Nitrogen-Centered Radicals: A Forgotten Species?

In the late 1990s I was working on a chemistry project involving cascade reactions of C-centred radicals (CCRs). At that time, N-centered radicals (NCRs) were little used compared to their C-centered relatives and I was wondering if things had changed much since then.

Considering that the first example of the Hofmann-Löffler-Freytag reaction was reported in 18831 it seems that the development of the chemistry of NCRs has been fairly slow. More recently, in 2008, Zard2 described NCRs as a forgotten species with significant synthetic potential in a comprehensive review of intramolecular NCR cyclisation. NCRs have suffered from limitations because traditional methods for their generation have relied on the synthesis of N-X precursors and required harsh conditions for bond homolysis (Scheme 1, path A). New developments in metal catalysis (path B) and visible-light photocatalysis (path C) have resulted in greater use of NCR chemistry (for C-N bond formation) and advances in this field were reviewed by Zhang3 last year.

Micheal 1

 Scheme 13 Strategies in C-N bond formation based on NCR chemistry

Generation of NCRs using visible-light photocatalysis is particularly attractive due to low catalyst loading and mild conditions, with most reactions occurring at room temperature without the need for highly reactive radical initiators. This technology has been widely applied to radical amination chemistry in the last three years. An interesting example of this approach entitled ‘’Visible-Light-Mediated Generation of Nitrogen Centered Radicals: Transition Metal Free Hydroimination and Iminohydroxylation Cyclisation Reactions’’ was published by Leonori.4 One of the transformations described is a 5-exotrig cyclisation of iminyl radicals to give pyrrolines without the use of a transition metal catalyst (Scheme 2). A diverse range of oximes were converted to the corresponding pyrrolines, in good yields, and bicyclic heterocycles were also prepared using this methodology.


Micheal 2 

Scheme 24 Visible-light mediated hydroimination

The mechanism of this reaction involves single electron transfer (SET) reduction of the aryl oxime ether (A) by a visible-light exited photocatalyst (*PC), followed by N-O bond fragmentation to give iminyl radical (D) which can then cyclise onto the pendant alkene (Scheme 3). The role of cyclohexadiene (CHD) is to reduce the intermediate radical (E), followed by a second reduction of the photocatalyst (PC), which is then irradiated (PC®PC*) completing the catalytic cycle. Cyclic voltammetry studies suggested that that the key single electron transfer (SET) reduction, by excited state organic dye eosin Y, would only be efficient when using nitro-substituted aryl oxime ethers (1a-1d) with suitable reduction potentials. The 2,4-dinitro-aryl oxime (1a) was found to be the best substrate.

Micheal 3.png

Scheme 34 Proposed photoredox cycle and electrochemical studies. EY = eosin Y, ppy = 2-phenylpyridine

It was found that a different activation mode could be used to generate the iminyl radical without the presence of a photocatalyst (Scheme 4). When a solution of the aryl oxime ether (2a) and triethylamine in acetonitrile was irradiated with visible-light in the presence of CHD the previously observed hydroimination product (3a) was obtained, and also an unexpected iminoalcohol (4a). When CHD was not in the reaction mixture iminoalcohol (4a) was the main product. The mechanism proposed for this process involves formation of an unusual electron donor-acceptor complex (5) which undergoes SET giving the dipolar species (6). This species can then fragment, and undergo 5-exo-trig cyclisation to give radical (7). If the radical is not reduced by CHD it can be oxidised by attacking the nitro group (8) which after N-O bond homolysis, and hydrogen atom abstraction, gives the iminoalcohol (4a). Evidence for the source of the oxygen was provided by generation of 2-NO-4-NO2C6H3OH (10) from the reaction which was in contrast to 2,4-dinitrophenol that was produced from the original hydroimination.

Micheal 4

Scheme 44 Initial findings and proposed reaction mechanism for the iminohydroxylation

The chemistry of NCRs has been developing in recent years, but they still remain less well used than CCRs. Significant problems, such a functional group compatibility, need to be solved before they can become routinely used for C-N bond construction alongside traditional ionic chemistry. Visible-light photocatalysis seems very likely to play a role in future developments.

Blog written by Michael Annis


1. J. L. Jeffrey and R. Sarpong, Chem. Sci., 2013, 4, 4092

2. S. Z. Zard, Chem. Soc. Rev., 2008, 37, 1603

3. T. Xiong and Q. Zhang, Chem. Soc. Rev., 2016, 45, 3069

4. J. Davies, S. G. Booth, S. Esaffi, R. A. W. Dryfe and D. Leonori, Angew. Chem. Int. Ed. 2015, 54, 14017


Chemical tools on trial: recent misleading information cases

For this week’s blog, I have picked it a still-hot and enjoyable Nature article by Monya Baker.1

How many times have you had screening results which make no sense to you end in frustration and unexplicable conclusions? How many times have you encountered that your chemical tools (hits) are neither as you thought, behaved oddly or even conducted regular quality controls on them and enough analytical tests? Read more to recognise unexpected setbacks.

