Novel method for assessing cardiotoxicity

One of the most common reasons for failure of compounds in drug discovery programmes is cardiac toxicity. Therefore assessing this in the early stages of any drug discovery programme is key. Currently the earliest indicator for cadio-tox is interaction between compounds and a voltage gated potassium channel hERG (the human Ether-à-go-go-Related Gene) channels. Generally electrophysiological techniques are used in hERG, although fluorescent-based kits are now available.  Although hERG is the most common interaction for cardiotoxicity, this does not measure other potential interactions that could result in cardiotox. Sirenko et al. have taken advantage of the recent emergence of inducible pluripotent stem cells (iPSC) to explore the effects of compounds on cardiomyocytes in more detail.

Culture of iPSC is not as straightforward as immortalised cells, however the cells were obtained from a commercial source (Cellular Dynamics) saving the differentiation process and these cells are able to be reproducibly differentiated into a large quantity and cryopreserved until use. The use of iPSC derived cardiomyocytes for toxicity testing has the clear advantage that these cells express full cardiomyocyte functionality and even have the beating characteristic expected of primary cardiomyocytes. Since this beating is analogous to the beating of a heart, and ion channel block can present as drug-induced arrhythmias, it is not hard to see how this can be used to identify cardiotoxic compounds. At this point most groups would have struggled to have the technology available to analyse these beatings, but since the authors were from Molecular Devices, they conveniently had a FLIPR tetra and image xpress high content screening (HCS) platform along with the technical knowledge to enable these beatings to be quantified.

Sirenko et al. were able to quantify these beatings on both systems. On the HCS, they used time-lapse images and quantified the beating using their own differential algorithm that measured the number of beats per minute. On the FLIPR, they used the Calcium 5 Assay Kit which enabled the beating to be measured using Ca2+ influx in beats/min. Since the HCS could not acquire images as quickly as the FLIPR, the HCS approach was less sensitive than using the FLIPR and less work was performed using this method, however, they were still able to obtain IC50’s for known agonists adrenaline and isoproterenol.

The increased sensitivity and throughput possible using the FLIPR, enabled more studies to be performed using it. As such compound-induced dose-dependent atypical beating patterns induced by known cardio-toxic compounds such as hERG, Ca2+ and Na+ channel blockers were measured. They were also able to validate an automated analysis algorithm that was used to calculate the effect of a variety of compounds in high-throughput format, with positive or negative chronographic effects such as digoxin and propranolol. Furthermore, due to the increased sensitivity of the FLIPR it was also possible to quantify components of each individual Ca2+ peak (i.e. amplitude, peak width, decay time etc.) rather than just the number of beats. This meant they were able to measure more compound specific effects that have more physiological deviations such as QT prolongation, i.e. cisapride that increased both peak spacing and peak width.

A large number of drug withdrawals from the market as well as late stage clinical trials are from cardiac toxicity. These withdrawals are clearly very expensive, but more importantly potentially very damaging to health. The use of hERG testing clearly identifies many cardiotoxic compounds, however, the use of iPSC cardiomyocytes and measuring Ca2+ influx and beating enables many more facets of cardiac toxicity to be measured. The study by Sirenko demonstrates the use of this technique to identify compounds already known to effect different facets of cardiac toxicity. The real test, however, would be to put compounds that were withdrawn from the market, or clinical trials due to cardiac toxicity that were missed by other tox screens. If the technique set out by Sirenko et al., were to have picked up this toxicity it would demonstrate a step forward in early determination.

Pinacol Arylboronates from Aromatic Amines

Boronic acids and esters are extremely versatile intermediates which have the potential to be transformed into a variety of other functional groups. We have highlighted a couple of examples of these in Trifluoromethylation of arylboronic acids by Cu-mediated CF3 radicals and Borylation feast.

Wang has just published an interesting method for the synthesis of pinacol arylboronates from aromatic amines. Unlike most other methods for introducing a pinacol boronate ester motif this chemistry is metal free and instead uses tert-butyl nitrite under Sandmeyer-type reaction conditions (Scheme 1).

