large pharmaceutical companies to invest in the development of new therapies.

Open Source Drug Discovery The challenges associated with finding medicines for malaria has led to the development of some more collaborative ways of working to find new drugs. The pharmaceutical company GlaxoSmithKlein (GSK) published the structures of 13 500 compounds (taken from its vast libraries of potential drugs) that were found to be active against malaria.[iii] This was the first time that a pharmaceutical company had shared a large set of data and placed it into the public domain so that researchers all over the world could use the results as starting points for the discovery of new antimalarials. More recently, other companies have followed suit and published the structures of compounds that kill the malaria parasite.[iv] Finding new medicines is a difficult and expensive process; recent statistics have shown that on average it takes 12 years and 2.6 billion USD to bring a drug to market.[v] OSM hope that removing any secrecy from the scientific process might expedite the discovery of drugs and reduce costs by eliminating any unnecessary duplication by researchers in different groups. The other wonderful thing about working in the open is that it elimates many of the barriers to participation, this means that anyone can take part, including the Undergraduate and High School students who have contributed to OSM so far (The University of Sydney, Massachusetts College of Pharmacy and Health Sciences, Lawerence University, Haverford College, The University of Edinburgh, Sydney Grammar School and more to come!)

The Triazolopyrazines

The triazolopyrazines are a group of compounds, first investigated by the pharmaceutical company Pfizer,  that were found to kill the malaria parasite. The compounds possess a common ‘core’ (Figure 2, shown in black) which is substituted with different groups of atoms at two positions (Figure 2, shown in blue and green). Medicinal chemists synthesised many molecules of this type and then sent them to biologists who tested their activity. The activity data was used by chemists to design different organic groups to attach to the core in an attempt to optimise the properties of the molecule by increasing activity and enhancing other properties required for a suitable medicine.

 

Figure 2: The core structure of the triazolopyrazine family of antimalarials. 

The first tests performed by biologists were in vitro experiments, meaning that they were tests on cells harvested from their normal environment and observed in glass plates. In theses tests a solution containing a potential new drug is administered to a plate containing Plasmodium falciparum and then biologists measure how effective the molecule is at killing the parasite. These preliminary experiments are really important in determining which molecules are active and inactive, but they don’t provide a full picture of how the molecules will behave in humans. Later experiments on some of the initially promising triazolopyrazine compounds showed that there were some properties that needed to be improved to maximise their chances as a new malaria medicine. Firstly, the solubility of the compounds needed to be increased so that they could be better absorbed in the body. Secondly, the molecules need to be more metabolically stable, so that they are not broken down by the body before they have chance to kill the parasite.

The SSP Project 2015

Over four weeks students worked in small groups (under the guidance of one of four demonstrators) and synthesised a brand new antimalarial compounds following a route developed (and shared online) by OSM.

Figure 3: SSP Synthesis Project 

In week one, students were provided with starting material 1 and each demonstrator group was given one of four different aromatic aldehydes (2 in Figure 3, and shown in more detail in Figure 4). A condensation reaction united 1 and 2  to form 3, which was  purified by recrystallisation in week 2. In week 3, compound 3 was treated with an oxidant to form bicyclic compound 4 and then in the final week, students displaced the chlorine present in 4 with an alcoholic side chain (Figure 3, shown in blue) to make their target molecules (5).

Figure 4: SSP Aromatic Aldehydes 2015

We combined samples from students who had made the same compounds (each of the four final structures are shown in Figure 5) and purified them in the research lab to make sure that they were clean enough for biolgical testing. Our measurements confirmed that the compounds were pure and so they were vialled up and sent off for testing against the parastie whilst we eagerly awaited the results.

Figure 5: Biological Data SSP 2015

The data showed that phenyl substituted MMV689970 was inactive against the malaria parasite, as were two of the chlorophenyl compounds (MMV689969 and MMV689968). Pleasingly, 4-chlorophenyl substituted MMV663915 was found to be a highly active compund, requiring a very low concentration of the compound to kill the malaria parasite. In fact, MMV663915 turned out to be one of the most active compounds synthesised in the OSM project to date!

