Uppsala University

Project Thesis

Suzuki coupling of functionalized arylboronic acids to a 2-Amino-5-(4-bromophenyl)-1,3,4-thiadiazole scaﬀold

Supervisors: Author: Dr. Wei Berts Fredric Ingner Dr. Jonas Malmström

February 21, 2015 Abstract Seven arylboronic acids, all but one containing functional groups, were chosen for Suzuki cross coupling to a predetermined scaffold for a contract project. The scaffold was synthesized and catalytic ability was assesed for three palladium catalysts: Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), (1,1’- Bis(diphenylphosphino)ferrocene)palladium(II) dichloride (Pd(DPPF)Cl2) and PEPPSI-iPr. Library reactions proceeded with Tetrakis since it was found to be the only catalyst capable of producing product during trials. All reactions were worked up by flash chromatography, except one where preperative HPLC had to be used. Reactions and purification steps were analyzed crudely with TLC and more thoroughly through LC-MS with ESI. Structural analysis in form of 1H-NMR was made of the scaffold and two products. It was found that all but one of the seven reactions proceeded to completion with 0.1 eq of catalyst under alkali conditions at 80o C.

1 Acknowledgements I would like to thank Dr. Fredrik Lehmann for allowing me to do my thesis work at OnTarget Chemistry. I am very grateful to have been supervised by Dr. Wei Berts and Dr. Jonas Malmström, without whom this thesis would not have been possible. I also extend my gratitude to all of the employees of OnTarget Chemistry who have made me feel very much at home and as a part of the team. Last but not least, I would like to thank my family, friends and girlfriend for showing me great support through adversity and rough times.

2 Contents

1 Introduction 4 1.1 Suzuki coupling ...... 5 1.2 General mechanism ...... 6 1.2.1 Ligand dissociation and oxidative addition ...... 6 1.2.2 Transmetalation ...... 7 1.2.3 Reductive elimination ...... 8 1.3 Aim ...... 8

2 Experimental 9 2.1 Materials and method ...... 9 2.1.1 Preparation of the scaﬀold 3a ...... 9 2.1.2 Catalyst screening ...... 9 2.1.3 Substrate scope - General method ...... 10 2.1.4 Substrate scope - Puriﬁcation ...... 10

3 Results and discussion 12 3.1 Scaﬀold synthesis ...... 12 3.2 Catalyst screening ...... 12 3.3 Library reactions ...... 13 3.4 General discussion ...... 21 3.5 Conclusions ...... 22

Appendices 25

I Puriﬁcation techniques 25 I.I Flash chromatography ...... 25 I.II Preparative HPLC ...... 25

II Preperative HPLC results 26

III HPLC results 27

IV NMR results 37

3 1 Introduction

There exists a steady demand for new pharmaceutical and other tailored organic molecules. A key factor in order to meet this demand in a sustainable and economic way is to efficiently develop and produce these molecules. Metal catalysis has proven to be an important tool in this development and can often be favored to stoichiometric "old fashion" C-C bond forming reaction in terms of selec- tivity and efficiency. In metal catalyzed reactions it can however be more difficult to foresee the outcome of a reaction and an empirical approach is often taken, as will be done in this project.

The molecules synthesized in this paper are intended for a contract project. No further information regarding the project can be disclosed due to conﬁdentiality agreement.

In this paper, seven selected boronic acids will be coupled to a scaffold through C- C bond formation. The reaction of choice is a palladium-catalyzed reaction commonly referred to as the Suzuki cross coupling reaction. A general reaction depicting the scaffold and selected boronic acids are shown below in Figure 1 and Figure 2. A more detailed explanation of Suzuki cross coupling and the reaction mechanism is found in section 1.1. Br R OH OH N + B Pd(PPh3)4/K2CO3(aq) N N N S R DME S 1c-n H2N H2N 3a 2c-n Figure 1: General reaction between scaffold and boronic acid substrate F F

OH O OH OH F OH B B B B OH O OH OH OH 1c-1 1c-2 1c-3 1c-4

OH OH OH B Cl B B N OH OH OH

1c-5 1c-6 1c-7 Figure 2: The seven arylboronic acids 1c-n selected for coupling

The main tasks of the project will focus on synthesis of the scaﬀold, ﬁnding a suitable catalyst and substrate scope.

4 1.1 Suzuki coupling Suzuki coupling, also known as Suzuki-Miyaura coupling or the Suzuki reaction, is a palladium catalyzed reaction in which a halide is coupled to an organoboronic com- pound, commonly an boronic acid or ester, thus resulting in the formation of a new C-C bond.

The Suzuki cross coupling reaction was ﬁrst presented in 1979 by Akira Suzuki and Norio Miyaura[12]. In 2010, Akira Suzuki was, along with Richard F. Heck and Ei-ichi Negishi, awarded the nobel prize in chemistry "for palladium-catalyzed cross couplings in organic synthesis"[15]. Today, the Suzuki reaction is the most utilized cross coupling reaction in both academic and industrial applications[6].

Suzuki coupling is widely used in drug development since it allows for a broad selection of functionalized groups whereas most other organometallic reactions, for example Grignard reactions[8], will attack reactive functionalized groups. Though there are other d-metal coupling reactions which also have a large tolerance for functional groups, for example the Stille coupling[19], Suzuki coupling is often prefered due to a number of following reasons. Suzuki coupling reactions, in contrast to other d-metal coupling reactions, are usually carried out under mild conditions[8]. Studies have shown satisfying reaction condition with water as solvent[5][20][4] which paves for a greener route than the use of organic solvents, this is of great importance for industrial scale application. Boronic acids and their by-products are considered non-toxic and easy to work- up which makes them preferable to, for example, organotin compounds used in Stille coupling. One of the great beneﬁts of using Suzuki cross coupling is that boronic acids are readily available in an extensive variety of structures[6][22].

5 1.2 General mechanism The Suzuki cross coupling reaction can be summarized as a three-step catalytic cycle which is shown in Figure 3. The reaction steps and other mechanistic aspects will be disclosed in the following paragraphs.