The author goes through a few examples where researchers have encountered, for example, that chemical probes hitting specific targets suddenly became inactive when alternatives suppliers came onto stage. A futher realisation that one isomer was presented in different proportions if it was mixed with its other enantiomer (mirror-image like compounds sharing the same molecular formula) altering its activity leading to discover that sometimes, researchers do not know what they have in their hands if too much trust is put on commercial sources or the lack of further structural corroborations.2

When screening large compound libraries and identifying hits, the presence of false positives, impurities, degradation, mixed-up and messed-up stocks could also lead to infructouos crusades. Especially when liquid stocks are kept for months or years, at variable temperatures, the effect of poor solubility, side-reaction impurities, solvent effects and cockails of natural product mixtures amongst others. 3

Suggested online resources like: Chemical Probes Portal, Probe Miner and Probes and Drugs Portal 4 have been created to be used as a good first port of call toolbox with advice and information on which chemicals or drugs to use as well as public assessments.

The infamous case of BOSUTINIB, an approved cancer drug which was sold by nearly 20 suppliers with the chemical substituents wrongly located (please see ‘Spot the difference’ cartoon) has created havoc. jose

Both bosutinib and the other mis-identified isomer target cell-signalling proteins but with different potencies putting under trial numerous papers which reported data on the isomer of bosutinib rather on itself.5

Another researcher, Kim Janda caused an uproar when in its group they could not synthesise a molecule described to boost cell production of a powerful natural antitumor protein, TRAIL. The distributors had perpetuated the mistake in the original publication and after even clinical trials and patents were issued, he filled a patent with the proposed new active version of the TIC10 structure. 6

Another case emphasises how activity showed a big drop after small changes in contrast with bigger changes. Scientists realised that the present of Zn in their preparation was responsible for this activity without effect from the organic molecule whatsoever. Copper or Palladium from its use in catalytic reactions have been reported showing false positives.7

Even the present of NMP (N-methyl-2-pyrrolidone) a polar solvent used in some cases for keeping chemical probes in solution has recenlty showed anticancer activity in control tests, with potential implications in misleading the potency of the substances contained on it. 8

A list of common-sense leading practises to suppress these errors are given in the paper:

  • Use and buy chemicals by their CAS number (unique identification to chemical structures)
  • Request a detailed quality control certificate from external suppliers
  • Place enquires about the synthetic method used
  • Perform independent or in-house analysis upon receipt of the starting chemicals and reagents
  • Structural forms, chiral compounds, potential ambiguity in their chemistry substitution, then determine their optical purity and apply chiral chromatography techniques to isolate single forms.
  • If possible, previous in-house synthesis and determination with structural characterization.

Mismatching results, not-well documented suppliers, dubious chemical insertions (not well validated) as well as poor purities and the presence of contaminants (even at low concentrations) are always to blame for cases like those described in the article over the years. Albeit the final responsibility lies on the chemist’s team who need to be reassured by exploring and conducting further unequivocal tests.

The views represented in this blog are the author’s own.

Blog written by Jose Gascon


  2. Huber, K. V. M. Nature, 508, 222–227 (2014).
  3. Bisson, J. et al. J. Med. Chem. 59, 1671–1690 (2016)
  4. See: Chemical Probes Portal ( ), Probe Miner ( and Drugs Portal ( )
  6. ,Oncotarget, 2014, 5(24): 12728–12737
  7. Hermann, J. C. et al. ACS Med. Chem. Lett. 4, 197–200 (2013)
  8.  Shortt, J. et al. Cell Rep. 7, 1009–1019 (2014).









Pronucleotides Assemble! A multifunctional catalyst designed to stereoselectively synthesise prodrugs

Nucleosides have emerged as a key chemical class in successful antiviral and anticancer drugs with nearly half of currently marketed drugs possessing these cores.1 However, the biologically active nucleoside phosphates, which are generated in vivo by phosphorylation, are poor drug candidates due to issues of permeability and stability. One pronucleotide strategy developed to address these challenges has been described by McGuigan. His ProTide platform introduces a 5’-arlyoxy phosphoramidite to the drug candidate which can result in improved cell permeability and rate of phosphorylation compared with non-phosphoramidite containing nucleosides.2,3

However the introduction of the phosphoramadite in the prodrug can have a significant effect on the potency, toxicity and rate of metabolism, effects which can be associated with the stereochemistry at the phosphoramidite phosphorus. Unlike for carbon chemistry, where stereocontrol is a sophisticated branch of asymmetric catalysis, p-chiral chemistry is far less developed. Although methods do exist to steer products towards a preferred p-chiral isomer, such as dynamic kinetic asymmetric transformation (DYKAT) using chiral auxiliaries or desymmetrisation of achiral species,3 these approaches have significant drawbacks with the former suffering from poor selectivities and low catalytic turnovers, whilst the latter is often a complicated multistep synthesis.

In addition to the stereo-isomeric challenges during prodrug assembly there also remains the challenge of chemoselectivity for 5’ versus 3’ phosphoramidation. With these challenges in mind, I wanted to highlight an excellent paper published by a team from the process research and development group of Merck & Co., USA, where they report the first good example of a catalyst being designed to address the issue of stereo- and chemoselectivity in the synthesis of pronucleotide prodrug candidates. In their paper they focused on a hepatitis C virus RNA polymerase inhibitor currently in late stage clinical trials (MK-3682, Figure 1).4

Figure 1Figure 1: General phosphoramidation scheme and the best in-class catalysts developed to effect the coupling. Yield is the total yield of phosphoramidite isolated, chemoselectivity for 5’ vs 3’ is represented by ratio 5’:3’and d.r is the ratio of P(R) to P(S).