The SSP results were warmly welcomed by the OSM team, even the ones that turned out to be dud compounds. In order to develop a model to predict active compounds it's really important to have data for compounds that don't work as well as we might have hoped, as well as the superstars of course! Additionally, one of the best things about being part of an open research project is the fact that we can tell the world which compounds don't work very well, so that no one else wastes time or money working on the same molecules.

Experts from OSM were intrigued by the huge differences in activity between the compounds, particularly between isomers containing a chlorine group. OSM wanted to find out more about which aromatic groups would provide potent antimalarials and wondered whether a substitution in the 4-position (opposite to the bond to the triazolopyrazine core) was of particular importance. This was a job for the SSP Class of 2016!

The SSP Project 2016

In March 2016 a new group of enthusiastic SSP students were ready to build on the work of their 2015 colleagues. A different set of aromatic aldehydes were selected, each substituted with either fluorine or chlorine at the assummed-to-be-important 4-position (Figure 6). Three of the compounds contained an additional fluorine or chlorine in differentt locations around the ring. The students wanted to examine whether it was the presence of a group at position 2 or 3 that had killed the activity in the 2015 compounds, or whether it was the absense of a group at postion 4.

Figure 6: SSP Aromatic Aldehydes 2015 

Changing the substituents around the aromatic aldehyde can affect lots of the physical properties of the molecule such as solubility (how greasy vs polar the molecule is) and the molecular weight (the mass of the molecule). It's also important to think about the size of the different substituents, as this can affect how well a molecule is able to fit in a binding site inside a biological system, and can therefore influence how well a molecule can interact with a biological target.

Figure 7: Atomic Radii of Hydrogen, Fluorine and Chlorine 

The students successfully made their target molecules and OSM received the biological results for these compounds in June 2016 (Figure 8).

Figure 8 Biological Data SSP 2016

Once again the data was extremely useful. Compounds MMV693151 and MMV693152 were both substituted in the 3- and 4-positions and were found to be only moderately active. It was interesting to compare this result to that for the inactive 3-chloro substituted 2015 compound (MMV689968, Figure 5) as OSM learnt that  substitution at the 4-postition is important to maintain activity. Also, as fluorine is similar in size to hydrogen, if we compare MMV689970 with MMV693151 one might infer that the size of the subsituents is less important than other properties that they confer, we see a jump from no activity (MMV689970, Figure 5) to moderate activity (MMV693151, Figure 8). Compound MMV693153, substituted in the 2- and 4-position, was found to be active and fairly good at killing the malaria parasites, again confirming the importance of a substituent at the 4-position. 4-Fluoro substituted MMV693154 supported this theory further as it was also found to be active in the assay.


The SSP Project 2017? 

Members of the OSM team across the USA, UK, Australia and NZ are looking at the results obtained as part of the SSP laboratory class and using this data to assist in the design and synthesis of new compounds. OSM particularly want to work on making the active SSP compounds (and others in the project) less greasy, and these analogs will be made and tested in the next few months. The next set of SSP compounds will be designed when all of this information has been taken into full consideration...watch this space!

 

Get involved!

If you are an undergraduate student or lab coordinator, a PI, a high school teacher or a high school student then OSM would love to hear from you.

As we are an open project, we prefer open communication, so:

Tweet us @O_S_M, find us on G+ or Facebook or come and look at our GitHub thread and see what 'issues' are ongoing in the project.

You can also check out our landing page opensourcemalaria.org and if you would really, really prefer to email then that's ok too: opensourcemalaria@gmail.com.

