Modular, stereocontrolled Cβ–H/Cα–C activation of alkyl carboxylic acids

Significance The combination of two newly emerging methods for chemical synthesis enables access to molecular space that was previously challenging or impossible to access. Thus, a C–H activation of ubiquitous carboxylic acids followed by their decarboxylative functionalization provides modular access to difunctionalized carbon frameworks with distinctly controlled stereochemistry. Application of this strategy to simplify the synthesis of medicinally important entities and to discover potent antimalarial compounds is described.


General procedures
General Procedure A1: Asymmetric sp 3

A1.1: Installation of Directing Group
The amides were prepared according to a previously reported procedure. 1 The carboxylic acid (1 equiv.) was suspended in anhydrous CH2Cl2 (0.36 M) under argon atmosphere. Oxalyl chloride (1.2 equiv.) and N,N-dimethylformamide (0.06 equiv.) were added carefully to the mixture (gas evolution observed). Upon completion, monitored by TLC, the solution was concentrated in vacuo to afford the acid chloride, which was used immediately in the next reaction without characterization. Then, the acid chloride was added to a vigorously stirred solution of 2,3,5,6-tetrafluoro-4-(trifluoromethyl)aniline (ArFNH2) (1.1 equiv.) in toluene (1 M).
The reaction mixture was stirred for 12 hours under reflux, then it was cooled to room temperature and concentrated under vacuum, followed recrystallization in EtOAc:Hexanes to give the desired product.

A1.2: C-H Activation
Arylations were carried out according to a previously reported procedure. 2 The amide (1 equiv.), Pd(OAc)2 (10 mol%), ligand L * (12 mol%), and Ag2CO3 (2 equiv.) were weighed in air and placed in a sealed tube (100 mL) with a magnetic stir bar. To the reaction mixture, aryl iodide (2 equiv.) and HFIP (0.1M) were added. The reaction mixture was first stirred at room temperature for 10 minutes and then heated to 80 °C for 36 hours under vigorous stirring.
Upon completion, the reaction mixture was cooled to room temperature and filtered through Celite ® using CH2Cl2. The solvents were removed in vacuo and the resulting mixture was purified by a preparative TLC using Hexanes:EtOAc (5:1) or Toluene:EtOAc (30:1) as the eluent. In the 13 C NMR analysis, peaks that correspond to those of the polyfluoroarylamide auxiliary appeared as nearly invisible, complex sets of multiplets; they are omitted in the following spectroscopic analysis.

A1.3: Removal Directing Group
A sealable pressure flask was charged with MeOH (0.025 M) and amide. Et2O•BF3 (35 equiv.) was added and the reaction vessel sealed. The reaction mixture was heated to 100 °C for 12 hours under vigorous stirring. Upon completion, the reaction mixture was cooled to room temperature.
The solvent was removed in vacuo, H2O and EtOAc were added, organic layers were removed, and the aqueous layer was then extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated. Purification by column chromatography (Hexanes:EtOAc = 2:1 to 1:1) afforded the corresponding ester.
The ester was dissolved in THF (10 mL). The solution was cooled to 0 °C, and a cold solution of LiOH•H2O (2 equiv.) in H2O (10 mL) was added. The reaction was maintained at 0 °C for 1 hour.
The reaction was acidified with 2 M HCl and extracted with EtOAc. The combined organic layers were dried over MgSO4 and concentrated. The residue was purified by column chromatography (Hexanes:EtOAc:AcOH = 10:10:1).

