Article pubs.acs.org/OPRD
Process Development of a Potent Neuroprotector Agent: Collismycin A Javier López-Ogalla,* Gonzalo Saiz, and Francisco E. Palomo Noscira S.A., Avenida de la Industria 52, 28760 Tres Cantos, Madrid, Spain S Supporting Information *
ABSTRACT: An efficient synthetic process for the natural product of marine origin, collismycin type A, a potent neuroprotector agent, has been developed. This new synthetic route avoids chromatographic steps, implies an improvement cost, and provides easy access to large scale.
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INTRODUCTION Collismycins are 2,2′-bypiridine molecules which have been isolated from Streptomyces species, by Gomi et al. Their structures were elucidated by spectral analyses and chemical conversion.1 Collismycins exhibit several biological activities, such as antifungal activity against some species such as Saccharomyces cerevisiae and Candida albicans.2 Gomi et al. have shown that some collismycins present cytotoxicity against P388 mutine leukaemia cells, and Shindo et al. have suggested that collismycin A and its isomer B could have an anti-inflammatory activity.3 Noscira has also described that collismycin exhibits protective properties against oxidative stress in cells and may therefore be useful for treating diseases or conditions induced by oxidative stress, especially neurodegenerative diseases.4 Among them Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most frequent progressive neurodegenerative diseases affecting millions of people in the world.5 Therefore, an interesting approach for developing new pharmaceutical compounds for treating neurodegenerative diseases may be the design of compounds which inhibit cellular oxidative stress.
material. Collismycin A is obtained after eight steps with an overall yield of 7%, being necessary to perform a total of four chromatographic purifications along the synthetic route (Scheme 1). Scheme 1
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The synthesis starts from 4-methoxy-2,2′-bipyridine N-oxide (2), which can be easily prepared from 2,2′-bypiridine by a known three-step sequence.7 The synthesis involves functionalization at carbon in position six of compound 1. Metalation of 4-methoxy-2,2′-bipyridine N-oxide using LDA at −70 °C and BrCN as electrophile is undertaken in order to obtain a bromine N-oxide. This molecule is subsequently reduced with PBr3, leading to 6-bromo-4-methoxy-2,2′-bipyridine (3). In a second sequence of reactions, the obtained brominebipyridine is subjected to another metalation under the same conditions (LDA at −70 °C), but using methyl disulfide as electrophile to introduce a methylthio moiety at C-5 after chromatography.8 To reach the target molecule, collismycin A, the functionalization of C-6 is carried out through a bromine− lithium exchange strategy. The chelate BuLi-TMEDA performs this exchange, and the obtained lithium derivative is then
ANTECEDENTS AND RESULTS The collismycin A can be obtained from natural sources. Usually, collismycins A and B (Figure 1) are obtained by fermentation with cultures of Actinomycetes strains WAS 1410.2 Nevertheless, the main disadvantage of this procedure is the limitation in yield and product isolation. The first total synthesis of collismycin A was described by Trécourt et al.,6 by using 2,2′-bipyridine (1) as starting
Figure 1. 2,2′-Bipyridine compounds structurally similar to collismycin described in the literature. © XXXX American Chemical Society
Received: November 2, 2012
A
dx.doi.org/10.1021/op3003129 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Table 1. Conditions used in the N-oxidation and nitration
quenched in the presence of DMF to give the aldehyde 4. Reaction of this aldehyde with hydroxylamine leads to collismycin A. Nevertheless, repetition of this synthesis in order to scale up to a multigram scale was very complicated. In this context and taking into account the therapeutic interest of collismycin A, we considered the need for developing a reproducible synthesis leading to the product in multigrams with acceptable performance and with a purity of 99.5% without performing chromatographic purifications. Thus, we have developed a synthetic route that leads to collismycin A with an overall yield above 20% (21% including all stages, and 43% if the stage equivalent to Trecourt’s sequence is not considered) with a purity of the final compound of 99.5% (Scheme 2). This procedure also leads to new intermediate bipyridine derivatives useful for obtaining new analogues of interest within the family of collismycin.9
A
reagent
%
B
reagent
%
1 2 3
H2O2, AcOH CH3CO3H, CHCl3 mCPBA, CH2Cl2
65 65 82
1 2
HNO3/H2SO4 KNO3/ H2SO4
61 40
Scheme 3
Scheme 2
C-4 position of bipyridine, HNO3 or KNO3 in H2SO4 were used as nitrating reagents, and the HNO3/H2SO4 mixture gave the best results in the synthesis of 4-nitro-2,2′-bipyridyl N-oxide (6). Then, we proceeded to the replacement of the nitro group by the methoxy group following the same methodology of Trecourt’s group which uses anhydrous sodium methoxide in methanol. Under these conditions the 4-methoxy-2,2′-bipyridyl N-oxide (2) was obtained without any chromatographic purification, with a 49% overall yield over the three stages. In order to ensure the safety of the nitrate intermediate 6, a calorimetric study was performed. As shown in Figure 2, the decomposition of the compound releases a moderate energy at a temperature of 250 °C, well above the reaction temperature in the synthesis of methoxy derivative 2. We continued with the process in order to prepare an intermediate selectively leading to methylthio in the