Lewis8Scheme 1 – Different routes to arylboronic acids or arylboronates

This paper presets optimised reactions conditions which build on Wang’s initial communication and removes the need for benzoyl peroxide (BPO). The best temperature examined for the reaction was at 80°C. It was found that at this temperature the addition of the radical initiator BPO had a negligible effect (Table 1). It was also shown that the use of toluene, DCE or dioxane and the 2 alternative alkyl nitrites all negatively affected the yield.

lewis9Table 1 – Optimised reaction conditions for 2-amino benzonitrile.

These optimised conditions were directly compared to those in Wang’s initial communication and it was seen that there was an increase in yield of between a 5% and 44% in all of the reactions attempted (Scheme 2).

lewis10Scheme 3 – Scope of para-substituted aniline derivatives

When the reaction of meta, ortho or multi-substituted anilines was attempted lower yields were observed when compared to the para-substituted analogues (scheme 4). The steric hindrance of the ortho-substituted anilines could explain the lower yields observed for these reactions. Also, in general, it was seen that electron rich aromatic rings gave lower yields than those with electron withdrawing groups. This could be explained by the mechanism that Wang goes on to propose and the extra stability that an electron withdrawing group would give in the transition state.

lewis11Scheme 4 – Reaction of meta, ortho or multi-substituted anilines

Whilst expanding the scope of this reaction to include other aromatic and heterocyclic amines the previous observations were confirmed with electron deficient compounds giving higher yields than electron rich (scheme 5). The authors report that the electron rich aromatic amines are prone to oxidation by t-BuONO resulting in lower yields of the boronated product. They also note a lack of stability of these heterocyclic boronate esters to silica gel column chromatography.

lewis12Scheme 5 – Other aromatic anilines

To overcome the problem of purification and isolation of these unstable boronate esters Wang developed a sequential borylation and palladium catalysed cross coupling reaction (scheme 6). This procedure was able to give an increased combined yield of 41% for flavone 6e over 2 steps compared to only 32% for the boronate ester intermediate 5db (scheme 5). The low yields of compounds 6r and 6s could be due to the inherent instability of the α-hetero boronate ester intermediates to proto-deboronate.

lewis13Scheme 6 – Sequential borylation and palladium catalysed cross-coupling reaction

Wang has proposed a possible radical mechanism for this reaction (scheme 7). The first step is the formation of a tetra-coordinate boron complex (A) of B2pin2 with tert-butoxide anion. A single electron transfer then occurs between the ate complex A and the aryldiazonium moiety to give radicals B and C. Intermediate B is then able to extrude N2 to give aryl radical D which is able to react with intermediate C to give the boronate ester product. To substantiate this mechanism Wang added 1.5 equivalents of a radical scavenger (TEMPO). He found that the reaction was sluggish and after 4 hours phenyl boronate ester was only formed in an 8% yield. In a control reaction he showed that no reaction was seen between TEMPO and B2pin2. In another reaction Wang heated a 1:1 mixture of BPO and B2pin2 at 100°C for 4 hours. This gave a 37% yield of phenyl boronate ester was formed which supports the aryl radical mechanism. However when he attempted to synthesise the aryl radical from PhI, Bu3SnH and AIBN and then to react this with B2pin2 only a 7% yield of the desired product was isolated. Due to this result Wang was not able to rule out the possibility of an anionic mechanism of nucleophilic aromatic substitution of the aryldiazonium salt by the boron ate complex.


Scheme 7 – Possible reaction mechanism

This paper builds substantially on the work that Wang had previously published with a simplification of the reaction conditions and further exploration of the scope of this reaction. He has been able to overcome the problem of purifying unstable boronate esters and showed that they can be taken through a Suzuki reaction successfully without the need for any work-up or purification. Overall this reaction provides a very simple, practical and scalable method to synthesise boronate esters from various aromatic amines.

