Rapid Catalyst Identification for the Synthesis of the Pyrimidinone Core of HIV Integrase Inhibitors

A microscale chemistry improvement engine: A pre-dosed microscale high-throughput experimentation additives platform enables rapid, serendipitous reaction improvement. This platform allowed one chemist to set up 475 experiments and analyze the results using MISER chromatography in a single day, thus resulting in two high-quality catalytic systems for the construction of the title compound 1. Support for a single-electron transfer mechanism was obtained. Microscale multiparallel chemistry platform technologies enable modern synthetic chemists to rapidly apply known potential solutions for a given synthetic transformation to new, high-complexity synthetic problems. This paradigm is typically most successful for solving problems where a rich history of relevant literature precedents and a thorough mechanistic understanding of the target reaction already exist. For example, a Suzuki reaction platform employing an array of known effective phosphine ligands, reaction solvents, and inorganic bases can be screened to uncover optimal substrate-specific reaction conditions with a high probability of success. However, many synthetic problems arise for which rationally designed platforms are not available, and cannot be designed owing to a lack of precedent or understanding. Nevertheless, microscale high-throughput experimentation (HTE) tools can be used to maximize the opportunity to serendipitously improve reaction performance, and several recent, high-profile reports have highlighted some initial progress in this area.1–8 Herein, we describe a broad-based, microscale additive-screening platform that was designed to minimize complexity and cost/time bottlenecks in the discovery of new ways to improve the performance for a wide variety of reactions. By using this approach, a single chemist was able to set up and analyze 475 different reaction conditions in a single day to identify new catalysts for the preparation of high-value pyrimidinone heterocycles, which are at the core of HIV Integrase inhibitors.9, 10 Follow-up investigations of identified conditions combined with quantum mechanical calculations led to the proposal of a single-electron transfer (SET) activation mechanism for this catalysis, thus illustrating how the pursuit of serendipitous solutions can sometimes lead to improved understanding. We envisioned creating a 96-well plate additives platform containing 95 different, highly practical pre-dosed compounds which might activate a reaction through a variety of different mechanistic pathways; there was one empty well for a comparative control reaction. The additives that were chosen are displayed in Figure S1 of the Supporting Information. Ideally, this reaction improvement engine would be inexpensive and simple to use, while providing results that would be a robust predictor of scale-up performance. To minimize reagent cost, we utilized very small 250 μL HPLC-vial inserts (microvials)11 as reaction vials, thus permitting reaction screening at approximately 20 μL of solvent (ca. 1 mg substrate per reaction). This is about 4–5 times smaller than the 1 mL vials that are the current HTE standard in industry and academic laboratories, and in our opinion represents the present lower limit that still enables robust chemistry development. The platform setup simply requires weighing out the substrates, reagents, and solvent into a single vial, then distributing this mixture to the additives plate using a pipettor, a process that only takes a few minutes to complete. Finally, after reaction incubation, we envisioned using MISER chromatography12, 13 for sample analysis, as this method enables an entire 96-well plate to be evaluated in about 1 hour, and produces a convenient graphical output that facilitates hit identification. We were interested in applying this platform to the synthesis of 2-substituted-5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxylic acid (pyrimidinone) derivatives of type 2 (Scheme 1), an important class of compounds with well-documented biological activity.9, 10, 14–16 Currently, the most effective approach for the synthesis of pyrimidinones employs a two-step process which involves a Michael addition of an N-hydroxy amidine to an acetylynic diester with a subsequent thermal dissociation/recombination event to afford the key pyrimidinone scaffold, typically in 15–50 % overall yield.15–20 This thermal rearrangement requires elevated temperatures (140 °C) to convert both E and Z isomers into product, thus leading to significant decomposition and low to moderate yields. To date, there has been no report of using catalysis to improve the yields of these reactions, or to decrease the reaction temperatures. Catalyst screening for the synthesis of pyrimidinone 2 a from Michael