A1.4: C-H Activation -free carboxylic acid
A5 was prepared according to a previously reported procedure. 3 A 2-dram vial equipped with a magnetic stir bar was charged with Pd(OAc)2 (4.4 mg, 10 mol%) and L4 (8.8 mg, 20 mol%) in HFIP (0.25 mL). The appropriate cyclopropanecarboxylic acid substrate (0.20 mmol), Ag2CO3 (82.7 mg, 0.30 mmol, 1.5 equiv.), Na2CO3 (31.8 mg, 0.30 mmol, 1.5 equiv.) and aryl iodide (0.40 mmol, 2 equiv.) were then added. Subsequently, the vial was capped and closed tightly. The reaction mixture was then stirred at the rate of 200 rpm at 80 °C for 16 hours. After being allowed to cool to room temperature, the mixture was diluted with EtOAc, and 0.1 mL of acetic acid was then added. The mixture was passed through a pad of Celite ® with EtOAc as the eluent to remove any insoluble precipitates. The resulting solution was concentrated, and the residual mixture was dissolved with a minimal amount of acetone and loaded onto a preparative TLC plate. The pure product was then isolated using preparative TLC with EtOAc:Hexanes (1:4 to 1:1) as the eluent and 1% v/v of acetic acid as the additive.

A2.2: C-H Activation
C-H arylation was preformed according to a previously reported procedure. 4 A sealed tube was charged with SI-1 (1.0 equiv.), dibenzyl phosphate (20 mol%), AgOAc (2.0 equiv.), aryl iodide (3.0 equiv.), and Pd(OAc) 2 (10 mol%). The reaction vessel was evacuated and refilled with inert atmosphere (N 2 , 3x). DCE (1.0 M) was then added and placed in an oil bath preheated to 110 ºC for 24 hours. Then, the reaction mixture was cooled to room temperature and the crude material was transferred to a round-bottom-flask using a 9:1 MeOH:H2O solution (8 mL of solution per 1 mmol reactant), followed by the addition of K2CO3 (3.0 equiv.). The mixture was allowed to stir at ambient temperature for up to 16 hours before solvent removal in vacuo. The resulting crude material was dissolved in CH2Cl2, filtered over Celite ® and concentrated in vacuo.
The resulting crude material was dissolved in 3:7 i-PrOH:CHCl3, washed with water (1x) and with brine (1x). The resulting organic layer was dried over Na2SO4, filtered, concentrated in vacuo before purification by flash column chromatography under the specified conditions.
The following arylated amides were prepared according to the General Procedure A2.2:

A2.3: Removal Directing Group
The carboxyl acid A6 was prepared according to a previously reported procedure. 4 The arylated product SI-2 (1.0 equiv.) was added to a round bottom flask and dissolved in MeCN (0.2 M). Boc2O (3 equiv.) was added, and the reaction mixture was heated to 50 ºC and stirred for 15 minutes, then DMAP (0.1 equiv.) was added, and the mixture was stirred for another 2 hours at 50 ºC. The solvent was removed in vacuo and the residue was dissolved in THF:H2O (2:1, 0.1 M).
After cooling the biphasic mixture to 0 ºC, 30% aq. H2O2 (10 equiv.) was added followed by LiOH (6 equiv.). The reaction mixture was allowed to reach room temperature, stirred for 1 hour and then heated to 50 ºC for 20 hours. The reaction mixture was separated, and the aqueous layer was washed with Et2O (3x). The resulting aqueous solution was acidified with 1 M HCl, exhaustively extracted with 3:7 i-PrOH:CHCl3 (5x) and dried. Concentration in vacuo gave the acid A6 as a solid which was used directly in the following step without further purification. The amides were prepared according to the previously reported procedure. 5 A round-bottom flask equipped with stir bar was charged with HOBt (1.2 equiv.), carboxylic acid (1 equiv.) and CH2Cl2 (0.2 M), after 5 minutes stirring at room temperature, EDCI·HCl (1.2 equiv.) was added and the solution was stirred for further 72 hours. The reaction mixture was diluted with CH2Cl2, aq. sat. NaHCO3 was added and the aqueous layer was extracted with CH2Cl2 (3x), dried over Na2SO4 and purified by column chromatography.

A3.2: C-H Activation
The arylated compounds were prepared according a previously reported procedure. 5 A sealed tube was charged with amide (1.0 equiv.), AgOAc (2.0 equiv.), aryl iodide (3.0 equiv.) and Pd(OAc) 2 (10 mol%). The tube was flushed with argon and sealed (for tetrahydrofuran scaffolds)*, then placed in an oil bath preheated to 110 °C and stirred for 38 hours. The reaction mixture was then allowed to cool down to room temperature and EtOAc was added. The resulting solution was filtered through a pad of Celite ® , eluting with further EtOAc (2x). The solvent was removed in vacuo, and the crude material was purified by flash column chromatography.