Figure 3: Catalytic cycle of the Suzuki cross coupling reaction

1.2.1 Ligand dissociation and oxidative addition As previously mentioned, the Suzuki reaction uses a palladium based catalyst. There are several catalysts available, all containing different ligands with different structures and properties. In general, a ligand should be a good σ-bond donor since this facilitates oxidative addition, the first step in the catalytic cycle. A ligand should also be bulky since this benefits reductive elimination, the last step in the cycle[7].

This paper will focus on a catalyst called Tetrakis(triphenylphosphine)palladium(0), Pd(PPh3)4, which is the most commonly used catalyst[13].

In the ﬁrst step of the catalytic cycle, oxidative addition, the halide is bonded to the palladium complex thus oxidizing the palladium. However, in order for oxidative addition to take place, a non-occupied coordination site is needed[23]. This is achieved through ligand dissociation where the loss of ligands are in equlibrium with the solution[3], see Figure 4.

6 Figure 4: Ligand dissociation and oxidative addition with oxidation states[3]

The figure shows how palladium transforms from a four-coordinate saturated 18 electron confirmation to a two-coordinate unsaturated 14 electron confirmation. The unsaturated complex contains two available coordination sites which enables for oxidative addition of the halide. The result is the stable trans-σ-palladium(II) complex which is also shown between the first and second step in Figure 3[13].

1.2.2 Transmetalation

During the transmetalation step the halide on palladium is substituted with the R2 group of the boronic acid. Although several diﬀerent routes have been proposed, a full explanation of the mechanism is yet to be found. It is however known, empirically, that a base needs to be added in order for the transmetalation step to proceed at a notable rate. There are two suggested main routes describing the role of base[1], these are shown in Figure 5 below.

Figure 5: Two main pathways describing the role of base in transmetalation[2]

7 In Path A, the base forms an negatively charged "ate" complex with the boronic acid. Boric acid and the halide anion is then excluded in the transmetalation step when the R2 switches from the boronic acid and binds to the palladium. In Path B, the base instead substitutes the halide on the palladium before undergoing transmetalation. The halide anion is thereby excluded already in the ﬁrst step. Boric acid is then excluded during the transmetalation step.

1.2.3 Reductive elimination Reductive elimination is the last step in the catalytic cyle. This step forms the ﬁnal desired C-C bond between R1 and R2 thus completing the cross coupling reaction. An illustration of the reductive elimination step is shown below in Figure 6.

Figure 6: Reductive elimination step

When the R-groups are ejected, palladium regains an electron pair. This lowers the oxidation state for palladium from Pd(II)→ Pd(0). The catalyst is thereby regenerated with two available coordination sites.

John P. Wolfe and Jie Jack Li describes in their book Palladium in Heterocyclic Chem- istry under which conditions reductive elimination works best. The following is a direct quote from Chapter 1 of the book[9]:

"The reductive elimination step can often be facilitated by the use of catalysts bear- ing bulky, monodentate phopshine ligands, and is believed to be most rapid when the two coupling partners have opposite electronic properties."

As described, bulky monodentate phosphine ligands is found in the catalyst "Tetrakis" which will be compared to other catalysts later in this paper. The opposite electronic properties of the selected boronic acids will, subsequently, also be tested due to their diﬀerent functionalizations.

1.3 Aim The aim of this project is to ﬁnd a general synthetic route for the coupling reactions and to examine whether the reactions shows any discriminatory eﬀects that might be linked to functionalization of the boronic acids.

8 2 Experimental

2.1 Materials and method Recorded MS spectrum used electrospray ionisation with positive fragmentation. The alkali HPLC methods (B1090X and SX1097X3)* used a NH4HCO3 (pH 10) buffer solution and is run on a Xterra C18 column, 3.5 µm pore size. Acidic method used 0.1% TFA buffer and is run on an ACE C8 column. Columns had same dimensions and flow-rate (50x3.0mm, 1mL/min) and both used acetonitrile to reduce polarity. Purity is determined at 305nm (SX1097X3), HPLC spectrums are recorded at 220nm (B1090X) if nothing else is stated.

*B1090X consists of a 2.5 minute run with a gradient reaching from 10% to 90% acetonitrile. SX1097X3 takes 4 minutes and has a gradient from 10% to 97% acetonitrile.

Flash chromatography used 40-63 µm 60 A silica gel. Preparative HPLC was performed with an Glison preperative HPLC system using an ACE, C8, column (21.2x100 mm, 25mL/min). Fractions were collected at λ=305 nm for peak signals of 100 mV or higher. Gradient starts with 20% at 1 minute and reaches 50% after 12 minutes. At 12 minutes, the column was washed with 100% acetonitrile and fractions were collected for an additional 6 minutes. Reactions were continuously followed with TLC.

A brief explanation of the puriﬁcation techniques used can be found in Appendix I.

2.1.1 Preparation of the scaffold 3a 4-bromobenzoic acid (1a) (4.0g, 20mmol) and thiosemicarbazide (2a) (1.8g, 20mmol) were added to a roundbottom flask. POCl3 (6mL) was added. A syringe filled with CaCl2 was placed through a septa on a condenser and the reaction was refluxed for 1h at 75oC. After cooling, water (6mL) was added and the reaction was further refluxed at 110oC for 4h. After cooling, NaOH (10-15mL, 50%) was added untill pH reached 8. The reaction mixture was filtered through celite, rinsed with cold water and then recrystallized in ethanol to obtain the final product 2-Amino-5-(4-bromophenyl)-1,3,4- thiadiazole (3a) as a fluffy white solid with a needle-like structure. Yield (batch 1): 2.2g, 43%. ESI [M+H]+=257 m/z. Retention time (HPLC): Product 3a at 1.372 min. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.76 (m, J= 8,4 Hz, 2.0 Hz, 2H), 7.73 (m, J= 8,4 Hz, 2.0 Hz, 2H), 7.53 (br s, 2H)

2.1.2 Catalyst screening 1.5 eq of (2,4-dimethoxyphenyl)boronic acid (1b) (100mg, 0.590mmol) was added to three seperate test tubes. Dimethoxyethane (4mL), 2M K2CO3 (200µL) and 3a

9 (100mg, 0.390mmol) was added to each test tube. 0.05 eq catalyst (0.02mmol) was added under stirring, one catalyst for each of the three test tubes. The tubes were left o + to react under stirring at 80 C over weekend. For Pd(PPh3)4: ESI [M+H] =314 m/z

2.1.3 Substrate scope - General method 1.5 eq of each boronic acid 1c-n (0.59mmol) was added to seven individual test tubes along with dimethoxyethane (4mL), 2M K2CO3 (200µL) and 1 eq of 3a (0.39mmol). 0.1 eq of Pd(PPh3)4 (45mg, 0.039mmol) was added under stirring and the tubes were left to react under stirring at 80 oC over night. The resulting mixture was ﬁltered through celite, rinsed with small amounts of MeOH and DCM, and then rotavapored yielding a solid. The products were then puriﬁed according to section 2.1.4.