Using mechanistic studies, computational modeling and an understanding about the enzymatic mechanism of P-O bond formation in the phosphorylation of nucleosides, the team successfully developed several small molecule organic catalysts that mimic the concomitant series of activation modes used by enzymes to effect the P-O bond formation. Early studies identified carbamates as a privileged class for controlling stereo- and chemoselectivities with catalyst (R)-B being the best of the first generation catalysts developed. Using computational modeling the carbamate was theorized to carryout 3 roles; leaving group activation, general base catalysis and oxyanion stabilization via a pentavalent transition state, giving rise to a 2.3kcal/mol differentiation between the desired R-stereochemistry and the S-stereochemistry at the phosphoramidite phosphorus (Figure 2). Catalyst I, the best of the catalysts reported, evolved from a conscious effort to increase the transition state differentiation by decreasing the entropy of the system via linkage of catalyst (R)-B. As highlighted in Figure 1 the linked catalyst, Catalyst I, was able to achieve excellent yields and high selectivities for both the desired 5’ product (99:1 in favour of the 5’ product) and with a d.r (of 99:1) in favour of the desired P(R) isomer.

Figure 2Figure 2: Transition state model showing multiple catalyst modes of action. Reproduced from reference 4.

This work demonstrates an excellent step forward in the controlled synthesis of pronucleotide prodrugs by continuing to employ rational design beyond the discovery phase SAR, well into the late stage development of the prodrug. Moreover the published work is an elegant example of the power of using an interdisciplinary approach to solve chemical problems via a rational design cycle.


Written By Jason A. Gillespie



  1. P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet, Nat. Rev. Drug Discov. 12, 447–464 (2013).
  2. Cahard, C. McGuigan, J. Balzarini, Mini Rev. Med. Chem. 4, 371–381 (2004).
  3. J. Sofia et al., J. Med. Chem. 53, 7202–7218 (2010).
  4. A. DiRocco et al., Science, 356, 426–430 (2017).

CrispR technology: Designer babies and Swine Donors

Nisha 1


The last decade has seen tremendous progress in ‘gene-editing’ techniques to make them more accurate, fast and replace the potential hit-and-miss methods of genetic engineering over the past few decades. The new technique called CrispR (pronounced Crisper!!) that emerged from pioneering work in bacterial cells in the early 2000s has evolved over the years to find applications in biotechnology, fundamental research and medical science.

The preliminary CrispR toolkit involved a RNA molecule called gRNA (guide-RNA) that is homologous to the area of DNA you wish to modify and a nuclease Cas9. The gRNA, as the name suggests, guides Cas9 to the region of interest to create a double strand break (DSB). The native cellular repair machinery: NHEJ (Non-homologous end joining) or HR (Homologous recombination), then repairs and introduces the necessary changes using a template DNA. Once the basic methodology of this technique was established, CrispR was used widely for gene-editing in almost every living organism from cells to bacteria, insects to plants and animals to now human embryos Doudna & Charpentier 2014; Sander & Joung 2014).

Nisha 2

Two studies published in the recent months show promising results with CrispR-mediated gene editing in embryos. The first study at Oregon Health & Science University (OHSU) and Salk Institute in the US, focuses on using CrispR for genome-correction to avoid transmitting genetic disorders in human embryos (Ma et al. 2017). The authors were keen on targeting genetic disorders that manifest in late-adulthood such as MYCBP3 gene mutation, which leads to hypertrophic cardiomyopathy (HCM). This genetic disease occurs in one in 500 and is a common cause of death in young athletes. The scientists CrispR-ed the mutated allele of MYCBP3 in embryos from IVF donors and replaced it with the wild-type maternal allele instead of a synthetic DNA template. By injecting the Crisp-R-Cas-9 cocktail along with the sperm (from a HCM male patient) into a wild-type oocyte (source of maternal DNA), they achieved increased correction rates and avoided mixed populations in the resulting embryos. Additionally, whole genome sequencing identified very few off-target effects of the gene editing, promising a potential break-through in germ line gene therapy. For ethical reasons these embryos could not be implanted into a uterus to develop a baby.

Yet another promising venture for CrispR was in the organ-transplant arena, where scientists in China have successfully CrispR-ed the retroviral gene in pigs (Niu et al. 2017). The study has attracted huge media attention as it promises xeno-transplants from pigs to humans without the fear of an immune attack. Traditionally such transplants are rejected in humans, as the PERV gene from pigs leads to retroviral infection in humans. The scientists attempted to target 25 copies of the PERV-gene (Porcine Endogenous Retrovirus) all at once using CrispR in adult cells, that would be later fused with ova (female reproductive cell) to grow into embryos and implanted in sows. Although, they met with disappointment in their initial trails due to ‘shredded-genomes’ in the targeted cells, Laika – the PERV inactivated piglet, was born after using a specific cocktail that kept the Crisp-R cells alive despite the aggressive gene-editing. Many other similar studies are underway exploring organ transplants from swine donors, by modifying their genome to ‘humanise’ them for successful transplants (Petersen et al. 2016; Martens et al. 2017) . While controversies continue in the field, if gene editing is required to replace anti-viral drugs in such a transplant, CrispR surely shows immense potential in the clinic.