A3.3: Removal Directing Group
The carboxyl acids were prepared according to the previously reported procedure. 5 A culture tube equipped with a stir bar was charged with the arylated compound (1.0 equiv.), DMAP (3.0 equiv.) and MeCN (1 M), followed by dropwise addition of (Boc)2O (20.0 equiv.) at room temperature. Then, the tube was placed in an oil bath preheated to 110 ºC for 1 hour. The mixture was then cooled to room temperature and another equivalent of (Boc)2O (10.0 equiv.) and DMAP (3.0 equiv.) was added and the reaction mixture stirred at 70 ºC overnight. The culture tube was then allowed to cool to room temperature and the solvent concentrated in vacuo. The residue was purified by Silica gel column chromatography to afford the Boc-protected intermediate.
To a solution of Boc-amide (1.0 equiv.) in THF:H2O (0.1 M, 3:1) were added LiOH•H2O (2.0 equiv.) and 30% H2O2 (5.0 equiv) at 0 ºC. The reaction mixture was stirred for 20 minutes at 0 ºC, then was allowed to warm up to room temperature and stirred for 18 hours. After completion, the mixture was extracted with Et2O to remove the organic impurities, the aqueous layer was acidified with 1M aq. HCl to pH=3 and extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo to afford the carboxylic acid, that was used in subsequent steps without further purification. aq. NaOH. Then the reaction mixture was stirred at room temperature overnight. After completion, organic impurities were removed by extracting with Et2O. The aqueous layer was acidified to pH = 1 with 3 M HCl, then extracted with EtOAc (3x), dried over Na2SO4 and concentrated in vacuo to afford the desired product 13, which was used without further purification.
The organic layer was separated, and the aqueous layer was further extracted with EtOAc (3x).
The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. Flash column chromatography (Silica gel, Hexanes:EtOAc) afforded SI-3.

A4.3: Removal Directing Group and synthesis of RAE
To a solution of compound SI-4 (1.0 equiv.) and DMAP (3 equiv.) in MeCN (0.08 M) was slowly added (Boc)2O (20 equiv.) at room temperature. The reaction mixture was stirred at 70 ºC for 6 hours. After cooling to room temperature, the reaction mixture was concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, 2:1 Hexanes:EtOAc) to yield compound SI-5 as a brown oil.
To a solution of compound SI-5 (1.0 equiv.) in THF:H2O (0.08 M, 3:1) were added LiOH•H2O (2 equiv.) and 30 % H2O2 (5 equiv.) at 0 ºC. The reaction mixture was stirred for 20 minutes at 0 ºC, then allowed to warm up to room temperature and stirred for 18 hours. After completion, the reaction was extracted with Et2O to remove the organic impurities and then the aqueous layer was acidified with 1 M HCl to PH=6 and extracted with EtOAc (3x). The combined organic layers were washed with brine (3x), dried over Na2SO4 and concentrated in vacuo to afford A14 as a white solid, which was used without further purification.

General Procedure B: Synthesis of Redox-Active Esters
Redox-active esters were prepared according to the previously reported procedure. [6][7][8][9]11,[13][14][15] A round-bottom flask or culture tube was charged with (if solid) carboxylic acid (1.0 equiv.) and N-hydroxytetrachlorophthalimide or N-hydroxyphthalimide (1.0 -1.1 equiv.). CH2Cl2 was added (0.1-0.2 M), and the mixture was stirred vigorously. Carboxylic acid (1.0 equiv.) was added via syringe (if liquid). DIC (1.1 equiv.) was then added dropwise via syringe, and the mixture was allowed to stir until the acid was consumed (determined by TLC). Typical reaction times were between 0.5 and 12 hours. The mixture was filtered (over Celite ® , Silica gel, or through a fritted funnel) and rinsed with additional CH2Cl2:Et2O. The solvent was removed in vacuo, and purification by column chromatography afforded the desired redox-active ester.