2.1.4 Substrate scope - Puriﬁcation

3 Preparation of 2c-1: The solid was dissolved in approximatly 4 mL of DMSO, diluted with 1 mL of acetonitrile and a few drops of 1% triflouroacetic acid buffer. Purifica- tion was made through a Gilson reversed-phase preperative HPLC system using water/acetonitrile + 0.1% TFA. 20-50% acetonitrile gradient from 1-12 min, 25mL/min flow, collecting at λ=305 nm. Final product was exsiccated under vacuum. Yield: 12.0mg, 10%. Purity: 89.4%. Retention times (HPLC, acidic method B1090A): Prod- uct 2c-1 at 1.430 min. The substrate 1c-1 can not be found in the spectrum and does not produce any fragments in MS (rt=0.489 min for B1090X reference). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.06 (m, 2H), 7.89 (m, 2H), 7.87 (m, 4H), 3.88 (s, 3 H).

Preparation of 2c-2: The solid was dissolved in DCM + 0.1% TEA by drop-wise addition of MeOH. The product 2c-2 was puriﬁed by ﬂash chromatography using DCM+ 0.1% TEA with a MeOH gradient of 1.2-2.0% with 0.4% increments every 250mL. Upon reaching 2.0%, an additional 250mL 3% MeOH eluent was used. Final product was exsiccated under vacuum. Yield: 13.1g, 11%. Purity: 98.8%. Product 2c-2 mass fragment: ESI [M+H]+=310 m/z. Retention times (HPLC): Product 2c-2 at 1.824 min. Substrate 1c-2 at 1.519 min.

Preparation of 2c-3: The solid was dissolved in DCM + 0.1% TEA by drop-wise addition of MeOH. The product 2c-3 was puriﬁed by isocratic ﬂash chromatography using DCM+ 0.1% TEA with 3.0% MeOH. Final product was exsiccated under vacuum. Yield: 13.5g, 11%. Purity: 82.4% (or 95.4%, see result section). Product 2c-3 mass fragment: ESI [M+H]+=322 m/z. Retention times (HPLC): Product 2c-3 at 1.649 min. Substrate 1c-3 at 1.245 min.

Preparation of 2c-4: The solid was dissolved in DCM + 0.1% TEA by drop-wise addition of MeOH and was then rotavapoured onto silica. The product 2c-4 was puri-

10 ﬁed by ﬂash chromatography. First by using 250mL 2.0% MeOH (DCM+ 0.1% TEA.) and then with an additional 250mL 3.0% MeOH. Final product was exsiccated under vacuum. Yield: 49.8mg, 50%. Purity: 97.8%. Product 2c-4 mass fragment: ESI [M+H]+=254 m/z. Retention times (HPLC): Product 2c-4 at 1.513 min. Substrate 1c-4 at 0.884 min.

Preparation of 2c-5: The solid was dissolved in DCM + 0.1% TEA by drop-wise addition of MeOH and was then rotavapoured onto silica. The product 2c-5 was puri- ﬁed by ﬂash chromatography using DCM + 0.1% TEA with a MeOH gradient of 1.2%, 1.5% and 1.7% with 250mL eluent each. Final product was exsiccated under vacuum. Yield: 50.8mg, 43%. Purity: 97.5%. Product 2c-5 mass fragment: ESI [M+H]+=302 m/z. Retention times (HPLC): Product 2c-5 at 1.720 min. The substrate 1c-5 can not be found in the spectrum (reference time at 1.370 min).

Preparation of 2c-6: The solid was dissolved in DCM + 0.1% TEA by drop-wise addition of MeOH and was then rotavapoured onto silica. The product 2c-6 was puri- ﬁed by ﬂash chromatography. First by using 250mL 2.0% MeOH (DCM+ 0.1% TEA.) and then with an additional 250mL 3.0% MeOH (DCM + 0.1% TEA). Final product was exsiccated under vacuum. Yield: 41.9mg, 40%. Purity: 74.0% (or 98.2%, see result section). Product 2c-6 mass fragment: ESI [M+H]+=268 m/z. Retention times (HPLC): Product 2c-6 at 1.624 min. The substrate 1c-6 at 1.175 min. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.81 (m, J = 8.6 Hz, 2H), 7.74 (m, J = 8.6 Hz, 2H), 7.62 (m, J = 8.6 Hz, 2H), 7.42 (br s, 2H), 7.30 (m, J = 8.6 Hz, 2H), 2.35 (s, 3H).

Preparation of 2c-7: The solid was dissolved in DCM + 0.1% TEA by drop-wise addition of MeOH. Purification by flash chromatography was attempted using DCM + 0.1% TEA with a MeOH gradient of 0-0.6%, increasing MeOH content with 0.2% every 200mL eluent. Product fractions were collected, rotavapoured onto silica and the column was re-run with 0.2-1.2% MeOH gradient, 0.2% steps every 200mL. Product- containing fractions were left to evaporate whereupon only trace amounts of product could be found. The column was flushed with 2.0% MeOH (incl. DCM + 0.1% TEA) which showed that residual product was still present. No product was able to be retrieved and purity was not determined. Product 2c-7 shows mass fragment for ESI [M+H]+=278 m/z. Retention times (HPLC): Product 2c-7 at 1.318 min. Scaffold 3a at 1.352 min. Substrate 1c-7 at 0.35 min (no visible MS-fragments).