 

 References

 WHO World Malaria Report 2015

[ii] http://www.mmv.org/malaria-medicines/five-species

[iii] Gamo F-J.; et al. Nature. 2010, 465, 305

[iv] http://openwetware.org/wiki/OpenSourceMalaria:Triazolopyrazine_%28TP%29_Series

[v] D. W. Light and J. R. Lexchin, British Medical Journal 2012, 345, e4348. (DOI: 10.1136/bmj.e4348)

 

Attached Files
6th November 2014 @ 00:27

The OSM consortium are excited to have some new members to the team; some excellent chemists from Sydney Grammar School. Erin Sheridan and Trent Wallis are working with a group of twelve students to synthesise some building blocks for the Series 4 Triazolopyrazines.

The target compounds were decided following discussion on GitHub and the students are now ready to start on their synthese. Erin and Trent are going to create a separate lab book/blog as part of the consortium's ELN.

There will be more news to follow, hopefully from the students themeselves as they sign up for the ELN, GitHub and Twitter. So watch this space!

There will be prizes awarded to the students who maintain the best ELN, so look out for some great experimental descriptions, photographs, videos and more!

 

GitHub Welcome

Linked Posts
9th January 2014 @ 03:45

The OSM team started work on the fourth series of compounds, the triazolopyrazines, in September 2013. The team is focused on the synthesis of compounds featuring ether or amide linkages to the triazolopyrazine core (see below).

The most elegant and efficient way to synthesised the amide compounds would be to use a common intermediate A, required for an established route to ether compounds of type B. Direct carbonylation of A could lead to the synthesis of carboxylic acids/ester intermediates (C) for diversification into different amides. Direct amide formation should also be possible, but for the moment bulk synthesis of useful intermediates would be preferable.

The reaction of aryl-X compounds to form carboxylic derivatives was pioneered by Heck et al. in the 1970s but great progress on metal catalysed carbonylation of aryl-x groups has been made in the last ten years, in particular with respect to Pd-catalysed reactions. (See ACIE review by Anne Brennführer, Helfried Neumann, and Matthias Beller DOI: 10.1002/anie.200900013).

The team received some details of preliminary attempts at carbonylations of compounds of type A by the CRO (see slide below).

Slide04.png

The CRO were not successful in their efforts but the OSM team are confident that carbonylation of A could be achieved. For the moment, we don't have enough synthetic resources to explore this reaction and are therefore working on amide synthesis starting from compound 1 (as seen on this slide) as this synthesis will hopefully not require as much optimisation as the carbonylation and can also be performed by undergraduates - the use of expensive transition metal catalysts and CO are not always possible.

Therefore, we are looking for potential collaborators who would like to develop the direct carbonylation of triazalopyrazine chlorides. If you think that you or your lab can help please join the team. Comment below or tweet us at @O_S_M. We prefer to avoid email but if that's easier then drop us a line at opensourcemalaria@gmail.com.

Many Thanks

The OSM Team


Linked Posts
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7th January 2014 @ 08:18

The OSM team are currently trying to synthesise some triazalopyrazines (series 4) that were found to be promising antimalarial starting points by big pharma.

I (Alice in Mat Todd's group at the University of Sydney) am currently attempting to resynthesise MMV670652 in order to confirm the activity of the racemate and subsequently determine the activity of each enantiomer following resolution.

I have been following a procedure provided by the CRO who had worked on these compounds. (The synthetic route and target are described in more detail here and preparative data found within:

FINAL REPORT MMV670652.doc
 

The CRO used Freon gas to difluoromethylate AEW 103 (alcohol SM shown below) but unfortunately, due to limitations on the availability of Freon-22 in Australia, the team need to find an alternative method for this transformation:

 

A review by Jinbo Hu, Wei Zhang and Fei Wang (DOI: 10.1039/b916463d) revealed a variety of methods for selective difluoromethylation and included a chapter on electrophilic difluoromethylation. 

'The most widely used method for the incorporation of a CF2H group into nucleophiles (such as oxygen-, nitrogen-, sulfur-, phosphorus-, and carbon-nucleophiles) is the reaction of the corresponding nucleophile with a proper difluorocarbene reagent.'