bipyridine ring A (Scheme 4). In contrast to the methodology used by Trecourt, we decided to synthesize compound 9 which bears a diisopropylamide group instead of a Br at the C-6 position of the bipyridine, thus avoiding in this way the formation of product mixtures during the introduction of the electrolyte S2Me2 and as a result avoiding chromatographic purification of the product. Thus, the required nitrile was prepared by cyanation of compound 2 following the methodology described by the group of Antonini et al.10 This methodology is performed in one step, using diethyl cyanophosphonate as reagent. The introduction of the nitrile group at C-6 evolves toward reduced bipyridine 7, which precipitates in the reaction medium and is isolated by filtration in high yield. With the intention of developing a safe process, the reaction was monitored with indicator strips11 of cyanhydric acid without observing any liberation of cyanhydric gas. The hydrolysis of nitrile 7 was performed in an aqueous basic medium of 4 N sodium hydroxide, to give the sodium carboxylate derivative 8 in quantitative yield. This sodium carboxylate was used in the next reaction step without neutralizing the corresponding carboxylic acid, due to the
The first three steps of the synthetic sequence are the same as those of the route of Trecourt et al. Therefore, initial efforts were focused on developing a scalable route to the nitro Noxide intermediate 6, which has been reported by other authors.7 The intermediate 6 was synthesized on a 1 kg scale, and it was necessary to optimize the N-oxidation reaction and subsequent nitration at C-4 to obtain the nitro derivative 6 (Table 1 and Scheme 3). H2O2, CH3CO3H, or mCPBA were used as reagents for the formation of the mono N-oxide product. The best results were obtained by using mCPBA in CH2Cl2 to give 2,2′-bipyridyl Noxide (5) with yields greater than 80%. For the nitration at the B
dx.doi.org/10.1021/op3003129 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Figure 2. Differential scanning calorimetry (DSC) of compound 6.
Scheme 4
results obtained from the titration study. This titration showed that the pyridine nitrogen has a higher pKa than the sodium carboxylate, so that pyridines are protonated before the corresponding carboxylic acid. On this basis we decided to continue without neutralizing the sodium carboxylate (Figure 3). The amidation reaction was performed in one pot, synthesizing first the acid chloride followed by subsequent addition of diisopropylamine to afford the amide 9. The overall yield of the last three steps is 81% (Scheme 4). The last steps in the synthesis include different key stages. One of these key steps consists of selectively functionalizing the C-5 in ring A of the bipyridine. The reaction was performed by using dimethyl disulfide as electrophile and n-BuLi as base at −78 °C. The reaction was monitored by HPLC−MS, being necessary to stop the reaction at −78 °C to avoid formation of subproducts. The thiomethylated compound 10 was isolated with a 95% yield and 92% purity, sufficient to continue with the synthesis. The reaction was performed by using, in every case, n-BuLi 1.6 M as base and THF as solvent, making variations in the number of equivalents of base, the volume of the solvent used, and the reaction temperature (Table 2). It was observed that the best conditions for thiomethylation consisted of utilizing 1.3 equiv of n-BuLi 1.6 M in hexane at −78 °C. The volume of the solvent used in the reaction does not appear to be a critical
Figure 3. Titration of compound 8. The experiment was performed by comparing the titration curves of 2,2′-bipyridine (1) (A) with compound 8 (B). Both compounds were dissolved in a mixture MeOH/HCl 0.1 N (80:20 v/v) as titrant, using an aqueous solution of 0.1 N NaOH.
parameter, and therefore 11 vol of solvent were employed. The yield of this step was between 87% and 95%. Then, diisopropylamide 10 was reduced to the corresponding aldehyde 4 by using DIBAL-H as reducing agent at low temperature (Scheme 5). A small study was carried out about C
dx.doi.org/10.1021/op3003129 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
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CONCLUSION A new efficient and scalable process for the synthesis of collismycin A has been developed. The process can be performed on multigram quantities for preclinical study phases. This new protocol avoids all chromatographic purifications of synthetic intermediates and final product, thus optimizing the isolation of compounds by washings or crystallization. This new methodology appears to be a viable route to obtain the compound on kilogram scale. Future work on this new process will focus on finding ways to avoid the steps requiring cryogenic conditions.
Table 2. Reaction conditions for thiomethylation of compound 9 amount 7 (g)
entry
base (equiv)
THF (vol)
T (°C)
yield (%)
1
0.5
1.1
10
−78
70
2
1.0
1.5
10
−78
70
3
1.0
1.5
10
−78 to rt
NA
4 5 6 7 8
1.0 10.0 63.0 125.2 138.0
1.3 1.3 1.3 1.3 1.3
10 10 16 11 11
−78 −78 −78 −78 −78
92 87 87 95 95
Article
comments unclean reaction; chromatography unclean reaction; chromatography unclean reaction; chromatography clean reaction clean reaction clean reaction clean reaction clean reaction
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EXPERIMENTAL SECTION All commercial reagents were used as supplied unless otherwise indicated. The progress of the reaction and analysis of the final compounds were performed by HPLC and thin layer chromatography in comparison with reference substances. Organic solutions were concentrated and/or evaporated to dryness under vacuum in a water bath (