11 3 Results and discussion

3.1 Scaffold synthesis The scaffold 2-Amino-5-(4-bromophenyl)-1,3,4-thiadiazole (3a) was synthesized according to the experimental procedure listed in section 2.1.1, Figure 7, by cyclization from 4-bromobenzoic acid (1a) and thiosemicarbazide (2a) aided by POCl3. After crystallization from ethanol the product was isolated in 43% yield for the first batch. The product was analyzed with LC-MS and NMR, see Appendix III: Figure 11 and Appendix IV: Figure 29 respectively. A second batch was produced, yield was not determined. Product purity was never determined although NMR and LC-MS data shows no notable contaminations. Br O S OH POCl N NH2 3 N + H2N NH 75oC S Br 2a H2N 1a 3a Figure 7: Reaction for the preparation of the scaffold 3a

3.2 Catalyst screening

The suitability of the three catalyst Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), (1,1’-Bis(diphenylphosphino)ferrocene)palladium(II) dichloride (Pd(DPPF)Cl2) and PEPPSI- iPr was examined by a trial cross coupling reaction between the scaﬀold 3a and 2,4- Dimethoxyphenylboronic acid (1b). The three reactions were analyzed with LC-MS after one hour. Spectra for Pd(PPh3)4 and Pd(DPPF)Cl2 are found in Appendix III: Figure 12-13, (The spectrum of PEPPSI-iPr is identical with the spectrum of Pd(DPPF)Cl2 and will not be displayed.) It was found that only Pd(PPh3)4 managed to produce the sought after product 2b. The spectrum of Pd(PPh3)4 shows residues of both starting materials 3a and 1b which indicates that the reaction had not gone to completion. The reaction was therefore con- tinued to run over weekend and were then analyzed with LC-MS. However, no diﬀer- ences could be found between the spectrum of the one-hour run compared to weekend run. The mass fragment of 279 m/z in the Pd(PPh3)4 spectrum belongs to the oxidized ligand specie triphenylphosphine oxide and will be consistent throughout the substrate scope.

12 H3C O CH3 Br O H3C O CH3 O N Pd.Cat./K2CO3(aq) N N S + HO B DME OH N H N S 2 1b 3a H2N 2b

Figure 8: Reaction of choice for the trial of diﬀerent catalysts[16][17][18]

Pd(PPh3)4 was selected as the only suitable catalyst. The catalyst molar equivalence was doubled to 0.1eq to ensure that conversion was not limited by the amount of catalyst and the experiment was repeated once more for Pd(PPh3)4. The reaction was followed with LC-MS which indicated that the reaction had come to a halt after two hours, still with residual 3a and 1b. No attempt of puriﬁcation was made and neither yield nor purity was examined for the trial reactions.

3.3 Library reactions Results of the library reactions are summarized in Figure 9 where the amount of recovered product, purity and yields are listed for all entries.

Several of the reported yields in the following section are believed to be severely biased due to issues during puriﬁcation. No internal standards were used during recording of the LC-MS spectra in the library reactions which makes it diﬃcult to assess the credibility of the experimental yields. An estimated yield is calculated in order to compensate for these errors. This estimated yield is based on the ratio of product to ligand intensity thus acting as a crude substitute for an internal standard. However, some assumptions are required before these yields can be used in discussion of the results, these are listed below.

It has to be assumed that all products have the same absorbency at the measured

13 wavelength since accounting for the diﬀerences in absorption between products require referenced absorption data of the products, which is not available. The samples are assumed to be perfectly homogeneous so that ligand to product ratio remains consistent throughout the solid samples. This is important in order to obtain an accurate representation of the full sample from the small quantity analyzed with LC-MS. Additionally, the ligand concentration is assumed to be constant between all samples and all products are considered to be fully dissolved during the measurements.

A point of reference, a reaction, has to be chosen from which the estimated yields of the other reactions can be decided. The synthesis of 2c-1 is chosen as reference point since it was purified through preparative HPLC and can therefore be regarded to have the least amount of human bias. The product 2c-1 of the first entry was produced by cross coupling of the scaffold 3a and substrate 4-methoxycarbonylphenylboronic acid (1c-1) according to the experimental procedure listed in section 2.1.3. The product was, after reacting overnight, isolated by preparative HPLC in an 10% yield according to the purification procedure listed in 2.1.4. Reaction mixture and purified product was analyzed by LC-MS, see Appendix III: Figure 14-17. Spectrum of the preparative HPLC run can be found in Appendix II: Figure 10. Isolated product was additionally analyzed with NMR, see Appendix IV: Figure 30, and purity was determined by LC-MS to 89.4%.

It is evident from the reaction mixture spectrum, Figure 14, that the product 2c- 1 merges with the ligand peak under alkali conditions, method B1090X. However, as shown in Figure 15-16, it was possible to isolate the product peak under acidic conditions, method B1090A. Subsequently, isolation through preparative HPLC was performed under acidic conditions. A small injection of the sample, ~0.5 ml, was required for a trial run in order to set the parameters for the preparative HPLC. The isolated product recovered from the trial run was saved and later added to the larger batch of isolated product. Despite optimization, as shown in Figure 10, the product peak is trailing and merging with a peak of unwanted material which causes losses in yield. This, combined with solubility issues during sample preparation, is believed to be the main reason for the poor experimental yield of 10%. An indication of the experimental yield being severely biased is that the LC-MS spectrum of the reaction mixture does not show any residual starting material 3a. Since neither the LC nor MS spectrum display any evidence of that side-reactions, pronounced enough to substantiate the major loss in yield, has taken place it is assumed that the starting material 3a has mainly been converted to the sought after product 2c-1. The possibility of the scaﬀold 3a participating in any reaction yielding products that are not visible in the LC spectrum nor produces visible MS fragments can be discarded due to the relative stability and conjugation of 3a. It can therefore be assumed that the low experimental yield is not due to low conversion but to other factors such as handling errors, solubility issues and several product

14 containing fractions being discarded due to unsatisfying separation. The same reasoning will be applied in the discussion of 2c-2 to 2c-6, that is all but 2c-7, since none of these show any residual 3a in their corresponding spectra. All of the experimental yields are therefore believed to be more or less biased.

Even though the experimental yield of 2c-1 is expected to be severely shifted from the true value it still remains the entry with the least amount of human bias, which is the reason for it being used as a reference for the estimated yield calculations. Errors in this yield therefore entails serious changes in the estimated yield and it is important to view the estimated yield as a size reference instead of as a numeric value. No estimated yield could be calculated since the normalization factor is based on the ligand to product 2c-1 ratio.

