Synthesis of Pyrazoles and Indazoles by Cu/O2 catalysed C-H activation

In a recent paper by Huanfeng Jiang a new practical synthesis of both pyrazoles and indazoles was described. By using a simple copper / oxygen catalytic system direct C-H bond amination was achieved.

Huanfeng started investigating this reaction by varying the copper catalyst, additive, solvent and reaction temperature to attempt to optimise the reaction conditions (table 1).

lp table 1Table 1: Optimisation of reaction conditions

With the optimal condition of Cu(OAc)2 (10 mol %) and DABCO (30 mol %) in DMSO at 100°C under O2 (1 atm) the scope of this reaction was explored (table 2 and 3).

As can be seen in table 2 the reaction gave pyrazoles in a >80 % yield where R1 = aromatic / olefinic, R2 = aromatic / olefinic / alkyl and R3 = aromatic / olefinic / alkyl. A wide range of functionalities were tolerated including fluorides, chlorides, bromides, nitriles, trifluoromethyls, carbonyls, sulphonamides and ethers. The only functionality investigated that was not tolerated was a free NH2. Huanfeng states that the geometry of the hydrazone double bond in the starting material is not important and believes that at elevated temperature in the presence on DABCO this would isomerise.

lp table 2Table 2: Synthesis of Pyrazoles

 After further optimisation of the reaction conditions it was found that the addition of 1 equivalent K2CO3 and an increased temperature of 120°C gave the best yields of indazoles. This reaction, as expected, tolerated the same functionalities as the pyrazole series. However, the potential to produce, presumably difficult to separate, mixtures of regioisomers makes this synthesis less desirable. It was noted that in the case of unsymmetrical aryl group that electron-rich substrates were favoured in the insertion step and that steric factors might also affect the regioselectivity.

lp table 3Table 3: Synthesis of Indazoles

The methodology to synthesise pyrazoles was then extended further to start from an aryl enone (scheme 1). Yields for this one-pot procedure (85 %) were comparable to the one step synthesis from a hydrazone (92 %).

lp scheme 1Scheme 1:  One-pot Pyrazole synthesis

To further the applicability of this chemistry Huanfeng functionalised a pyrazoles with NBS and used this building block in a Sonogashira reaction, Suzuki-Miyaura reaction and an amidation reaction (scheme 2).

lp scheme 2Scheme 2: Pyrazole derivatisation

To probe the mechanism of the C-H amidation several experiments were conducted using stochiometric quantities of an electron-transfer scavenger (1,4-dinitrobenzene), a radical clock (diallyl ether), or a radical inhibitor (hydroquinone, TEMPO) (scheme 3). The reaction proceeded in each case and when using diallyl ether no cyclised product was observed. These results suggest that this is not a radical mediated transformation.

lp scheme 3Scheme 3: Mechanism probing experiments

Based on these preliminary mechanistic studies Huanfeng has postulated the following mechanism (scheme 4). Initially olefinic hydrazone 1 or aryl hydrazone 3 would react with Cu(OAc)2 to form an Cu-N adduct A. The nitrogen from this Cu-N adduct could then undergo an intramolecular electrophilic substitution followed by aromatisation via C to give the pyrazole / indazole. The reduced copper species (Cun-2) could then be reoxidised by oxygen to complete the catalytic cycle.  Alternatively metallacycle B could be formed by electrophilic metalation or C-H bond activation followed by reductive elimination and aromatisation.


lp scheme 4Scheme 4: Proposed catalytic cycle

In conclusion this synthesis uses cheap and readily available reagents and has been shown to proceed in high yields even when preformed in air (72 % yield, table 1 entry 14).  This methodology circumvents the need to prefunctionalise starting materials with (pseudo) halides or directing groups and therefore has the potential to reduce the step count and open up previously more challenging or unavailable pyrazoles or indazoles.

Cancer Genome Landscapes

Vogelstein et al have published a very informative review on the genomic landscapes of cancer.

As the cost of genome sequencing has fallen 100 fold in the last ten years it is becoming commonplace to sequence the exomes of sets of 100+ tumours, which is allowing us to study the genomic make up of tumours.

The number of mutations a tumour has is dependent on its type:  solid adult tumours have between 33-66, where as childhood cancers far fewer.

FP1Exceptions to this are lung cancers in smokers and melanomas that have far more mutations due to the mutagenic impact of the carcinogen (smoke/sunlight) that initiates them. Tumours with defects in DNA Damage Response also accrue disproportionally large number of mutations.

Interesting, the number of mutations observed in solid adult tumours in self-renewing tissues (e.g. colon) are proportional to the age of the patient implying that these mutations may be present at the pre-neoplastic stage.

It is estimated that between two to eight sequential alterations that develop over the course of 20 to 30 years are actually causative of the cancer.  These are termed  “driver mutations” and occur in driver genes.  Each alteration causes a selective growth advantage to the cell in which it resides.  The other mutations occur because the cancer is genetically unstable, are termed passenger mutations, and confer no selective advantage to the cell in which they reside,

Driver genes are genes that contain driver mutations and there are two types:

Tumour suppressors that confer a selective advantage to the cell when the are “broken”

Oncogenes who confer a selective advantage to the cell, if they are “activated”.

Vogelstein et al. estimate how many driver genes exist using the 20:20 rule. In tumour suppressors at least 20% of the mutations cause truncation of the gene product, where in oncogenes at least 20% of the missense mutations occur in a single position along the polypeptide chain. (see figure 2)


PIK3CA and IDH1 are oncogenes, where as RB1 and VHL are tumour suppressors.

Using the 20:20 rule Vogelstein et al.identify ~140 genes whose intragenic mutations contribute to cancer (so-called Mut-driver genes).