Owing to the recent discoveries this gene-editing tool is now under the scrutiny of ethical committees and policy makers, as they fear a domino effect and the advent of ‘designer babies’. Nature, earlier this month published a report that marked down this technology due to the unintended mutations it caused in-vivo in a mice experiment (Schaefer et al. 2017). The study asserts the importance whole genome sequencing if these tools were to be applied in ‘real-people’. Although geneticists are not unaware of the potential pitfalls in genome editing, if Crisp-R will survive these hurdles only time will tell.

Blog written by Nisha Peter


Doudna, J.A. & Charpentier, E., 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science (New York, NY), 346(6213), pp.1258096–1258096.

Ma, H. et al., 2017. Correction of a pathogenic gene mutation in human embryos. Nature, 72, p.1117.

Martens, G.R. et al., 2017. Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs. Transplantation, 101(4), pp.e86–e92.

Niu, D. et al., 2017. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science (New York, NY), p.eaan4187.

Petersen, B. et al., 2016. Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes. Xenotransplantation, 23(5), pp.338–346.

Sander, J.D. & Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4), pp.347–355.

Schaefer, K.A. et al., 2017. Unexpected mutations after CRISPR-Cas9 editing in vivo. Nature Methods, 14(6), pp.547–548.

Fabulous Fluorine in Medicinal Chemistry

Since the FDA approved the steroid fludrocortisone, the first fluorine-containing drug, in 1955, the number of fluorine-containing drugs appearing on the market has rapidly risen to approximately 25% of drugs.1 These include the blockbuster drugs Prozac, Lipitor and Prevacid, Fig. 1. This may seem like an unusual trend considering fluorine-containing natural products are quite rare, so why is it that fluorine is so abundant in drugs? When you consider the unique chemical properties that fluorine has, the reason for a drug designer’s love of fluorine becomes clearer.

Catherine 1

Fig. 1: Examples of fluorine-containing drugs

Fluorine is a small, highly electronegative and lipophilic atom. The incorporation of fluorine into a molecule is an increasingly popular strategy to improve drug potency. Fluorine substitution can enhance potency and impact target selectivity by affecting pKa, modulating conformation, hydrophobic interactions and lipophilicity. Fluorine is also frequently used to improve drug metabolism. More recently, radiolabelled fluorine drugs have been used in positron emission tomography (PET) for imaging and diagnosis purposes in medicine.2

Catherine 2.JPG

Fig. 2: Effects of fluorine in medicinal chemistry3

Fluorine is much more lipophilic than hydrogen, so incorporating fluorine atoms in a molecule will often make it more fat soluble. This means permeability across membranes is increased resulting in a higher bioavailability. Because of the strong electron withdrawing ability of fluorine, it’s inclusion in a molecule has a very strong effect on the acidity or basicity of proximal functional groups. Altering the pKa can strongly modify the binding affinity and the pharmacokinetic properties of a pharmaceutical agent. Often, fluorine is introduced to lower the basicity of a compound which aids in permeability.4

Fluorine can play an important and unique role in influencing molecular conformation. Sterically, fluorine is similar in size to a hydrogen atom, but the high electronegativity of fluorine results in a highly polarised C-F bond with a strong dipole moment and a low-lying C-F anti-bonding orbital available for hyperconjugative donation.2 This can cause fluorine-containing molecules to adopt a different preferential conformation compared to the non-fluorinated molecule which may result in increased binding affinity to a receptor.

One of the major challenges in drug discovery is that of low metabolic stability of compounds. Lipophilic compounds are susceptible to oxidation by liver enzymes, in particular cytochrome P450. Fluorine substitution at the metabolically labile site or at adjacent sites to the site of metabolic attack is a common strategy to improve metabolic stability. The inductive effect of fluorine should result in decreased susceptibility of adjacent groups to metabolic attack by cytochrome P450. Additionally, fluorine can modulate lipophilicity and restrict conformation, which may afford improved metabolic stability.3

PET imaging using 18F tracers is a rapidly developing area in medicinal chemistry. PET scans show biological processes which can give invaluable metabolic information. PET is used as a diagnostic tool, particularly in oncology, but also as an in vivo pharmacological imaging tool in drug development, especially in the areas of biodistribution and drug occupancy studies.4

One drawback of introducing fluorine substituents into drugs is that fluorination can be rather difficult and many processes create challenges in the manufacturing process. However, numerous new, safe and mild fluorinating reagents have been invented in recent years, many of which are commercially available, making the process much simpler.5,6

Blog written by Catherine Tighe


  1. Wang, J.; Sánchez-Roselló, M.; Aceña, J. L.; del Pozo, C.; Sorochinsky, A. E.; Fustero, S.; Soloshonok, V. A.; Liu, H. Chemical Reviews 2014, 114, 2432.
  2. Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Aceña, J. L.; Soloshonok, V. A.; Izawa, K.; Liu, H. Chemical Reviews 2016, 116, 422.
  3. Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Journal of Medicinal Chemistry 2015, 58, 8315.
  4. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Chemical Society Reviews 2008, 37, 320.
  5. Yerien, D. E.; Bonesi, S.; Postigo, A. Organic & Biomolecular Chemistry 2016, 14, 8398.
  6. Campbell, M. G.; Ritter, T. Organic Process Research & Development 2014, 18, 474.