Guidelines for Sonogashira cross-coupling reactions

As a synthetic organic chemist you will have built many carbon-carbon bonds and most probably in some occasions this will have been between sp2 and sp carbons. The so called Sonogashira cross-coupling reaction (Scheme 1), the most important of the latter carbon-carbon bond formations, is the reaction between an aryl or vinyl halides with a terminal acetylene catalysed generally by a palladium catalyst.

sono1Scheme 1.

 However, if one is to perform such reaction, the search for the ideal conditions can be difficult as addressed by a recent review (Chem. Soc. Rev. 2011, 40, 5084) where a search in SciFinder with the topic ‘Sonogshira’ revealed more than 1500 hits for the period 2007-2010.

Some general rules have previously been published by Hartwig in 2007 (Inorg. Chem. 2007, 46, 1936) which were derived from studies of the electronics and the bulkiness of the substrates and catalysts:

  1. The oxidative addition in Ar-X is promoted by electron-withdrawing groups at the aryl halide.
  2. Steric bulk of phosphines or NHC ligands coordinated to Pd promote the formation of a monoligated complex, which turns out to be highly active for oxidative addition.
  3. There is a pronounced steric effect in the transmetalation, while the ligand bite angle and the electronic effect are less important.
  4. Reductive elimination tends to be favoured by less electron donating ligands and steric bulk.

More recently Plentio has presented a guide on the performance of the Sonogashira cross-coupling (J. Org. Chem. 2012, 77, 2798) based on about 200 reactions (Scheme 2), where he correlates the stereoelectronic properties of the substituents in aryl bromides, acetylenes and phosphines.

 sono2Scheme 2.

  His conclusions can be summarized:

  •  The most important factor for choosing the ideal Pd/phosphine catalyst is the steric bulk of the phenylacetylene and Plentio reaches an ideal combination for the optimum steric bulk on the acetylene and phosphine. In short, the higher the steric bulk on the arylacetylene and the lower is the bulk of the phosphine ligand will promote the most efficient transformations.
  • Electron-withdrawing groups on the arylacetylene also increases the reactivity and this effect is more pronounced when the electron-withdrawing group is located on the acetylene rather than on the aryl bromide.
  • The more electron-rich the phosphine is, the higher the reactivity is.
  • The steric bulk on the aryl bromide, alpha to the bromo substituent, is more detrimental for the reaction than steric bulk on the acetylene.


Amyloid in Alzheimer’s Disease – The End of the Beginning or the Beginning of the End?

In terms of drug discovery, there are four general ways of identifying new drugs: 1) there is serendipity, where a chance preclinical or clinical observation is translated into a novel therapeutic (with the initially cardiovascular Viagra being an example, the clinical utility of which you could quite literally hang your hat on); 2) Iteration, in which a new drug is an improvement upon an existing drug (e.g. reduced side effects or better pharmacokinetics); 3) repositioning, whereby a drug approved or initially evaluated for Indication A proves efficacious in Indication B and finally; 4) hypothesis-driven drug discovery, in which drugs are targeted towards a pathway or protein specifically implicated (e.g. genetically or pathologically) in a disease process. This latter process is the most rational and intellectually satisfying and forms the basis of the multiple amyloid-related approaches to treating Alzheimer’s disease since the amyloid core at the centre of the hallmark senile plaques  as well as the genetics of familial cases of AD all point the scientific finger of guilt towards the amyloid pathway.

As regards the amyloid hypothesis of Alzheimer’s Disease, it is now a few months ago that we discussed the big summer of data that lay ahead with Bapineuzumab and Solanezumab. Well, the data has now been chewed over and digested and as the year draws to a close, it is a good time to be reflective and assess where the field stands. So, in alphabetical, chronological and clarity of what-happens-next? order let us first consider Bapineuzumab. The data for the first Phase III study, Study 301, was disappointing but not surprising since the AD patients were the ApoE4-carrier subpopulation that the Phase II study suggested were less susceptible to the potential benefits. Hopes were therefore pinned on data from the ApoE4-noncarrier Study 302. However, these data were unambiguously negative (follow the links for data of Studies 301 and 302 presented at the September meeting of the European Federation of Neurological Societies) with the complete lack of ambiguity resulting in the termination of the two additional ex-US and incomplete studies (Studies 3000 and 3001). So, all-in-all, quite an emphatic end of story for i.v. Bapineuzumab.