Interesting this is far fewer than the ~500 genes identified in the Cancer Gene Census as being causative of cancer.  They suggest that other genes (Epi-driver genes) that are altered by epigenetic mechanisms and cause a selective growth advantage, but the definitive identification of these genes has been challenging.

Although every individual tumor, even of the same histopathologic subtype as another tumor, is distinct with respect to its genetic alterations, but the pathways affected in different tumors are similar.  These driver genes function through a dozen signaling pathways that regulate three core cellular processes: cell fate determination, cell survival, and genome maintenance.


They also briefly discuss that currently most anti-cancer drugs available inhibit the activity of an enzyme. However, of ~140 driver genes identified, only 31 could be targeted in this manner.

Indeed the majority of Mut-driver genes encode tumor suppressors, not oncogenes. Drugs generally interfere in the function of a protein – conceptually very difficult to produce a drug that will restore the function of a protein.

They conclude that in the future, the most appropriate management plan for a patient with cancer will be informed by an assessment of the components of the patient’s germline genome and the genome of his or her tumor.  That the inherent heterogeneity of tumours and their metastases makes resistance to targeted therapies ‘inevitable’ and that it is important to research the efficacy of combination therapies. They also suggest that the information from cancer genome studies should be exploited to improve methods for prevention and early detection of cancer, which will be essential to reduce cancer morbidity and mortality.

Targeting leukaemias with a therapeutic human antibody

Currently marketed anticancer monoclonal antibodies (mAbs) recognise extracellular proteins or those expressed on the cell surface. Generally these are not tumour-specific, as oncogenic proteins tend to be nuclear or cytoplasmic. These intracellular proteins can however, be presented on the cell surface as T cell epitopes by the major histocompatibility complex (MHC). These epitopes are recognised by T cell receptors (TCRs). Therefore, the generation of ‘TCR-like’ mAbs that can recognise cell surface epitopes that are derived from tumours are an exciting potential cancer therapy.

Dao et al., recently published a paper in Science Translational Medicine, in which they have exploited phage display technology to produce a specific mAb (ESK1) against Wilms tumor 1 (WT1), an oncoprotein that is overexpressed in both leukaemia and a range of solid tumours such as ovarian cancer and mesothelioma, but is rare in normal tissues. It was also recently ranked as a top cancer target for immunotherapy by the NIH. ESK1 targets a 9mer WT1 derived peptide (RMF) that is processed and presented by HLA-A0201. RMF induces cytotoxic T cells that are able to kill WT1+ tumour cells in vitro.

ESK1 was shown to bind to acute myeloid leukaemia (AML) CD34+/CD33+ cells expressing HLA-A02 and WT1 but not to normal peripheral blood mononuclear cells. The specificity of the binding was confirmed with other cell types with no general cross-reactivity to healthy or leukemic cells that do not express WT1. Radioimmunoassay experiments then demonstrated that there was adequate RMF expressed on the surface of many cancer and leukaemia cells, whilst the levels of epitope on WT1 negative healthy cells are low.

mAbs can cause cytotoxicity in four ways: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), antibody-dependent cellular phagocytosis (ADCP) and inducing apoptosis. ESK1 was shown to be active in ADCC assays against the following cell types: JMN mesothelioma, BV173 leukaemia, ovarian carcinoma, colon carcinoma cell lines and AML cells. No other form of mAb-mediated cytotoxicity was observed.

The efficacy of ESK1 was then tested in vivo in mice that had been xenografted with BV173 acute lymphoblastic leukaemia (ALL) cells or BA25 acute lymphocytic leukaemia. Two intravenous doses of 100ug of ESK1, when administered in conjunction with human effector cells, suppressed the growth of  leukemic cells in both animal models. Prolonged or, in some cases, leukaemia-free survival were observed. Two control studies confirmed that the RMF/A2 epitope was required for the therapeutic effect observed. Further, no evidence of toxicity was observed in transgenic mice when given therapeutic doses of ESK1.


Survival of NSG mice with BV173 leukaemia. **P<0.01 for all treatment groups compared to untreated control animals or animals treated with isotype control hIgG (log-rank Mantel-Cox test).

The ESK1 antibody could therefore be promising as a new cancer drug, with a large clinical impact for those patients with WT1+ tumours or leukaemia with HLA-A02 expression.


Aggregation false positives in cell based assays?

An article dealing with the common problem of compounds that are false positives in screening assays was published recently (  One cause of compounds acting as false positives in screening assays is that they can self-aggregate, forming colloidal particles. This aggregation effectively sequesters the protein from its target and prevents activity. This has been a common problem in drug screening assays, particularly with soluble protein methods. In this publication the Shoichet lab at University of California, and others, have been investigating a further scope of the problem by examining  GPCR assays using a cell based format.

They took four compounds that were known to form aggregates and measured the activity against a variety of receptors using the Beta- Arrestin assay. The results show that these compounds were acting as antagonists against the receptors when they were stimulated with their agonist ligand, and this activity could be reversed with the addition of detergent or the use of centrifugation.

They also observed inverse agonism when the compounds were tested against the receptor in the absence of the activating ligand of the receptor, maybe via membrane perturbation.

It all highlights a type of assay artefact, which was thought to be more prevalent in soluble protein assays, can also have a bearing in cell based formats.The steps show by the authors (centrifugation and detergent usage) should be included to reduce the chance of false positives even if you are using a cell based method.

gw1Figure extracted from: Sassano, M. F., Doak, A. K., Roth, B. L., & Shoichet, B. K. (2013). Colloidal aggregation causes inhibition of g protein-coupled receptors. Journal of medicinal chemistry, 56(6), 2406–14. doi:10.1021/jm301749y