Letting the public loose in our labs

On Sunday 25th June 2017, University of Sussex hosted its first Community Festival. The local public were invited to explore the Falmer campus and get involved with a number of hands-on activities across the various schools and departments. There were taster activity sessions at the Sussex Sport facilities, nature walks around the campus, live jazz, talks and more. Thousands of people turned up for the day (1).


Sussex Drug Discovery Centre was represented on the programme by a delegation of our group – Trudy Myers (SDDC Co-ordinator), Jess Booth (Assay Development and Screening Biologist), Kay Osborn (Biology Technician), Lucas Kraft (PhD Student) and myself. Those who signed up to our activity were taken on a whistle-stop tour through the drug discovery process.

First of all, Jess gave an overview of what the drug discovery process entails (see Figure 1) and where the SDDC fits into that process.

Fiona 3.png

Figure 1 : the drug discovery process (2)

After this introduction, our guests donned lab coats and safety glasses and were given a tour of the group’s protein purification suite. Kay explained how the term “protein” is used in the context of drug discovery to describe tiny molecular machines in our bodies that carry out a variety of functions, and in some cases, cause disease symptoms. She showed the group some incubating flasks containing E. coli which is often used to produce a large quantity of these disease-causing protein. Then she explained how that protein can be later isolated from the bacteria cells via a technique called FPLC (fast protein liquid chromatography) (3) which separates proteins out by their different sizes.

Fiona 4

Next, the newly-briefed scientists were taken into the main biology lab where they were able to run their own biochemical assay. While some took to the pipettes quicker than others, all had fun pipetting reagents onto plates that had lots of different drug compounds in them to see if they got any hits – wells that turned pink indicated a successful compound that had bound to the protein and inhibiting its function while blue ones were unsuccessful ones.

Fiona 5

During the last part of our activity I showed our participants around the oncology chemistry lab. I pointed out many of the similar pieces of kit that they would find in their kitchen. We discussed how the results from the assay they had just run in the biology lab informs the work that is carried out by chemists and how we vary the procedures (or “recipes”) we use to make new drug compounds.

The feedback for the activity was overall very positive and our enthused guests went away knowing a lot more about the drug discovery process than when they arrived, which was our aim.

Fiona Scott is a first year PhD student at the SDDC.


  1. University welcomes local residents for fun-filled day of discovery. University of Sussex News. [Online] [Cited: 1st August 2017.]
  2. NMT Pharmaceuticals. [Online] [Cited: 1st August 2017.]
  3. Dermot Walls, Sinéad T. Loughran. Protein Chromatography Methods and Protocols. New York : Humana press, 2011. pp. 439-447.


Malaria taking control – increasing its chances for reinfection?

This week I attended the ISNTD Bites conference 2017 at the Institute of Child Health, London. I was really impressed with one of the talks given by Ailie Robinson on the work that she had conducted for her PhD at the London School of Hygiene and Tropical Medicine. Ailie has been investigating the influence of Plasmodium infection on the human volatile odour profiles in an endemic setting.

What caught my interest was something that I hadn’t previously considered with vector borne pathogenic diseases; which was how pathogens can affect its host in order to improve the likelihood of its host to be bitten by its vector and thus completing its life cycle.

Ailie presented her work where she had analysed the volatile odour profiles from three groups of participants with varying asymptomatic infections of malaria. The three groups investigated were: low density malaria (Plasmodium falciparum) infection, high density malaria infection or negative infection as a control.

Interestingly the gas-chromatographic-mass-spectrometry (GCMS) analysis of the volatile odour profiles from the people in the three groups showed that the high density infection group had significant increases in 3 organic compounds over the other 2 groups. To further investigate this interesting observation Ailie went on to present her work using gas-chromotography-electroantennography (GC-EAG); which is a technique that can be used to determine if the malaria causing mosquito (Anopheles gambiae) is attracted to a certain chemical; to show that all three of the observed molecules that were significantly increased in the high density infection group were highly attractive to the female mosquito. Inferring that the malaria parasite is by someway (either by expressing these chemicals itself or via a host response to infection) maximising its chances for reinfection.

Another interesting outcome from these experiments is the potential for the simple detection of asymptomatic malaria infection by using a breath test. See Ailie Robinson’s article here – ( I look forward to seeing her published data soon.

Blog written by Ryan West

Spirocycles in Drug Discovery

Medicinal chemists are constantly in search of molecules that explore new chemical space whilst looking for novelty amongst the plethora of patented molecules. Three-dimensionality is also key in maintaining solubility of new drug compounds.