General Procedure C2: Nickel-catalyzed Negishi Alkylation
The Nickel-catalyzed Negishi alkylation of redox-active esters was performed according to the previously reported procedure. 8 Please see this reference for graphical supporting information.

Preparation of Alkyl Zinc Reagents from Alkyl Bromides
Two round bottom flasks were flame-dried and allowed to cool under vacuum. Both flasks were then backfilled with argon from a balloon. One flask was charged with Mg turnings (1.5 equiv.) and I2 (0.01 equiv.

General Procedure C3: Nickel-catalyzed Negishi Alkenylation
The Nickel-catalyzed Negishi alkenylation of redox-active esters was performed according to the previously reported procedure. 9 Please see this reference for graphical supporting information.

Synthesis of Alkenylzinc Reagents from Alkenyl Grignard Reagents
From commercial Grignard reagents (5 mmol scale) The following Grignard reagent was purchased as solutions in THF from Sigma-Aldrich.
ZnCl2 (1.0 equiv.) and LiCl (1.25 equiv.) were flame-dried under vacuum. Upon cooling, the flask was placed under argon atmosphere. Anhydrous THF (1 M) was added, and the mixture was stirred vigorously and gently heated with a heat gun until all ZnCl2 and LiCl dissolved. At this time, commercial alkenyl Grignard reagent (1.0 equiv.) was added slowly via syringe to the ZnCl2 and LiCl solution. Upon complete addition, the mixture was stirred for at least 15 minutes before use.
The transmetallation was assumed to occur in quantitative yield.

Synthesis of Alkenylzinc Reagents from α,β-Unsaturated Alkenyl Bromide or Iodide
The following procedure was developed based on that of Knochel and coworkers. 10 A flask was charged with LiCl (2.0 equiv.) and Zn dust (2.0 equiv.). The flask was placed under vacuum and gently dried with a heat gun. Upon cooling, the flask was placed under argon atmosphere. THF (1 M) was added, and the mixture was stirred vigorously. TMSCl (0.05 equiv.) was added dropwise, and the mixture was heated to reflux with a heat gun. The mixture was then allowed to cool to room temperature, 1,2-dibromoethane (0.05 equiv.) was added dropwise, and the mixture was heated to reflux with a heat gun. Upon cooling, the flask was placed in a room temperature water bath. Next, if using an α,β-unsaturated alkenyl iodide the substrate (1.0 equiv.) was added dropwise. If using an α,β-unsaturated alkenyl bromide, the substrate was added in one portion via syringe. The mixture was then stirred at ambient temperature for 1 hour resulting in a green or red colored solution. Note: If the color does not change within 5-10 minutes, it is likely that the zinc insertion reaction did not proceed. If this is the case, gently heat the reaction mixture with a heat

General Procedure C4: Nickel-catalyzed Negishi Arylation
The Nickel-catalyzed Negishi arylation of redox-active esters was conducted according to a previously reported procedure. 11 Please see this reference for graphical supporting information.

Preparation of ZnCl2 Solution (1.0 M in THF)
A Schlenk flask equipped with stir bar was flame-dried and allowed to cool to room temperature under vacuum. The flask was backfilled with nitrogen, and ZnCl2 (1 equiv.) was added. The flask was placed under vacuum and heated in an oil-bath preheated to 150 ºC. After stirring for 2-12 hours, the flask was removed from the oil bath and allowed to cool to room temperature. The flask was backfilled with nitrogen, and THF (1 M) was added. The mixture was vigorously stirred until all ZnCl2 was dissolved (approximately 12 hours). However, if necessary, the solution can be used before all solids have completely dissolved without a decrease in yield of the subsequent reaction.