adduct 1 a using the additive platform. The pyrimidone core is highlighted in red. A MISER chromatogram for the 96 reactions run in 1,4-dioxane reveals which additives resulted in significant product formation (highlighted in green). To use the additives-screening platform for this project we first established, through temperature screening, that the highest temperature at which no pyrimidinone product 2 a was formed thermally from 1 a in a variety of solvents was 60 °C. The additive screening was carried out by dosing the substrate 1 a (2.5 μmol) and internal standard (0.25 μmol biphenyl) in 20 μL of five different solvents (o-xylene, 1,4-dioxane, methanol, NMP and DMF) to five of the 250 μL additives plates containing 20–50 % of the pre-dosed additives. Each reaction was then heated for 4 hours at 60 °C. Upon completion, the reactions were diluted with MeOH and the five plates were subjected to HPLC MISER analysis (see the Supporting Information).12, 13 Thus, with this platform and workflow in hand, 475 different reactions were evaluated with just 370 mg of material in a single day. Figure S1 in the Supporting Information shows the MISER chromatograms for the 96 reactions run in 1,4-dioxane. The reactions giving product are easily identified by the presence of a semiquantitative peak at the relevant mass. As expected, the control well (A01), which contains no additive, showed no product formation. Rewardingly, product 2 a was formed in significant quantities with a number of practical catalysts (Cu, Fe, Ni) and also several ligands. The assay yields (AY) of the successful leads were then determined by conventional quantitative HPLC analysis, with the best leads tabulated in Table 1. Well Additive 2 a AY % B08 FeCl2 32 B09 FeCl3 35 B10 Fe(acac)2 51 C05 NiCl2(PPh3)2 12 C06 CuCl 48 C07 CuCl2 20 C08 CuBr 44 C09 CuI 29 C10 Cu(acac)2 36 C11 dibromo-(1,10-phenanthroline)/CuII 74 D11 Cu powder <75 micron 34 H07 dppf 2 H09 XPhos 8 H11 phenanthroline 19 The best catalyst lead, dibromo(1,10-phenanthroline)/CuII (3; 50 mol %), from the initial screening in 1,4-dioxane afforded 74 % AY of the desired pyrimidinone 2 a (under noncatalyzed conditions at 140 °C, 50 % AY was obtained for product 2 a). We decided to further optimize this lead using multiparallel screening in 250 μL vials (Table 2). First, we found that the loading of 3 could be reduced from 50 mol % to 5 mol % in 1,4-dioxane without impacting the reaction yield (entries 1 and 2). Subsequently, we found that the dichloro analogue 4 performed identically to 3 (entry 3), which had limited commercial availability. We next evaluated several environmentally friendly solvents as alternatives to 1,4-dioxane (entries 4–8), and found that 2-methyltetrahydrofuran (2-MeTHF) and cyclopentylmethyl ether (CPME) were effective green replacements, with 2-MeTHF affording 2 a in 84 % AY (entry 8). We next evaluated the effects of temperature and loading with catalyst 4 on the reaction performance (entries 9–13). Reactions at 60 °C, 40 °C, and room temperature, at 5 mol % and 1 mol % catalyst loadings showed that the best reaction conditions in terms of yield, loading, and time were using 5 mol % of catalyst for 4 hours at 40 °C, thus giving 90 % HPLC AY (entry 10). Remarkably, overnight reactions at room temperature (entry 12) afforded complete conversion, and hence we had identified a practical catalyst that was able to reduce the reaction temperature by 120 °C. Entry Catalyst, loading T °C Solvent AY % 2 1 3, 50 mol % 60 1,4-dioxane 74 2 3, 5 mol % 60 1,4-dioxane 77 3 4, 5 mol % 60 1,4-dioxane 75 4 4, 5 mol % 60 CPME 79 5 4, 5 mol % 60 THF 56 6 4, 5 mol % 60 MeOH 40 7 4, 5 mol % 60 NMPa 42 8 4, 5 mol % 60 2-MeTHF 84 9 4, 1 mol % 60 2-MeTHF 74 10 4, 5 mol % 40 2-MeTHF 90 11 4, 1 mol % 40 2-MeTHF 78 12 4, 5 mol % RT 2-MeTHF 84 13 4, 1 mol % RT 2-MeTHF 70 The substrate scope for pyrimidinone formation was next explored with a series of electronically diverse and pyridine-containing Michael reaction adducts (1 b–g), each prepared by the method described by Culbertson (Table 3).20 The optimized procedure using catalyst 4 produced compounds 2 b and 2 c in excellent yields (entries 1 and 2). This catalyst also promoted the reaction with the remaining substrates 1 d–g at 60 °C, however the yields were not as high (entries 3–8). Entry Substrate Catalyst loading T °C Solvent 2 AY % 1 1 b 5 mol % 60 2-MeTHF 94 2 1 c 5 mol % 60 2-MeTHF 88 3 1 d 5 mol % 60 2-MeTHF 51 4 1 e 5 mol % 60 2-MeTHF 16 5 1 e 5 mol % 40 DCE 71 6 1 f 5 mol % 60 2-MeTHF 17 7 1 g 5 mol % 60 2-MeTHF 51 8 1 g 50 mol % 60 2-MeTHF 75 To improve the yields of 2 d–g, we next evaluated solvent, temperature, and catalyst loading effects on these substrates and found that the yield of compound 2 e could be improved to 71 % by using 5 mol % of 4 in 1,2-dichloroethane (DCE) at 40 °C (entry 5). Similarly, the yield