Oxetanes have long been known in medicinal chemistry as very useful bioisosteres or surrogates for carbonyl or gem-dimethyl motifs. They reduce lipophilicity and therefore increase solubility when compared to their gem-dimethyl counterparts and reduce metabolism and are less likely to be covalent binders in comparison with the corresponding carbonyls. They have also been shown to be excellent hydrogen bond acceptors,   Penny 1

Whilst oxetanes have been thoroughly explored in drug discovery as a single motif, their composition in spirocycles has been relatively unexplored. In 2008, Carreira et al compared parent amino heterocycles (eg. azetidines, pyrrolidines and piperidines) with the corresponding spirocycle incorporating oxetanes.

Penny 2

In all cases, the spirocycles were stable at pH 1-10 and were considerably less basic than their parent compounds due to their conformation. All have lower logD than their gem-dimethyl and carbonyl analogues. In the cases shown above, the spirocycles also exhibited far lower intrinsic clearance than the carbonyl or parent compounds.

Morpholine moieties are also regularly found in drug discovery programs and in final drug candidates, however, the spirocyclic surrogate seems to fall under the radar despite it being much more soluble, less lipophilic and more metabolically stable.

Penny 3

In addition, the oxygen lone pair is shown to be 1.3 angstroms further out than in morpholine and can therefore be regarded as an elongated morpholine (similar to piperidone but much more metabolically stable). This spirocycle could be used to probe chemical space within a binding site or could in fact have a stronger interaction within the binding site due to probing deeper within a given pocket of the site.                          Penny 4

Carreira et al later looked at the differences between morpholines, piperidines, piperizines and thiomorpholines compared with their spirocylic analogues in terms of their physico and biochemical properties. In general, the spirocyclic compounds had higher solubility, lower logD and were intrinsically more stable in human and liver microsomes compared to the parent compounds.

Penny 5

The authors proceeded to synthesise the antibacterial compound Ciprofloxacin with the piperazine motif replaced with either a piperizine-like spirocycle (Compound A) or the morpholine-like spirocycle (Compound B). Both compounds showed comparable MIC and most interestingly, neither A nor B showed any sign of metabolism in human microsomes whereas Ciprofloxacin showed slight metabolism.

Penny 6

Mykhailiuk et al published an interesting paper very recently which is what initially sparked this blog. They comment on the distinct lack of spirocyclic compounds in FDA approved molecules despite the number of patents surrounding such molecules slowly increasing in recent years. The authors describe spirocycles as an “overlooked motif for drug discovery”.  Their work surrounds the replacement of 2-substituted piperidines with 2-azaspiro[3.3]heptane surrogates. These compounds are not synthetically challenging as one might imagine and they originate from commercially available, relatively cheap starting materials with synthesis easily scalable to 50g of product in a single batch. They also demonstrate that the synthesis has scope and can tolerate a wide range of substituents.

The synthesis of the spirocyclic analogue of FDA-approved local anaesthetic Bupivacaine was completed by this group to compare this motif in a drug discovery setting. The results showed that despite their analogue exhibiting slightly higher plasma protein binding and in-vitro metabolism, in-vivo, their analogue showed faster onset and similar duration to Bupivacaine with lower systemic toxicity than the FDA approved drug.                               Penny 7Whilst spirocycles remain unrepresented in FDA approved molecules, it is clear that interest both within academia and industry is growing. With studies like these being published more frequently demonstrating the improved drug-like properties of these compounds when compared to their monocyclic counterparts, it is likely that it won’t be long before these motifs are seen in approved drugs.

Blog written by Penny Turner


Angew. Chem. Int. Ed., 2010, 49: 9052-9067

Angew. Chem. Int. Ed., 2008, 47: 4512-4515

Angew. Chem. Int. Ed., 2010, 49: 3524-3527

Angew. Chem. Int. Ed., 2017, 56: 8665-8869

Dopaminergic neurons display antigens: autoimmunity in Parkinson’s disease pathology

Parkinson’s disease (PD) is a chronic, progressive degenerative disorder of central nervous system.  Its pathology is characterised by selective loss of dopaminergic neurons in the nigrostrial pathway, and clinical manifestations are exhibited as motor impairments, including resting tremor, bradykinesia, and rigidity.  Current medications offer symptomatic relief but, to date, do not address the dopaminergic neuronal death. The lack of understanding of the etiology of this selective cell death still remains the major stumbling block in the development of neuroprotective therapies. Current research implicates a number of key molecular mechanisms compromising the function and survival of this specific subset of neurons, and these involve abnormal protein accumulation and phosphorylation, mitochondrial dysfunction, oxidative damage and deregulated kinase signalling.  Although the current hypothesis focuses on the toxic aftermath of α-synuclein protein deposits, an alternative theory pioneered by Dr. Sulzer’s group within the Department of Neuorology at Columbia University, implies a role of the immune system in PD pathology, more specifically suggesting that Parkinson’s is in fact an autoimmune disease.