Preparation of the Arylzinc Reagents
Phenyl Zinc reagent was prepared according General Procedure C4, from the commercially available phenylmagnesiumgrignard reagent purchased as solutions in THF from Sigma-Aldrich.
Arylzinc reagent SI-36 was prepared from SI-34 by a method developed by Knochel. 12 To an ovendried culture tube backfilled with argon (3x) was added SI-34 (1 equiv.) and THF (0.3 M). Then a solution of iPrMgBr•LiCl (1.3 M in THF, 1 equiv.) was added dropwise at −60 ºC. After stirring for 30 minutes, iodo-lithium exchange was completed (as determined by quenching an aliquot with water and confirming the disappearance of the aryl iodide by 1 H NMR). Then, a ZnCl2 solution (1.0 M in THF, 1 equiv.) was added to the mixture at −60 ºC. After stirring for 10 minutes at −60 ºC, the culture tube was put into an ice bath. The solution of ArZnCl•LiCl was stirred until it became clear and was titrated with I2 before being used for the coupling reaction.

Titration of Arylzinc Reagents
LiCl (5 equiv.) was added to a septum-containing screw-capped culture tube equipped with a stir bar. The culture tube was flame-dried under vacuum and cooled under flow of argon from a balloon. The cap was removed, I2 (1.0 equiv.) was quickly added to the culture tube, and the cap was replaced. The exact amount of I2 added was recorded. Anhydrous THF (0.1 M) was added, and the mixture was stirred for 5 minutes to afford a dark brown solution. A 1.00 mL syringe was filled with approximately 0.90 mL of ArZnCl•LiCl, and the solution was added dropwise via the syringe. The exact volume of ArZnCl•LiCl solution in the syringe was recorded (titration start point). Over the course of the titration the color changes from dark brown to light brown to yellow to colorless, indicating complete consumption of I2, and upon consumption of I2, the titration end point was recorded. The concentration of the ArZnCl•LiCl solution was then calculated. Typical concentrations of arylzinc reagents ranged from 0.18 M to 0.24 M in THF.
The following alkylzinc reagents were used according to General Procedure C4:
The volume of DMF used was calculated based on the titration of the arylzinc reagent solution.
DMF (anhydrous) was added via syringe, and the mixture stirred for 2 minutes at room temperature for solid TCNHPI-esters. TCNHPI-ester (if liquid) was dissolved in DMF (anhydrous) and added to the culture tube containing NiCl2•glyme or NiCl2•6H2O (20 mol%) and ditBuBipy L5 (40 mol%). The mixture was stirred for 2 minutes at room temperature. Then, arylzinc reagent in THF (3.0 equiv.) was added in one portion, and the mixture was stirred for 12-16 hours at room temperature. The mixture was diluted with EtOAc or Et2O and quenched with 1 M HCl (aq.). The reaction can also be quenched with H2O or half-saturated aq. NH4Cl solution for acid-sensitive substrates. The organic layer was washed with H2O and brine, dried over anhydrous Na2SO4, and concentrated in vacuo (CAUTION: some of the products are volatile). The crude material was purified by silica gel column chromatography or preparative TLC (pTLC).

General Procedure C5: Nickel-catalyzed Negishi Ethynylation
The Nickel-catalyzed Negishi ethynylation of redox-active esters was conducted according to a previously reported procedure. 13 Please see this reference for graphical supporting information.

Preparation of Ethynylzinc Chloride Solution
A flame-dried tube was charged with of freshly prepared 1 M ZnCl2/LiCl THF solution (2 equiv.), then ethynylmagnesium bromide (1.0 equiv., 0.5 M THF solution, Sigma-Aldrich) was added dropwise at room temperature. The resulting solution was stirred at room temperature for 30 minutes until it became homogeneous. The resulting ethynylzinc solution was then titrated with I2 before use.

Procedure for the Ni-catalyzed Decarboxylative Ethynylation
A culture tube was charged with TCNHPI redox-active ester (1.0 equiv.) and a stir bar.

S55
The Iron-catalyzed Kumada alkynylation of redox-active esters was conducted according to a previously reported procedure. 13 Please see this reference for graphical supporting information.

