Parallel Synthesis Technologies in Lead Discovery and Optimization

Chapter 10

Parallel Synthesis Technologies in Lead Discovery and Optimization: Strategies and Applications

Downloaded by PENNSYLVANIA STATE UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: November 23, 2004 | doi: 10.1021/bk-2005-0892.ch010

Robert J. Pasteris Stine-Haskell Research Center, DuPont Crop Protection, P.O. Box 30, Newark, DE 19714

Combinatorial and parallel synthesis technologies have become an integral part of many discovery research organizations. This paper will discuss DuPont's entry into this field and how our strategies to capture the best value from these technologies have evolved. Applications to both lead discovery and lead optimization programs will be presented with examples taken from DuPont's fungicide, herbicide and insecticide programs. Technologies illustrated include parallel solid phase and solution phase synthesis, mixture synthesis, multi-component reactions and the design and use of combinatorial libraries. The use of contract research organizations as a way to leverage internal resources will also be discussed.

© 2005 American Chemical Society In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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110 In the early 1990's, combinatorial chemistry and high throughput screening technologies were evolving rapidly and prompted new ways of thinking about discovery processes. DuPont Crop Protection was interested in exploiting these new technologies and partnered with our Central Research and Development organization to develop a core competency in these areas. Our approach was to explore and develop high throughput synthesis methods via this partnership and integrate selected technologies into our crop protection discovery programs via a combinatorial chemistry core team. The core team's focus centered on applications to hit and lead optimization and targeted discovery programs generating small, focused libraries of well characterized discrete compounds for in vivo testing. The team leveraged their capabilities by establishing and managing external collaborations to design and produce larger, diverse discovery libraries directed towards hit and lead generation. By taking this approach, DuPont Crop Protection was able to assess a wide range of new chemistry technologies and their value to lead generation and optimization programs while avoiding large internal capital investment. This paper reviews a few selected examples of applications of these technologies highlighting solution phase, solid phase and mixture techniques, which helped speed the discovery process.

Scytalone Dehydratase Inhibitors via a focused mixture strategy In order for Magnaporthe grisea to infect rice plants, it must melanize an infection structure to penetrate the leaf surface (1), thus making inhibition of fungal melanin biosynthesis an attractive approach for preventing rice blast disease. Scytalone dehydratase (SD) catalyzes the dehydration of scytalone and vermelone in this pathway (2). Figure 1 shows the structures of carpropamide (3) and diclocymet (4), two recently commercialized blasticides which inhibit this enzyme and compound 1, a proprietary class of potent SD inhibitors discovered by DuPont (5).

carpropamid

diclocymet

1

Figure I. Selected scytalone dehydratase inhibitors Inspection of these inhibitors show that they are all amides with the amine portion being highly conserved while the acid portion contains greater structural

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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diversity. As a complement to DuPont's SD structure-based design program, a target-focused mixture library strategy was implemented where a diverse set of commercially available acid chlorides were reacted with an equimolar mixture of five optimized amine moieties known to be tight binders at the SD binding site. The reactions were carried out in parallel using afiltrationblock and a basic amine resin to bind the HC1 formed in the reaction. Filtration and concentration of each reaction well gave the desired equimolar mixture of five amides. The mixtures were assayed for their level of SD inhibition and the most active mixtures were deconvoluted by synthesis of the five individual amides and assayed to identify the inhibitor structures. Thus, 3,4-dichlorobenzoyl chloride was the acid component which gave the most active mixture in this study. Deconvolution showed that almost all the activity was derived from the 3,3diphenylpropylamide component of that mixture. This compound 2 (Figure 2) was a 22 nM inhibitor of SD, but showed poor greenhouse level activity, most likely due to its high logP value. Replacement of the dichlorophenyl ring with the more hydrophilic dichloropyridine ring gave compound 3, a 1 nM enzyme inhibitor with good greenhouse activity against rice blast disease.