of compound 2 g was improved to 75 % when we increased the loading of catalyst 4 back to 50 mol % (entry 8). However, we were unsuccessful in improving the yields for compounds 2 d and 2 f in this way.21 Also present in the initial screen of additives, were several ligands (dppf, XPhos, and phenanthroline), which gave some conversion of 1 a into 2 a. Follow-up experiments revealed that these ligands gave product slowly and did not turnover as catalysts. We hoped, however that we might be able to identify a ligand that could act as an effective catalyst, so we employed a pre-dosed microscale ligand-screening approach (again in 250 μL vials) with which we could rapidly evaluate 112 sterically and electronically diverse, mono- and bidentate achiral phosphine ligands. The phosphine screen was conducted for 4 hours at 60 °C in total reaction volume of 20 μL of 1,4-dioxane containing 2.5 μmol of 1 a and 5 mol % of ligand per reaction. In this way a single ligand, 1,1′-(ferrocenediyl)phenylphosphine (5) was identified to have significant catalytic activity. This ligand gave excellent conversion and 78 % HPLC AY of 2 a at a 5 mol % catalyst loading (see the Supporting Information). We then applied this ligand to the synthesis of compounds 2 a–g while also evaluating seven solvents, and found that each substrate had a different solvent that was optimal (see the Supporting Information). Most importantly, we were able to identify a new catalyst that afforded 2 f in a very high, 88 % AY (Scheme 2). High-yielding reaction conditions for the synthesis of compound 2 f using catalyst 5. With identification of two different, novel catalytic systems, using 4 and 5, for the synthesis of pyrimidinones we were interested in determining how these small-scale screening results would translate to a 0.4 mmol (160×) scale. Indeed, the small-scale assay yields proved to be highly predictive of scale-up performance, as demonstrated in Table 4. Conditions Aa Conditions Bb Microvials AY % Scale-up AY % Microvials AY % Scale-up AY % 2 a 90 94 (85) quant. 90 (88) 2 b 94 92 (83) quant. 99 (94) 2 c 88 83 (80) 94 74 (74) 2 d 51 55 (50) 49 34 (30) 2 e 71 71 (73) 41 44 (40) 2 f 17 25 88 quant. (91)c 2 g 75 77 (75)c 46 43 With these successful synthetic results identified through serendipity, we were interested in determining if we could establish a mechanism for the reaction. It was previously proposed and supported through density functional calculations, that the thermal synthesis of the pyrimidinones involves a direct NO cleavage to form a polar radical pair (PRP) with a substantial preference for recombination.22 With our new catalysts, we surmised that the Cu and Fe may be working to reduce the energy of the transition state through a SET reduction mechanism. Quantum mechanical calculations23 at the B3LYP/6-31G(d) level showed that indeed the dissociation and recombination of the radical anion should be much more facile than that of the neutral species (Figure 1). Energy profile for SET catalysis mechanism. B3LYP/6-31G(d) free energies calibrated with G3(MP2)B3 (ΔG, kcal mol−1) for the Z and E isomers (values for E isomers in parenthesis) in the gas phase. While copper has a well-documented legacy as a SET catalyst,24 we were interested in determining whether the ligand 5 also proceeded through SET activation or whether it proceeded through a new mechanism. Experimentally, we determined that ferrocene itself is not a catalyst in this reaction, and also that other ferrocenyl ligands (dppf, dippf, dtbpf) gave product but were significantly less reactive than 5 (see the Supporting Information). The calculated oxidation potential of the catalyst 5 (E0calc Fc(PPh)+/Fc(PPh)=−0.62 V in acetonitrile vs. Fc+/Fc) suggests that 5 may be a better reducing agent than ferrocene itself, thus supporting its role as a SET catalyst. We intend to study the specific catalytic activity in more detail and to pursue other potential applications. Herein, we demonstrated how a pre-dosed, microscale additives platform can enable a single chemist to evaluate 475 discrete reactions in a single day, thus providing a convenient and powerful “improvement engine” for organic synthesis. Employing this platform and subsequent microscale optimization techniques rapidly led to the identification of robust substrate-specific procedures for a range of biologically important pyrimidinones which are at the core of HIV Integrase inhibitors. We envision that similar, generally useful platform tools will soon become more widely available, thus dramatically impacting chemistry development and enabling increased access to chemical diversity and lower-cost synthesis. Most importantly, we believe that such platforms will lead to the discovery of new and potentially useful chemical reactivity and reaction mechanisms. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.