Preparation of Substituted Alkynylmagnesium Bromide Solution
A flame-dried tube was charged with alkyne (1.0 equiv.) and anhydrous THF (1 M). After that, freshly titrated ethylmagnesium bromide (1.0 equiv.) THF solution was added dropwise at room temperature. Note: gas was generated during the addition. The resulting solution was stirred at 50 ºC for another 30 minutes. After cooling down to room temperature, the resulting Grignard solution was titrated with I2 before use.
The following alkynylgrignard reagents were used according to General Procedure C6:

Procedure for the Fe-catalyzed Decarboxylative Alkynylation
A culture tube was charged with TCNHPI redox-active ester (1.0 equiv.), FeBr2•H2O (20 mol%) and a stir bar. The tube was then evacuated and backfilled with argon from a balloon.

General Procedure C7: Nickel-catalyzed Boronic Ester Suzuki Cross-Coupling
The Nickel-catalyzed Boronic Ester Suzuki of redox-active esters was conducted according to a previously reported procedure. 14 Please see this reference for graphical supporting information.

Preparation of Nickel/ligand Solution
A culture tube charged with NiCl2•6H2O (1 equiv.) and diOMeBipy L8 (1.36 equiv.) was evacuated and backfilled with argon three times. DMF (0.05 M) was added and the resulting mixture was stirred at room temperature overnight to afford a pale green suspension.

Preparation of B2pin2/MeLi-Complex Solution
A flame-dried culture tube was charged with B2pin2 (1 equiv.) under argon atmosphere and anhydrous tetrahydrofuran (1 M) was added. A solution of MeLi (1.6 M in Et2O,1 equiv.) was added at 0 ºC and the resulting mixture was stirred at room temperature for 1 hour before use.

Procedure for the Ni-catalyzed Decarboxylative Cross-Coupling
A culture tube charged with TCNHPI redox-active ester (1.0 equiv.) and MgBr2

General Procedure C8: Nickel-Catalyzed Giese Conjugate Addition
The Nickel-catalyzed Giese Conjugate addition of redox-active esters was conducted according to a previously reported procedure. 15 Please see this reference for graphical supporting information.
A culture tube was charged with LiCl (3.0 equiv.). Note: Due to its hydroscope nature, LiCl can be difficult to weigh on small scale. In our experience excess LiCl is not detrimental to the success of the reaction. Next, NHPI RAE (1.0 equiv.), Zn powder (2.0 equiv.), and Ni(ClO4)2•6H2O (20 mol%) were added. A stir bar was added, and the culture tube was evacuated. The tube was backfilled with argon from a balloon, and Michael acceptor (2.0 equiv.) was added via syringe. To the reaction mixture was added MeCN (0.4 M), and the mixture was stirred overnight at room temperature. After at least 12 hours, a H2O and sat. aq. NH4Cl solution (1:1) were added. The mixture was extracted with EtOAc or Et2O (3x), and the organic layer was dried over MgSO4. The crude was purified by Silica gel flash column chromatography or preparative TLC (pTLC) to afford the desired compound.  How do I monitor the reaction?

Answer:
We use TLC analysis with UV visualization and staining (KMnO4) to monitor the reaction.
Usually, it takes 24 hours for completion of the C-H activation step.

Question 2:
Can I run the C-H activation reaction in round bottom flask adapted with condenser instead of sealed tubes?
Answer: For large scale reaction (>2 g substrate) on azetidine heterocycle scaffolds, the reaction was carried out using a round bottom flask adapted with condenser without impacting the yield.

Question 3:
Does the enantiopurity of my compounds erode under hydrolysis conditions?
Answer: No, we transformed the carboxylic acid products into the corresponding RAEs and determined their ee by chiral HPLC. The RAEs have the same the ee compared with the corresponding C-H activation products, which means enantiopurity doesn't erode under hydrolysis conditions

Question 4:
Why are different equivalents of Boc2O required for Boc-protection of aminoquinoline group?
Answer: This is due to different steric hindrance of arylated heterocycle. Four and five memberring heterocycles possess higher reactivity than six member-ring towards Boc-protection, which causes different loading of Boc2O in the Boc-protection step.