As mentioned, pathological features of PD include the loss of nigrostriatal dopamine neurons and the formation of Lewy bodies rich in fibrillar α-synuclein. This 140-amino-acid protein is abundantly expressed at a relatively high level throughout the brain (Iwai et al., 1995) and is thought to play physiological roles in the regulation of the dopamine transporter (Sidhu et al., 2004). However, misfolding of α-synuclein into protofibrils and higher-order oligomers (Uversky et al., 2002) leads to a toxin gain of function (Martin et al., 2006), which is associated with the pathogenesis of neurodegeneration (Giasson et al., 2000). Furthermore, this protein has been genetically linked to the early onset of familial PD (Kruger et al., 1998). However, the mechanism by which α-synuclein causes neurodegeneration remains unclear.

In addition to α-synuclein dysfunction, PD pathology is also characterised by a sustained microglial reaction throughout the disease progression Imamura et al., 2003). Microglial cells are the resident immune cells in brain and play a major part in the neuroinflammatory response (Soulet and Rivest, 2008). On an epidemiological level, the contribution of an inflammatory response in neurodegeneration is evidenced by the decreased risk of falls in PD patients on administering the non-steroidal anti-inflammatory drug (NSAID), ibuprofen (Gagne and Power, 2010). On a cellular and molecular level, the significant elevation in inflammatory cytokines has been found in both the cerebrospinal fluid and postmortem brain of PD patients (Mogi et al., 1994). These cytokines have been reported to induce the death of dopaminergic cells (Vivekanantham et al., 2015), and thus facilitating neurodegeneration in PD.

Dr. Sulzer’s group have recently identified the presence of antigens displayed on dopaminergic neurons in post-mortem brain tissues. It has long been argued that brain cells are safe from immune cell attack because they do not display these molecular markers for immune cell target recognition.  However, these new findings indicate that they can in fact be targeted.  Abnormal processing of self-proteins can produce epitopes, which are presented by major histocompatibility complex (MHC) proteins to be recognised by specific T cells that have escaped tolerance during thymic selection (Marrack and Kappler, 2012). Such actions by the acquired immune system have been implicated in autoimmune disorders, including type-1 diabetes. Although PD has not before been linked to autoimmunity, it does demonstrate altered protein processing. As previously described, activation of microglia and elevated cytokine levels have described in PD patients, indicating a role of the innate immune system.  But what evidence is there to implicate the acquired immune system?

Rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD was initially provided by Brochard, et al (2009). It was found that CD8+ and CD4+ T cells, but importantly not B cells, infiltrate the substantia nigra (SN) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration, which is consistent with postmortem human PD specimens. Further investigation concluded that T cell-mediated dopaminergic toxicity is almost exclusively arbitrated by CD4+ T cells (Brochard et al., 2009).  The Sulzer group from Columbia University, more recently, reported antigen presentation by MHC class I expression in dopamine neurons in the SN of adult human PD brains (Sulzer et al., 2017).  This was the result of activation by cytokines released from microglia.  CD8+ T cells kill neurons that present the appropriate combination of MHC class I and peptide (Cebrián et al., 2014).  On comparison of PD patients to age-match healthy controls, the group then identified two antigenic regions in α-synuclien (Fig. 1a).  The first near the N terminus elicited an apparent class II restricted IL-5 and IFNγ response (Fig. 1b–d). The second antigenic region was near the C terminus required phosphorylation of amino acid residue S129 that resulted in a markedly higher IL-5 responses in patients with Parkinson’s disease than in healthy controls. The Y39 antigenic region is noticeably close to the α-synuclien mutations that cause Parkinson’s disease (A30P, E46K, H50Q, G51D, A53T; Hernandez et al., 2016), and phosphorylated S129 residues, found in the second antigenic region, are present at high levels in Lewy bodies of patients with Parkinson’s disease (Fujiwara et al., 2002). Finally, blood tests have revealed that people with Parkinson’s show an immune response to these antigens, while people who don’t have the condition do not (Sulzer et al., 2017).


Figure 1α-synuclein autoimmune responses are directed against two regions. a, Sequence of α-synuclein. Antigenic regions are highlight with dashed line with amino acids Y39 and S129 shown in bold. b-d, Magnitude of responses expressed as SFC per 106 PBMC’s per peptide and participant combination. Left, response to all overlapping native α-synuclein 15-mer peptides in patients with PD (n=733) and control (n=372).  Right, response against specific 15-mers.  Grey shading indication the antigenic region containing Y39. e-g,  Magnitude of responses. Left, response to all native phosphorylated S129 α-synuclein 15-mer peptides in patients with PD (n=150) and control (n=72). Right, response against specific S129 peptides. Closed circles, patients with PD (n=19, † indicates peptides; n=25, all other peptides); open circles, control (n=12).  Two-tailed Mann-Whitney U-test; NS, not significant.

These findings are the first time the immune system has been associated with a major pathological role in Parkinson’s. They present an argument for the classification of PD as an autoimmune disorder.  However, what isn’t clear is which comes first: does the immune response directly causes neuron death, or does the disease result in a heightened immune response?  If suppression of this autoimmune response does indeed stop disease progression, these findings could provide an attractive target for therapeutic intervention.

Blog written by Victoria Miller


Brochard et al., J. Clin. Invest. (2009) 119, 182–192.

Cebrián et al., Nat. Commun. (2014) 5, 3633.

Fujiwara et al., Nat. Cell Biol. (2002) 4, 160–164.