2 A = CH 3 A=Ν

4

Figure 2. Selected SD inhibitors identifiedfrom a mixture strategy The success of this mixture strategy with 131 commercially available acid chlorides prompted us to repeat this approach using DuPont's proprietary inhouse acid collection from which a subset was selected based on knowledge of the nature of the binding site. Again, mixtures of five amides were prepared, assayed and deconvoluted to identify novel potent scytalone dehydratase inhibitors such as cyclobutane carboxamide 4. Compound 4 exhibited a Kj of 0.026 nM against the enzyme (6) and had excellent greenhouse and field activity against rice blast disease. As shown by these examples, the use of small focused mixture libraries was found to be a highly effective parallel synthesis approach by providing new, potent enzyme inhibitors while eliminating over 75% of the synthesis and testing effort that would have been required if each compound was prepared and tested individually.


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Dihydropyridine Miticide Lead Optimization The dihydropyridine 5 depicted in Figure 3 was purchased from a compound broker and found to be highly miticidal in greenhouse testing. This lead compound is prepared by a novel three-component reaction process involving an aniline, a ketone and the electron deficient olefin l,l-dicyano-2,2bis(trifluoromethyl)ethylene (BTF) (7). The chemistry is run most conveniently with two equivalents of BTF at room temperature in a water miscible solvent where addition of water precipitates the product. The first equivalent of BTF acts as a dehydrating agent to drive formation of an enamine intermediate, which adds across the double bond of the second BTF molecule, and ring closes onto one of the cyano groups to give the observed product. H C ^CH 3

X

3

5

Figure 3. Synthesis of lead compound 5 The high level of diversity possible in each of the three components coupled with our desire to rapidly develop a structure activity relationship and identify potential field candidates led us to choose a positional scanning approach to narrow each reactant set. We would then do combinatorial crosses of the best amines with the best carbonyl components with the best olefins to identify the optimally active analogs. The synthesis was carried out in parallel using solution phase liquid handling methods and filtration blocks to collect the precipitated products. Conceptually, the acetone component could be replaced with any carbonyl compound containing an adjacent methylene group which will undergo enamine formation. Carbonyl variations, which we found to readily undergo this reaction with 4-chloroaniline and BTF in dry acetonitrile, are aliphatic linear, branched and cyclic ketones, pyruvates, β-ketoesters, β-ketosulfones and 1,3-diones. Biacetyl did not provide product under these conditions. Aldehydes and acetophenones required pre-formation of the enamine intermediate prior to BTF addition and tended to proceed in lower yields. Eighty examples were chosen to test steric, electronic and lipophilic properties. In cases where the carbonyl component was unsymmetrical and could produce two isomeric enamines, multiple dihydropyridine products were formed.


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4-Chloroaniline was replaced by one hundred and fifty ortho, meta and/or para substituted anilines using acetone as both the carbonyl component and the solvent. Again, substituents were chosen to test steric, electronic and lipophilic properties and all gave smooth product formation. Five- and six-membered heterocyclic amines were also explored and tended to give lower yields of less pure products. In some cases, die only product isolated derived from direct addition of the aminoheterocycle to BTF. Aliphatic amines failed in this reaction even using pre-formed enamines. Replacement of the BTF component was less productive. Dicyanoethylenes derived from methyl or ethyl pyruvate do not form stable adducts with 4chloroaniline in the presence of acetone (7). Dicyanoethylenes derived from trifluoroacetophenones orfromchloral reluctantly gave addition products. Many other BTF replacements failed. The structure activity relationship developed for these dihydropyridines showed that optimal activity was derived from BTF, using anilines containing a halogen, cyano or CF group in the para position and small aliphatic and cyclic ketones and acetophenones. 3

Structures isoelectronic with the presumed enamine intermediate in this chemistry (7) were found to react readily with one equivalent of BTF in dry acetonitrile. As shown if Figure 4, amidines, guanidines, isoureas and isothioureas all gave the corresponding dihydropyrimidines at room temperature and showed good to excellent miticidal activity in greenhouse tests (8). Amide oximes required heating to form the BTF adduct and were not miticidal. Cyanoamidines would not condense with BTF. R^NH A r '