The story for Eli Lilly’s Solaneuzumab is, however, not quite so clear cut. To recap, the EXPEDITION1  and EXPEDITION2 were pivotal Phase III studies. The EXPEDITION1 study missed its primary end-point but showed a significant effect on a secondary end-point, namely cognition in mild AD. This secondary end-point was then used as the primary end-point in the EXPEDITION2 study, but there were no significant effects of Solanezumab. However, combining data from these 2 studies – one of which showed efficacy, the other one not – showed a significant effect in mild but not more advanced moderate AD patients. This is entirely consistent with the way the field has been moving, namely that amyloid-related treatments need to be as early in the disease process as possible. There was a brief flirtation with the prospect that because of the large unmet need regulators may find a way to approving Solanezumab based on the existing data. However, subsequent to discussions with the FDA, Eli Lilly accept that approval would require a new Phase III study in mild AD patients, although their press release does note that “It is possible that different courses of action could be taken in different jurisdictions.”. Given their recent run of bad luck with neuroscience Phase III compounds (the γ-secretase inhibitor Semagacestat in 2010 and then this year the mGlu2/3 agonist pomaglumetad methionil and Solanezumab), one can only admire the depth of the company’s neuroscience financial trouser pocket and their obvious commitment to the area.

If the Solaneuzumab data tells us that treating earlier is the way to go, then the ultimate extension of this approach is a prevention trial. Such a trial, which commences in the spring of 2013 and is being organised by the Banner Alzheimer’s Institute in Phoenix, is being conducted as part of the 5-year, $100 million Alzheimer’s Prevention Initiative and will focus on a family in Columbia with genetic mutation associated with on onset of Alzheimer’s disease in their late 40s. Subjects with the mutation will receive Crenezumab, an anti-amyloid antibody developed by Roche/Genentech and licensed from the Swiss company AC Immune. An additional “branch study” will also take place in the US and will include an additional 150 US mutation carriers. A second prevention trial is also due to start in early 2013 and will be conducted by the Dominantly Inherited Alzheimer Network Trials Unit (DIAN TU) and will evaluate the effects of three different drugs on subjects (160 carriers and 80 non-carriers) with AD-causing mutations.  These three drugs were selected from the more than a dozen drugs proposed by the 10 pharmaceutical companies that comprise the DIAN Pharma Consortium and include the anti-amyloid antibodies Solanezumab and Gantenerumab, a Roche antibody currently in a Phase III trial for very early, presymptomatic (prodromal) AD known exotically as SCarlet RoAD, with a third drug, the Lilly BACE inhibitor currently in Phase II, also being selected for potential inclusion.

So, there remains life in the amyloid hypothesis. But what about other approaches? Well, as we mentioned at the top of this article, drug repositioning (or drug repurposing) is an attractive potential alternative since it is a route accessible to research councils and academic centres (i.e., it lacks the huge development costs of novel therapies). Recently, a number of drugs currently in clinical use for other indications have been shown to have an effect on amyloid metabolism or the associated neuroinflammatory response in animal models, including, for instance, the anticancer (cutaneous T-cell lymphoma) drug Bexarotene, the antiepileptic drug Levetiracetam and the blood pressure drug Prazosin. Nevertheless, the extrapolation between effects in animal models and human is a large and tenuous one with, for example, Rosiglitazone producing marked effects in transgenic mice but there were no signs of efficacy in two  Phase III studies. So, despite claims that “Drug giants give up on Alzheimer’s cure” (The Independent, 19th September, 2012) it would appear that there still remains a major commitment to the development of new therapeutics for Alzheimer’s Disease and that recent developments in the field represent the end of the beginning rather than the beginning of the end.