Gagne & Power, Neurology (2010), 74, 995–1002.

Giasson et al., Science (2000), 290, 985–989.

Hernandez et al., J. Neurochem. (2016) 139, 59–74.

Imamura et al., Acta Neuropathol. (2003), 106, 518–526.

Iwai et al., Neuron (1995), 14, 467–475.

Kruger et al., Nat. Genet.(1998), 18, 106–108.

Marrack & Kappler, Cold Spring Harb. Perspect. Med. (2012) 2, a007765.

Martin et al., J. Neurosci. (2006), 26, 41–50.

Mogi et al., Neurosci. Lett. (1994), 165, 208–210.

Sidhu et al., FEBS Lett. (2004), 565, 1–5.

Soulet & Rivest, Curr. Biol. (2008), 18, R506–508.

Sulzer, Nature (2017) 0.

Uversky et al., J. Biol. Chem. (2002), 277, 11970–11978.

Vivekanantham et al., Int. J. Neurosci. (2015), 125, 717–725.

The Necessary Nitrogen Atom

In the modern drug discovery process, medicinal chemists strive for high-impact design elements during multiparameter optimisation of lead compounds into efficacious drug candidates. One of such well-known design elements, often referred to as “the magic methyl effect”, “the methyl walk” or “the methyl scan”, involves a replacement of a H atom with a Me group and, as the result, can lead to profound potency improvement (>100-fold).

The authors of the recently published perspective (DOI: 10.1021/acs.jmedchem.6b01807) discuss another common design element for multiparameter optimisation: substitution of a CH group with a N atom in aromatic and heteroaromatic ring systems. Such replacement, when going from a simple benzene ring to pyridine, results in profound changes in molecular and physicochemical properties, such as distribution of electron density, polar surface area, basicity, which in turn affects lipophilicity and solubility. In more complex chemical structures, these changes can impact upon a number of intra- and intermolecular, orbital, steric, electrostatic, and hydrophobic interactions, such as lone pair, dipole−dipole, hydrogen bonding, metal coordination, van der Waals, σ-hole, σ*S−X, and π-system interactions, which in turn can translate into modified pharmacological profiles.

The authors then illustrate an extensive number of drug discovery case studies from the recent literature, where a replacement of a CH group with a N atom resulted in ≥10-fold improvement in at least one key pharmacological parameter, as shown, for example, in Figure 1. The replacement of the C7 CH group in (1) with a N atom to give (2) resulted in a 300-fold improvement in biochemical potency, Cdc7 IC50 = 2700 and 9.0 nM for (1) and (2), respectively. This potency improvement was attributed to the different conformational preferences of the two analogues. The biaryl dihedral angle of >150° in indole (1) greatly differs from that of aza-indole (2) (dihedral angle of 0°), facilitated by a steric clash of H7 and H6′ in (1) and lone pair repulsion between N7 and N2′ in (2).


Figure 1. An example of a necessary nitrogen atom on potency improvement, likely due to different conformational preferences.

The effects of such replacement on basicity, lipophilicity, polar surface area, and hydrogen bonding capacity are relatively predictable; whereas effects on aqueous solubility, passive permeability, efflux profiles, active transport, protein binding, and metabolic stability can be more whimsical and counterintuitive. In some examples, no rational explanation could be given for the observed effect.

With regards to changes in potency, as the authors are cautious to point out, there is an approximately equal probability of increasing or decreasing potency by exchanging CH groups and N atoms, based on a matched molecular pair analysis (MMPA) of available data. However, these findings are very similar to those discussed for the magic methyl effect.

This perspective is not a manual for what pharmacological improvements will be realized upon the substitution of a CH group with a N atom, but rather as an extensive exemplar of the dramatic improvements that can be achieved under certain circumstances. The systematic N atom scan (N-scan) should be exploited where appropriate, particularly in cases where the preferred binding pose of the ligand is not known.

Some recommendations for the N-scan tactics are summarised below. The newly installed N atom might:

  1. Engage in a hydrogen bond with specific residues of the target receptor or receptor-bound water molecules that need to be satisfied
  2. Remove unfavorable van der Waals interactions the replaced CH group made with the target receptor
  3. Form unfavorable electrostatic interactions with an antitarget receptor
  4. Have a positive effect on the binding conformation of the ligand
  5. Mask a hydrogen bond donor in the ligand
  6. Reduce the basicity or HBA strength of an existing N atom in the ligand
  7. Evenly distribute the polar surface area of the ligand
  8. Properly tune the lipophilicity of the ligand
  9. Be shielded by other substituents or functionality in the ligand
  10. Stabilize chemically labile functionality in the ligand
  11. Replace a metabolically labile CH group in the ligand

Medicinal chemists have to constantly juggle the design of biologically active molecules with drug-like properties according to the rules of Lipinski and Veber, in order to achieve good permeability and absorption levels, which in turn lead to high oral bioavailability. These properties are particularly important when considering therapeutic targets located in the central nervous system (CNS) behind the blood−brain barrier (BBB). Keeping the number of hydrogen bond acceptors down is one of such requirements; however, in some cases, it may turn out that the introduction of one (or two) extra N atoms may be necessary.

Blog written by Irina Chuckowree