N

R^N^NH * -NH

H

Ar

Ar

Amidines

Guanidines

RjO^NH A

.NH

Ar

Isoureas

R^^NH ..NH

Ar

Isothioureas

R1^NH n

2

.N

R 0 2

Amide oximes

Figure 4. Dihydropyrimidines obtained via BTF condensations


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The positional scanning approach and parallel synthesis methods used in this optimization program were found to be highly effective, allowing a wide range of structure variations to be explored and the structure activity relationships to be quickly defined. The best set of aniline components were reacted with the best set of carbonyl components using BTF as the olefin to give a combinatorial array of products from which the field candidates in Figure 5 were chosen and tested against a range of economically important mite species.

Figure 5. Insecticidefieldcandidates.

N-Azoyl Phenoxypyrimidine Herbicide Lead Optimization Carotenoid biosynthesis has long been a target for herbicide discovery with many commercial herbicides acting at various enzyme targets along this pathway. DuPont discovered a class of phytoene desaturase inhibitors where an azole was linked to a phenoxypyrimidine via a carbon-nitrogen bond (9). Compounds such as 6 (Figure 6) showed excellent preemergent and early postemergent herbicidal activity against broadleaf and grassy weeds with wheat safety (10). Both parallel solution phase and solid phase synthesis methods were used to help define the structure activity relationships in this active area.

MCPBA DCM 75-85%

Figure 6. Solution phase synthesis of compound 6


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As shown in Figure 6, a regioselective synthesis route was developed where the more reactive 4-chloro group of compound 7 was first replaced by a methylthio blocking group. Introduction of the trifluoromethyl pyrazole heterocycle in the 2 position proceeds smoothly. Oxidation of the methylthio group converts it into a methylsulfonyl group, which is readily displaced by nucleophiles, such as phenols, to give the desired compounds. The last step has conveniently been carried out in parallel by using a strongly basic ion exchange resin to give clean products by simple filtration. Eighty examples were prepared by this method. This synthetic approach was modified to the traceless linker method outlined in Figure 7. A simple, inexpensive, high load thiol resin was easily prepared by heating a high load aminomethyl polystyrene resin 11 with γ-thiobutyrolactone for a few hours in toluene. The resulting thiobutyramide resin (TBA resin) (11) is a free-flowing, shelf-stable material which does not require protection of the thiol function and maintains its active thiol titer after a year of storage at room temperature in a sealed container.

|^ Η Ν

2

6 Toluene

1 1

1) Dl-CI-Pyrimidine. DlEA, DMF

P ^ N ^ ^ " ° , TBA resin

2) Azole, DBU. DMF 3) MCPBA, EtOAc

Figure 7. Solid phase synthesis method Sequential reaction of the TBA resin with a 2,4-dichloropyrimidine and an azole followed by MCPBA oxidation gives the highly fimctionalized resin bound reactive intermediate 12. Reaction of 12 with phenols not only introduces the third diversity component, but also releases the final product 13 from the resin. By using a combination of these solution and solid phase methods, the structure activity relationships were quickly developed and field candidate selection and patent exemplification was facilitated.


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Oxazolidine Scaffold-Based Discovery Library A proven method for discovery library synthesis is via a scaffold approach. The scaffold is a core structure or template upon which diverse functionality is appended. The scaffold should be small relative to the appended functionality and should be constructed by reliable chemistry from readily available inputs. Oxazolidines fit this profile - they are compact, heterocyclic rings easily constructed by treatment of an aldehyde or ketone with an amino alcohol. The ring nitrogen can be further substituted by reaction with acid chlorides, isocyanates, etc., providing a highly functionalized molecule. An efficient, robust solution phase synthesis protocol was developed to prepare many compounds based on this scaffold in high yield and purity. Figure 8 outlines the synthesis used to prepare a 2500 compound oxazaspirodecane microtiter plate based sublibrary. T s 0 H

η „,/ 2

3A Sieves or TMOF/CHjCN

o-V V

Isocyanate or Acid Chloride P-DIEA

9^

R2

R1

Parallel Synthesis Technologies in Lead Discovery and Optimization

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