One paper referenced (DOI 10.1021/jm900716v) used trimethylsilyl 2,2-difluoro-2-(fluorosulfonyl)acetate or 2,2-difluoro-2-(fluorosulfonyl)acetic acid (shown above) to enable difluoromethylation of 3,6-dimethyl-5-nitropyridin-2-ol (shown below):

The review focused on methods for phenolic difluoromethylation and when surveying the literature, examples of difluoromethylation of aliphatic alcohols were found to be scarce and use free radical methods for the introduction of the group. In September's online meeting, Joie Garfunkel (MMV) suggested the use of trimethylsilyl 2,2-difluoro-2-(fluorosulfonyl)acetate in combination with a copper catalyst in MeCN at elevated temperatures and pressures (Joie's colleagues had used this method at Merck).

The methods I have attempted so far involved the use of the diflurocarbene generating reagent trimethylsilyl 2,2-difluoro-2-(fluorosulfonyl)acetate. I followed the prep. outlined in the paper and began to explore the reaction with copper at room temperature and then in a sealed tube. So far, I haven't had great success with this reaction.

I'm going to try some more conditions and also the use of the more toxic 2,2-difluoro-2-(fluorosulfonyl)acetic acid but would be most grateful for any ideas or assistance in how to get this reaction to work.

Additionally, prefered methods for the trifluoromethylation of the same substrate would be greatly appreciated.

Please post below or tweet me @all_isee and watch this space!

Cheers, 

Alice



Attached Files
3rd November 2013 @ 23:53

The team want to synthesise some amides based on the data from the CRO on the series four triazolopyrazines. Some synthesis data on known compound MMV670652 was available from the CRO, however, experimental procedures are not available for the amides (example below and here).

 

Patrick Thomson proposed this route on his blog and Github:

 

Sabin Llona-Minguez suggested a synthesis that started from 2-chloro-6-methylpyrazine 18 (£30/g Apollo) which could be oxidised to form 14 in a single step. 

The advantage to Patrick/Sabin's route is that the chemistry could all be performed by students in an undergraduate teaching lab, which might be an ultimate aim for this route. Patrick also highlighed that compound 13 might not be readily available commercially, which could make Sabin's suggestion more attractive.

Joie Garfunkle's elegant proposal would allow the amides to be synthesised by divergence from a common intermediate used in the synthesis of other series 4 compounds. Joie's suggestion was to use Pd-catalysed carbonyation of common intermediate 2 to furnish the methyl ester 19, which could then be transformed into the desired amide.

Patrick Thomson suggested that a collaboration with a group at the University of Edinburgh (his own institution) who specialise in high pressure carbon monoxide reactions could help to develop synthesis via carbonylation of common Cl-intermediate 2.

Update 14th Nov 2013

Joie Garfunkle analysed the weekly reports from the CRO and deduced a common route used for the synthesis of amide compounds, see below:

This route contains several analogies to a suggested improvement to the synthesis of the triazolopyrazine core by Stefan Debbert when he cited an ACIE paper that employs Chloromamine-T as oxidant in combination with 2-MeTHF solvent rather than CH2Cl2 and PIDA (DOI: 10.1002/anie.201001999). The ACIE paper also employs a Pd-catalysed coupling of an aldehyde derived hyrazone with a 2-chloropyrazine prior to oxidative cyclisation.

This route also demonstrates the importance of starting material 14 - a commercial source or cheap/quick synthesis is required. Additionally, it suggests that Patrick's route could be problematic as the hydrazine may react with the carboxylic acid in preference to displacement of the chloride. References for each of the steps need to be found but so far this route looks quite robust.

I still think that carbonylation is the most attractive route long term as it makes use of common intermediates. However, if optimisation is required then it might be quicker to go for a route involving more steps if they can a) be performed by undergraduates and b) require less time spent on reaction improvement.

What do you think of the propsed routes? If you have experience with similar syntheses or you would like to suggest a new one, please do so by posting on Github, facebook or commenting below. Alternatively, (although we would prefer to avoid email) email opensourcemalaria@gmail.com and one of the team will post your idea to GitHub.

Cheers, The OSM team

 

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