Cross coupling reaction of the substrate 1c-1 using the same catalyst under similar conditions has been previously been reported with different scaffold, 3-Bromo-4- tosyloxycoumarin [10]. It was reported a 24% yield for 5%mol catalyst after 24 hours using a smaller amount of starting material. Since bromine of the scaffold in the referenced article has an ortho substituted keto and tosyl group which might contribute to steric, along with inductive, effects thus lowering the yield it is hard to correlate this yield to that of the product 2c-1. However, the referenced low yield for reaction with the substrate 1c-1 makes it more credible that a low yield also could be obtained for product 2c-1. Additional testing with the use of internal standard would be required in order to more thoroughly determine the amount of produced product.

The product 2c-2 of the second entry was produced by cross coupling of the scaffold 3a and substrate 4-tert-butylphenylboronic acid (1c-2) according to the experimental procedure listed in section 2.1.3. The product was, after reacting overnight, isolated by flash chromatography in an 11% yield according to the purification procedure listed in 2.1.4. Reaction mixture and purified product was analyzed by LC-MS, see Appendix III: Figure 18-19. Product purity was determined by LC-MS to 98.8%. The latter MS-fragment in the spectra is believed to belong to residual TEA and can be ignored.

With the same reasoning as for 2c-1, it can be concluded that an experimental yield of 11% underestimates the amount of product 2c-2 formed in the reaction and it is likely that the true value of formed product lies closer to the estimated yield of 72%. With the difficulties of normalizing the products for their relative differences in absorbency arising from conjugation and differences in substituents no further estimations will be made regarding the yield. Assuming that the estimated yield is closest to the true conversion, there can be found no evidence of the para positioned tert-butyl group interfering with the reaction due to bulkiness; inductive effects are negligible. However, external references using the same substrate and catalyst under similar conditions is needed to confirm this belief.

15 The product 2c-3 of the third entry was produced by cross coupling of the scaffold 3a and substrate 3-(trifluoromethyl)phenylboronic acid (1c-3) according to the experimental procedure listed in section 2.1.3. The product was, after reacting overnight, isolated by flash chromatography in an 11% yield according to the purification procedure listed in 2.1.4. Reaction mixture and purified product was analyzed by LC-MS, see Appendix III: Figure 20-21. Product purity was determined by LC-MS to 82.4%.

LC-MS spectrum of the crude reaction mixture does display several small traces which might indicate side-reactions. Since these peaks are very low in intensity along with not being accompanied by any larger MS fragments except for the residual 1c-3, lig- and and TEA, they would not be expected to constitute the remaining 89% that would missing from conversion for a 11% yield. With the same reasoning used for 2c-1 it can be expected that the calculated estimated yield of 88% is likely closer to the real amount of formed product. The tifluoromethyl substituent is the strongest electron withdrawing group used in the library reactions. Neither this nor the meta positioning of the substituent seems to have any notable effect on the systems ability to produce the desired product deeming from the estimated yield. However, external references using the same substrate and catalyst under similar conditions is needed to confirm this belief.

The product 2c-4 of the fourth entry was produced by cross coupling of the scaffold 3a and substrate phenylboronic acid (1c-4) according to the experimental procedure listed in section 2.1.3. The product was, after reacting overnight, isolated by flash chromatography in an 50% yield according to the purification procedure listed in 2.1.4. Reaction mixture and purified product was analyzed by LC-MS, see Appendix III: Fig- ure 22-23. Product purity was determined by LC-MS to 97.8%.

The experimental yield is also here believed to be largely biased, although not to the same extent as the previous entries due to the higher experimental yield. An experimental yield of 50% is significantly higher than that of the previous entries despite the product 2c-4 being difficult to separate due to the similar retention time of the ligand. The reason for the higher experimental yield is believed to be arbitrary since the lower reported experimental yields are believed to be faulty. Also, no con- nection based on functionalization or polarity can be found between the entries with highest experimental yield, 2c-4, 2c-5 and 2c-6, that would separate them from the rest of the entries. Suzuki and Miyaura themselves have conducted cross coupling reactions using the same substrate 1c-4 and catalyst under slightly different conditions. A cross coupling between the substrate 1c-4 and p-methyphenyl bromide, which is the most suitable comparison in regards to inductive effects and position of substituents, with 3%mol Pd(PPh3)4 catalyst in benzene reached 88% yield in 6h [14]. Deeming from the LC-MS spectum, the reaction running over night, the referenced

16 article and that 10%mol of catalyst was used for the substrate scope in this thesis it is assumed that both the experimental and estimated yield, of 66%, underestimates the amount of product formed in the reaction. A better estimation could be made if the relative absorbency of all products in the substrate scope was known and intensities were normalized from this. Judging from the obtained and referenced data, no substantial conclusions can be drawn about the eﬀects of non-substituted arylboronic substrates on the reaction other than it is possible to generate the sought after product without any substantial losses.

The product 2c-5 of the fifth entry was produced by cross coupling of the scaffold 3a and substrate 4-chloro-2-methylphenylboronic acid (1c-5) according to the experimental procedure listed in section 2.1.3. The product was, after reacting overnight, isolated by flash chromatography in an 43% yield according to the purification procedure listed in 2.1.4. Reaction mixture and purified product was analyzed by LC-MS, see Appendix III: Figure 24-25. Product purity was determined by LC-MS to 97.5%.

A larger amount of trace peaks are present in various intensities, some accompanied by notable MS fragments, which might indicate side-reactions. Side reaction products could arise from the substrate 1c-5 being chlorated and might therefore also be participating in the oxidative addition step forming additional cross coupling products. However, if these cross coupling reactions are present it will not be as extensive as for the brominated scaﬀold 3a since bromine has a lower bond dissociation energy and will therefore be the favored pathway in the oxidative addition [21]. Additional reason to believe that side reactions have occurred is that, when compared to reference data, no traces can be found of the substrate 1c-5, which was added in excess and therefore must have been consumed in side reactions. It could be possible that steric eﬀects of the ortho substituted methyl group decreases the rate of transmetalation thus promoting chlorine cross coupling reactions, which would not be sterically hindered, yielding more side reaction products. Estimating the amount of side reaction products from intensity will not be attempted since the products could have varying amounts of conjugation and substituents thus shifting the absorbance window making intensities irreliable. The MS spectrum could theoretically give information regarding relative sizes of the side reaction products and products such as homocouplings of the substrate 1c-5 could be expected to show typical M+2 fragment from the chlorine substituents; however no such M+2 fragment could be found upon examination of the spectrum. Since no referenced literature could be found regarding the substrate 1c-5 , it is hard to determine whether the experimental yield of 43% or the estimated yield of 88% is closest to the true amount of formed product.