Question 5:
Is it possible to decrease the loading of Pd catalyst in C-H activation?
Answer: In order to obtain high yield for the C-H activation step, we usually used 10 mol% Pd loading. However, for some highly reactive substrates such as pyrrolidine, the catalyst loading S59 could be decreased to 5 mol% affording the product in comparable yield.

Question 6:
Why was only arylation achieved for C-H activation step?

Answer:
We have performed some other transformation such as alkenylation, alkynylation, fluorination on heterocyclic substrates. Although the C-H activation reaction proceeded well, the following removal of directing group turns out to be problematic either because of the steric hindrance or elimination of HF to afford the corresponding unsaturated amide directing group byproduct.

Answer:
For the decarboxylative cross-couplings conducted in this manuscript, the yields were in general higher starting from TCNHPI redox-active esters compared to NHPI redox-active esters.
Moreover, the high crystallinity of the TCNHPI redox-active esters facilitate their purification and offer the possibility to upgrade the enantiomeric excess by recrystallization. In general, better enantioenrichment was achieved with the solvent system (Hexanes:EtOAc) compared to (CH2Cl2:MeOH).

Question 2:
Does the enantiopurity erode under certain DCC conditions?

Answer:
We have compared the ee of final products obtained after the DCC reaction with the corresponding RAEs. Both have comparable ee indicating that enantiopurity doesn't erode under DCC conditions.

Question 3:
I want to synthesize a large quantity of a decarboxylative cross-coupling product. Can I increase the concentration in order to lower the amount of solvent?

Answer:
When working on a larger scale we typically increase the concentration to 0.5-1 M without any detrimental effect on the yield. However, Suzuki couplings are very sensitive to concentration and should always be conducted under standard conditions.

Question 4:
Suzuki reactions seem to be the most convenient to setup, are there any limitations in terms of substrate compatibility?
Answer: Although Suzuki reaction is among one of the most robust reaction that we have developed, it still has some limitations. For example, alkyl boronic acids are completely unreactive for this reaction. For some heterocycles, such as pyrazole, imidazole, we need to use the corresponding Bpin esters instead of boronic acids.
The resulting solution was filtered through a pad of Celite ® , eluting with further EtOAc (2 x 30 mL

Experimental Procedures and Characterization Data for the synthesis of BRD8468 analogs (Figure 5-b)
Compound SI-44 16

Compound 76
Route A:
The organic layers were combined and dried over Na2SO4, filtered and concentrated in vacuo to afford the desired compound 83 (1.5 mg, 98%) as a colorless oil.

Instrumentation
Parasitized erythrocytes in screening media, and SYBR Green lysis buffer are dispensed using a MultiFlo from BioTek. Compound transfer is carried out by using the Labcyte ECHO Acoustic Liquid Handler. SYBR Green fluorescence is read on an Envision Multimode Reader, and parasitemia is determined using a light microscope (Primovert) with a Zeiss A-plan 100´/1.25 Oil Immersion objective.

Plasmodium falciparum propagation and parasitemia determination
Parasitized erythrocytes from each respective strain are passaged when parasitemia levels reach 8-10% via dilution into fresh culture medium to a final concentration of 1% parasitemia.
Cultures are maintained in 10 mL volumes in 25 cm 2 tissue culture flasks (Fisher Cat. # 10-126-39). The freshly diluted culture is gassed with a low oxygen mixture (96% nitrogen, 3% carbon dioxide, 1% oxygen) and incubated at 37˚C. Complete media is either added or exchanged every 1-2 days and parasitemia is monitored every 1-3 days, as needed, depending on the growth kinetics. The percent of parasitemia is estimated by obtaining a 1 µL blood smear. The smear is fixed onto the slide by placing in methanol for 30 seconds, stained in 10% Geimsa stain, and the percent of parasitized erythrocytes vs uninfected erythrocytes is determined by microscopy with a light microscope.
Maintenance requires daily media changes and fresh blood every two weeks. Cultures are gassed for approximately 30 seconds to 1 minute using a blood gas mixture to maintain a gas composition of 96% nitrogen, 3% carbon dioxide and 1% oxygen and incubated at 37°C.