The product 2c-6 of the sixth entry was produced by cross coupling of the scaﬀold 3a and substrate 4-methylphenylboronic acid (1c-6) according to the experimental procedure listed in section 2.1.3. The product was, after reacting overnight, isolated

17 by flash chromatography in an 40% yield according to the purification procedure listed in 2.1.4. Reaction mixture and purified product was analyzed by LC-MS, see Appendix III: Figure 26-27. Isolated product was additionally analyzed with NMR, see Appendix IV: Figure 31, and purity was determined by LC-MS to 74.0%.

The LC-MS spectrum of the reaction mixture shows some indication of side-reactions, however these side reactions are not seemingly extensive enough to substantiate cross coupling products equivalent to the additional 60% loss of yield. The experimental yield is therefore believed to be severely biased. The estimated yield of 51% does also seem quite low given that the substituent is a para positioned methyl group and is not expected to contribute notably to any steric nor inductive effects which might affect the biaryl formation. Also, the bulkier substrate 1c-2 managed to produce its corresponding product with the estimated yield of 72% despite the relatively bulky tert-butyl substituent. There is no reason to believe that the methyl substituted substrate would produce a lower yield than the bulkier tert-butyl substrate and therefore the difference in estimated yield is believed do arise from another factor. A possible factor could be differences in solubility during LC-MS analysis. However, with a small difference in retention time, 12 seconds, between products it is not credible that their solubilities would give rise to such large differences in estimated yield. Another factor might simply be due to differences in absorbency. No reference could be found of any cross coupling reaction using the same substrate 1c-6 and catalyst with a similar scaffold which makes it difficult to verify the credibility of the yields given.

The product 2c-7 of the seventh entry was produced by cross coupling of the scaffold 3a and ﬁrst substrate 4-(cyanomethyl)benzeneboronic acid (1c-7) according to the experimental procedure listed in section 2.1.3.

The reaction was stopped after running over night even though the LC-MS spectrum, see Appendix III: Figure 28, only showed a small amount of produced product with both starting materials 3a and 1c-7 still present. The stopping of the reaction was justified by that all other entries had managed to produce their corresponding products with notably higher yields given the same amount of time, but also since the trial reactions only required two hours to reach maximum conversion at the given conditions. It was therefore presumed that the maximum amount of the product 2c-7 already had been produced for the given conditions. Purification by flash chromatography was attempted despite the low conversion, see 2.1.4 for procedure. This was necessary, not only in order to calculate the yield due to lack of internal standard during measurements thus making quantification very difficult, but also since samples of the products were requested for studies outside of this project. No product was obtained despite several attempts of purification since the product has a retention time which overlaps with the scaffold 3a, thus making separation difficult.

18 Successful cross coupling has previously been reported for the same substrate 1c-7 under similar reaction conditions using the same catalyst Pd(PPh3)4. The reported data shows that 100% conversion was reached in 6h with only 5%mol catalyst, while only 31% conversion after 5h for 1%mol [11]. However, the substrate scope in this thesis was performed with 10%mol catalyst and further increase would not be appropriate. The most feasible approach to increase the conversion would be to experimentally op- timize temperature, base and solvent for the system.

Estimated yield calculations was made based on the ligand intensity of the sample which, given the assumptions stated in the beginning of this section (3.3), would give an estimate of the amount of product created by the reaction. Despite no yield was experimentally determined the estimated yield was calculated to roughly 24% which is still a low yield. A reason for this may be due to such a simple issue as the sample not being homogeneous and a sample containing mainly ligand was measured in LC-MS thus underestimating the yield. Another reason may be related to the product 2c-7 having additional π-electrons thereby adding to conjugation and shifting the absorption-window from that expected of the other substrates thus showing a lower yield than expected. However, conjugation eﬀects are also present in 2c-1 which was used as reference for the yield estimations and would not serve to explain the remaining starting material. The reasons for the residual starting material can only be speculated in, although there is no pronounced evidence of it being related to the nitrile substituent since, as mentioned before, similar reactions have been referenced to proceed well under slightly altered conditions. It can be concluded that the strength of deactivation by the functional groups does not directly relate to the ability of carrying out the reaction since the triﬂuoromethyl group of 2c-3 is more deactivating that the nitrile group and still manages to produce a higher yield.

19 Br R OH OH N + B Pd(PPh3)4/K2CO2(aq) N N N S R DME S 1c-n H2N H2N 3a 2c-n

Entry Boronic acid Product Amount (mg) Yield % a)Estimated b)Purity % Yield %

N N S OH O c) 1 B H2N 12.0 10 10 89.4 OH O 2c-1 1c-1

N N S OH 2 B H2N 13.1 11 72 98.8 OH 2c-2 1c-2

F F F F N N S OH F d) 3 B H2N 13.5 11 88 82.4 OH 2c-3 1c-3

N N S OH 4 B H2N 49.8 50 66 97.8 OH 2c-4 1c-4 Cl

N N S OH 5 B Cl H2N 50.8 43 88 97.5 OH 2c-5 1c-5

N N S OH e) 6 B H2N 41.9 40 51 74.0 OH 2c-6 1c-6 N

N N S OH f) 7 B N H2N – – 24 – OH 2c-7 1c-7

a) Yields estimated from ligand intensities using 2c-1 as reference. b) Purity determined at 305 nm. c) Puriﬁcation was unsuccessful for 2c-1. d) 95.4 % if "blob" at 3.384 min is disregarded. e) 98.2 % if "blob" is excluded in calculations, f) 2c-7 was puriﬁed by preperative HPLC.