Plasmodium falciparum cultured in 1536-format
A MultiFlo (BioTek) is used to dispense parasitized erythrocytes, uninfected erythrocytes and RPMI screening media into 1536-well plates (789092-A by Griener Bio-One) in a final volume of 8 µL containing 0.3% parasitemia and 2.5% hematocrit. The plates are incubated for 72 hours at 37˚C in a gas chamber in the presence of the low oxygen blood gas mixture.

Assay Protocol
In brief, cultures of the respective P. falciparum strains to be used for screening are prepared with screening media (complete media without human serum but supplemented with 0.5% Albumax II) and fresh erythrocytes. Compounds are transferred via the Labcyte ECHO Acoustic Liquid Handler into the assay plates. Parasitized erythrocytes and fresh erythrocytes are prepared with screening media and dispensed into the assay plates containing compound for a final parasitemia of 0.3% and 2.5% hematocrit. The assay plates are directly transferred and cultured in a gas chamber at 37˚C in the presence of the low oxygen blood gas. After 72 hours and daily gas exchanges, the assay plates are removed from the incubator and SYBR Green lysis buffer is added S238 to each well using the MultiFlo. Plates are incubated for an additional 24 hours at room temperature for fluorescence signal development. Fluorescence intensity is read on an Envision Multimode Reader.

Preparation of compound stocks and dilutions
Compounds are solubilized from powders in 100% DMSO at a final concentration of 10 mM and stored at room temperature by the Calibr Compound Management Group (CMG). Compound dose-response activity plates were prepared by adding 30 µL 10 mM stock solution to columns 1 and 13 in a 384-well compound plate. Compounds were transformed into 12-point , 1:3 dilutions by serially transferring 10 µL of the prior well to 20 µL DMSO.

Treatment of parasitized human erythrocytes with compound
Compounds are transferred directly into the 1536-well assay plates using the Labcyte ECHO Acoustic Liquid Handler, followed by the addition of a mixture of parasitized human erythrocytes and fresh uninfected human erythrocyte in screening media.

Plasmodium falciparum invasion of human erythrocytes
Based on the measured parasitemia levels, each culture is added to screening media to yield a final parasitemia of 0.3% and 2.5% hematocrit blood. The dispense step is performed with a MultiFlo and the assay plates are immediately covered using a custom metal lid and placed in a gas chamber purged with a low-oxygen gas mix and incubated at 37°C. The total assay volume is 8 µL.

Determination of Plasmodium falciparum proliferation by nucleic acid detection
After a 72-hour incubation (equivalent to 1-2 cycles during the blood stage) at 37°C under 95% humidity, a prepared mixture of the lysis buffer (5 mM EDTA, 1.6% Triton X-100, 20 mM Tris-HCl, 0.16% Saponin) in water and SYBR Green detection (0.1%) reagent is dispensed at 2 µL per well using the MultiFlo. Cultures are incubated for an additional 24 hours at 25°C before measuring fluorescence intensity using the Envision with a 505 Dichroic Bottom Mirror.
Excitation and emission filters are 485 nm and 530 nm, respectively.

Data analysis
Data are normalized based on maximum fluorescence signal values for DMSO-treated wells (no inhibition by compound) and the minimum fluorescence signal values for wells containing the highest concentration of inhibitor control compounds, for example, atovaquone at a final concentration of 12.5 µM. Data are analyzed on a plate-by-plate basis and compared to reference compounds that are always included on every plate, typically artemisinin, mefloquine and S239 atovaquone. The half-maximal effective concentration (EC50) values are obtained using the GeneData curve fitting model. The standard logistic regression model is applied for curve fitting.
The quality of the assay run is assessed by the performance of the reference compounds where the EC50 must be within 3-fold of the standard reference values for the assay plate to pass requisite data quality needs. The z-factor for this assay ranges from 0.5-0.8. Additionally, all compounds are typically assayed in duplicate (independent assay plates) and EC50 values ideally must not vary by more than two-fold between plates.