Figure 9: Summarized results from substrate scope 3.4 General discussion The calculated estimated yield is very crude which makes it difficult to draw any conclusions from, although it might act as a guide for the amount of bias present for each entry. This however is highly dependent on that the stated assumptions actually does reflect the systems sufficiently, which it does not. It is for example known that the absorbency is not constant but does change, although it is not know to what extent this does affect the calculations. This can nor yet be accounted for since the absorbance of the products is needed for the given wavelength, but not known. Also the homogenity of the samples retrieved for LC-MS is of great concern as non-homogen samples will result in gross errors for the calculations.

A large challenge in the laboratory trials was to separate remaining ligand from the products. At first, contaminant arising from the ligand was assumed to be triphenylphosphine, PPh3, since this is the ligand used in the catalyst Pd(PPh3)4. However, MS confirmed that the contaminant was the oxidized state of the ligand, triphenylphosphine oxide, which is notorious for being hard to remove. It eludes at the same rate as many of the products which makes extraction difficult and many product containing fractions had to be discarded due to them containing this contaminant. This in turn had large implications on the product yield.

Another challenge during the laborative trials was that the products have very poor solubility in most solvents, except from DMSO or DMF which are very tricky to get rid of after isolation and often involves methods which include several extraction steps. Since the reactions were proceeding on the milligram scale, only very small amounts of these solvents could be used. This resulted in not fully dissolved samples which likely is one of the main reason for the poor yields.

A third reason for the poor yield could simply be that the starting material has been consumed in side reactions. Suzuki cross coupling reactions are for example known to produce homocoupled products where the scaﬀold or boronic acids couple to itself. However, the LC-MS chromatograms does not show any pronounced evidence of that homocoupled products were to have been formed during the substrate scope.

21 3.5 Conclusions All of the library entries managed to produce their respective products despite the varying functionalizations of the boronic acids. The reaction yields are likely severely biased by human interaction and experimental yields can only be viewed as lower boundaries of how much product has been converted. Estimated yields and referenced yields for similar reactions are required in order to make reasonable estimations regarding the amount of product produced in each entry. Additional experiments including internal standards are required in order to determine the eﬃciency and yield of the corresponding entries and to more thoroughly investigate the functional group dependence.

LC-MS results showed that the scaffold 3a was fully consumed in all but the first reaction which indicates that the reactions had been completed in the sense that no more product could have been formed. Evidence is however found of side reactions consuming the scaffold 3a and/or substrate. Confirming that reaction was feasible under the given parameters, reaction conditions could be further optimized to allow for a larger tolerance of functional groups or increasing yields of the selected substrates. The conditions at which the reactions were preformed involved 10% molar equivalence of catalyst which could likely be scaled down significantly and still have the reactions proceeding well. Other parameters to assay would be varying the temperature, solvent and base.

22 References

[1] Ataualpa A. C. Braga, Nelson H. Morgon, Gregori Ujaque, and Feliu Maseras. Computational characterization of the role of the base in the suzuki-miyaura cross- coupling reaction. J. AM. CHEM. SOC. 9, Volume 127(NO. 25):9299, 2005.

[2] Ataualpa A. C. Braga, Nelson H. Morgon, Gregori Ujaque, and Feliu Maseras. Computational characterization of the role of the base in the suzuki-miyaura cross-coupling reaction. J. AM. CHEM. SOC. 9, Volume 127(NO. 25):9300, 2005. Scheme 2.

[3] Jonathan Clayden, Nick Greeves, and Stuart Warren. Organic chemistry. Oxford University Press, Great Claredon Street, Oxford OX2 6DP, second edition, 2012. page 1079.

[4] Nancy E. Costa, Adrea L. Pelotte, Joseph M. Simard, Christopher A. Syvinski, and Amy M Deveau. Discovering green, aqueous suzuki coupling reactions: Synthesis of ethyl (4-phenylphenyl)acetate, a biaryl with anti-arthritic potential. Journal of Chemical Education, Volume 89(Issue 8):1064–1067, 2012.

[5] Carmela Crisostomo-Lucas, Ruben A. Toscano, and David Morales-Morales. Syn- thesis and characterization of new potentially hydrosoluble pincer ligands and their application in suzuki-miyaura cross-coupling reactions in water. Tetrahedron Letters, Volume54(Issue24):3116–3119, 2013.

[6] Dennis G. Hall ed. Boronic Acids: Preparation and Applications in Organic Syn- thesis, Medicine and Materials. Wiley-VCH, Somerset, NJ, USA, September 2012. page 214.

[7] Dennis G. Hall ed. Boronic Acids: Preparation and Applications in Organic Syn- thesis, Medicine and Materials. Wiley-VCH, Somerset, NJ, USA, September 2012. page 217.

[8] Sambasivarao Kotha, Kakali Lahiri, and Dhurke Kashinat. Recent applications of the suzuki–miyaura cross-coupling reaction in organic synthesis. p9634, Depart- ment of Chemistry, Indian Institute of Technology–Bombay, Powai, Mumbai 400 076, India.

[9] J.J. Li and G.W. Gribble. Palladium in Heterocyclic Chemistry: A Guide for the Synthetic Chemist. Tetrahedron Orgnaic Chemistry Series. Elsevier Science & Technology Books, 2007.

[10] Renhua Fan Liang Zhang, Tianhao Meng and Jie Wu. General and eﬃcient route for the synthesis of 3,4-disubstituted coumarins via pd-catalyzed site-selective cross-coupling reactions. J. Org. Chem., 72(19):7279–86, 2007.

23 [11] Hans-Ulrich Blaser Matthias Beller. Organometallics as Catalysts in the Fine Chemical Industry. Springer Heidelberg, 42 edition, 2012. page 119-124.

[12] Norio Miyaura and Akira Suzuki. Stereoselective synthesis of arylated (e)-alkenes by the reaction of alk-1-enylboranes with aryl halides in the presence of palladium catalyst. J. Chem. Soc., Chem. Communy, page 866, 1979. DOI: 1039/C39790000866.

[13] Norio Miyaura and Akira Suzuki. Palladium-catalyzed cross-coupling reactions of organoboron compounds. Chem. Rev., Volume 95:2460, 1995.

[14] T.; Suzuki A. Miyaura, N.; Yanagi. The palladium-catalyzed cross-coupling reaction of phenylboronic acid with haloarenes in the presence of bases. Synthetic Communications, 11(7):515–517, 1981.

[15] Nobelprize.org. The nobel prize in chemistry 2010. Nobel Media AB 2013. Web. 12 May, 2014.

[16] Image of Pd(dppf) from wikipedia. http://en.wikipedia.org/wiki/(1,1’- bis(diphenylphosphino)ferrocene)palladium(ii)_dichloride.

[17] Image of PEPPSI-iPr from Sigma-Aldrich. http://www.sigmaaldrich.com/catalog /product/aldrich/669032.

[18] Image of Tetrakis from Sigma-Aldrich. http://www.sigmaaldrich.com/catalog /product/aldrich/216666.

[19] Hans J. Reich. Stille coupling. http://www.chem.wisc.edu/areas/reich/chem547/6- transmet%7B04%7D.htm, 2012.

[20] Christoph Roehlich, Andreas S. Wirth, and Klaus Koehler. Suzuki coupling reactions in neat water as the solvent: Where in the biphasic reaction mixture do the catalytic reaction steps occur? Chemistry - A European Journal, Volume 18(Issue 48):15485–15494, 2012.

[21] Tom D. Sheppard*. Metal-catalysed halogen exchange reactions of aryl halides. (Emerging Area) Org. Biomol. Chem., 7:1043–1052, 2009.

[22] Akira Suzuki. Recent advances in the cross-coupling reactions of organoboron derivatives with organic electrophiles, 1995-1998. Journal of Organometallic Chemistry, Volume 576:147–168, 1999.

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24 Appendices

I Puriﬁcation techniques

I.I Flash chromatography Flash chromatography is an example of adsorption chromatography and separates molecules based on the solutes affinity towards the stationary phase; the stronger the solute adsorbes, the longer the retention time. The method uses pressurized gas to drive the elute through the column at a notably higher speed than traditional chromatography, thus the name flash chromatography. Molecules with the same equilibrium between adsorption at the stationary phase and solubility in the mobile phase will, ideally, elude at the same speed. This causes molecules to align in different "bands" throughout the column. Retention times and width of these bands can be regulated by shifting the equilibrium through variations in composition and polarity of the mobile phase.

I.II Preparative HPLC Preparative HPLC is the automated alternative to flash chromatography. If equipped with an autoinjector, preparative HPLC makes for an extremely useful choice when a large array of samples has to be separated. It could also be an alternative if two bands are too close together to be efficiently separated by flash chromatography.

Some disadvantages, in respect to ﬂash chromatography, are that the columns are much more expensive and that the yield and amount of sample that can be separated in a run is lower.

25 II Preperative HPLC results

Figure 10: Zoomed in spectrum of preperative HPLC for entry #7, 9-12 min, 305 nm (and 254 nm). Fractions 2 and 3 were collected.

26 III HPLC results

Product peaks, in the library reactions, have been marked with a circle for easier iden- tiﬁcation.

Scaﬀold synthesis

Figure 11: LC-MS Spectrum of recrystalized 3a (batch 1) at 254 nm, method B1090X

27 Catalyst screening

Figure 12: LC-MS Spectrum of Pd(PPh3)4-catalyzed reaction at 220 nm (B1090X)

Figure 13: LC-MS Spectrum of Pd(DPPF)Cl2-catalyzed reaction at 220 nm, method B1090X

28 Product 2c-1

Figure 14: LC-MS Spectrum of crude 2c-1 at 220 nm, method B1090X

Figure 15: LC Spectrum of crude 2c-1 at 305 nm, method B1090A

Figure 16: LC Spectrum of crude 2c-1 at 220 nm, method B1090A

29 Figure 17: LC-MS Spectrum of puriﬁed 2c-1 at 305 nm, method ST1097A3

30 Product 2c-2

Figure 18: LC-MS Spectrum of crude 2c-2 at 220 nm, method B1090X

Figure 19: LC-MS Spectrum of puriﬁed 2c-2 at 305 nm, method SX1097X3

31 Product 2c-3

Figure 20: LC-MS Spectrum of crude 2c-3 at 220 nm, method B1090X

Figure 21: LC-MS Spectrum of puriﬁed 2c-3 at 305 nm, method SX1097X3

32 Product 2c-4

Figure 22: LC-MS Spectrum of crude 2c-4 at 220 nm, method B1090X

Figure 23: LC-MS Spectrum of puriﬁed 2c-4 at 305 nm, method SX1097X3

33 Product 2c-5

Figure 24: LC-MS Spectrum of crude 2c-5 at 220 nm, method B1090X

Figure 25: LC-MS Spectrum of puriﬁed 2c-5 at 305 nm, method SX1097X3

34 Product 2c-6

Figure 26: LC-MS Spectrum of crude 2c-6 at 220 nm, method B1090X

Figure 27: LC-MS Spectrum of puriﬁed 2c-6 at 305 nm, method SX1097X3

35 Product 2c-7

Figure 28: LC-MS Spectrum of crude 2c-7 at 220 nm, method B1090X

The mass spectrum does not show the mass fragment of 3a. However, this was displayed during computer editing thus making it able to distinguish the product peak from 3a.

36 IV NMR results

Scaﬀold synthesis

Figure 29: (top) NMR Spectrum of 3a (batch 1) at 400 MHz, 7.45-7.85 ppm shift. (bottom) Simulated spectrum from ACD/Labs online HNMR Predictor 1H NMR (400 MHz, DMSO-d6) ppm 7.76 (m, J= 8,4 Hz, 2.0 Hz, 2H), 7.73 (m, J= 8,4 Hz, 2.0 Hz, 2H), 7.53 (br s, 2H).

37 Product 2c-1

Figure 30: 1H-NMR Spectrum of 2c-1 product at 400 MHz, full scale and 7.8-8.2 ppm

1H NMR (400 MHz, DMSO-d6) δ ppm 8.06 (m, 2H), 7.89 (m, 2H), 7.87 (m, 4H), 3.88 (s, 3 H)

38 Product 2c-6

Figure 31: 1HNMR spectrum of 2c-6, 400 MHz, solv. DMSO-d6

1H NMR (400 MHz, DMSO-d6) δ ppm 7.81 (m, J = 8.6 Hz, 2H), 7.74 (m, J = 8.6 Hz, 2H), 7.62 (m, J = 8.6 Hz, 2H), 7.42 (br s, 2H), 7.30 (m, J = 8.6 Hz, 2H), 2.35 (s, 3H).