Multigram scale synthesis of synthetic cannabinoid metabolites

Linköping University | Department of Physics, Chemistry and Biology Master thesis, 30 hp | Educational Program: Chemistry

Spring term 2021|LITH-IFM-A-EX—21/3985—SE

Multigram scale synthesis of synthetic cannabinoid metabolites

Ahmad Hussamadin

Examiner: Peter Konradsson Supervisor: Tobias Rautio, Xiongyu Wu

Abstract

As of today, synthetic cannabinoids are one of the biggest groups of new psychoactive substances. These substances can be used as substitutes for the psychoactive drug cannabis, avoiding the legal restrictions on cannabis. Furthermore, a variety of synthetic cannabinoids are synthesized with either significant or very minor structural differences, making the detection of said novel drugs hard to keep up with and is therefore of great importance to have standards which help in the identification of the intake of the parent synthetic cannabinoid.

In this project, several metabolites of synthetic cannabinoids with indole/indazole cores with different side chains was synthesized. The general strategy used in this project was to N-alkylate the desired core followed by amide coupling with L-tert-leucine methyl ester or L-Valine methyl ester hydrochloride which resulted in 8 potential synthetic cannabinoid metabolites.

Abbreviations

ACN Acetonitrile

CB1 Cannabinoid receptor type 1

CB2 Cannabinoid receptor type 2

DCM Dichloromethane

DMF Dimethylformamide

EtOAc Ethyl acetate

HCl Hydrochloric acid

KHSO4 Potassium bisulfate

LC-MS Liquid chromatography–Mass spectrometry

MeOH Methanol

MgSO4 Magnesium sulfate

NaH Sodium hydride

NaOH Sodium hydroxide

NMR Nuclear magnetic resonance

NPS New psychoactive substances

TBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate

TEA Triethylamine

TFA Trifluoroacetic acid

TLC Thin-layer chromatography

UNODC EWA United Nations Office on Drugs and Crime Early Warning Advisory

Δ9THC Δ9-tetrahydrocannabinol

Table of Contents

1 Introduction .................................................................................................................................... 1

1.1 Background ............................................................................................................................. 1

1.2 Synthetic cannabinoids ........................................................................................................... 1

1.3 Pharmacology, metabolism, and effects of synthetic cannabinoids ...................................... 2

1.4 Aim .......................................................................................................................................... 2

1.5 Targeted metabolites .............................................................................................................. 2

2 Synthetic approach and mechanism ............................................................................................... 5

2.1 N-Alkylation ............................................................................................................................. 5

2.2 Amide coupling ....................................................................................................................... 6

3 Results and discussion .................................................................................................................... 7

3.1 N-Alkylation ............................................................................................................................. 7

3.2 Amide coupling ....................................................................................................................... 8

3.3 Methyl (S)-2-(1-(5-hydroxypentyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (3b) 8

3.4 NMR data ................................................................................................................................ 9

4 Conclusions ................................................................................................................................... 10

5 Acknowledgments ......................................................................................................................... 11

6 Experimental ................................................................................................................................. 12

7 References .................................................................................................................................... 21

8 Appendix ....................................................................................................................................... 22

1

1 Introduction 1.1 Background New psychoactive substances (NPS) is the common name used for the group of drugs that previously were known as ‘‘designer drugs’’, ‘‘legal highs’’, ‘‘herbal highs’’, or ‘‘bath salts’’ [1,2]. As of 2020, 1047 unique substances have been reported to the United Nations Office on Drugs and Crime Early Warning Advisory (UNODC EWA). [2] These drugs are sold to teenagers and young adults because they are cheap, easily available to get on the Internet and are near impossible to identify because they are not detectable by standard drug screening tests [1,3,4]. The term NPS applies to a wide variety of substances and are divided into subgroups which include aminoindanes, phenethylamines, tryptamines, synthetic cannabinoids, synthetic cathinones and synthetic opiods. The majority of NPS are stimulants (36%) followed by synthetic cannabinoid receptor agonists (29%). [2] In 2019, UNODC EWA reported that 28% of the NPS fatalities were associated with synthetic cannabinoid usage. [5]

1.2 Synthetic cannabinoids Synthetic cannabinoids are usually metabolized very quickly in the body, making their detection complicated. It is especially difficult when the parent substance is undetected. It is therefore important to research and develop efficient synthetic routes to produce metabolite standards to provide for the toxicological and metabolic studies to understand the effects that the substances have on the body which requires multigram amounts of the compounds to investigate in detail. These synthetic routes can potentially also serve as a basis for other novel synthetic cannabinoid as well as hasten the production of the standards with good yields. [6,7]

Synthetic cannabinoids have some common structural features that defines them as seen in Figure 1. The four parts that define the structure of synthetic cannabinoids are the heterocyclic core, the linker, the linked group, and the lipophilic tail. The core is usually aromatic moieties such as indole or indazole. The tail is a flexible alkyl chain that can for example be a pentyl or butyl chain, with or without a terminal fluorine atom. The linker, which can be an ester or an amide that connects the core to the linked group which can be amino acids such as leucine and valine or organic compounds such as adamantane and naphthalene. [8,9,10]

NH

O

O

N

N

O

F

F

NH

O

O

OO

NH2

O

N

N N

The cores:

The linked groups:

The linkers:

The tails:

N

Figure 1. Illustration of the common structural features of synthetic cannabinoids showing different possible parts that can be used.

2

1.3 Pharmacology, metabolism, and effects of synthetic cannabinoids Cannabinoids are categorized into three groups: phytocannabinoids, endocannabinoids, and synthetic cannabinoids. Phytocannabinoids is the type of cannabinoid that are produced by plants. Most notable phytocannabinoids are from the genus Cannabis (Δ9THC). [11] Endocannabinoids are the type that is produced in humans endogenously which have various function throughout the central nervous system and serve as intercellular lipid messengers. [11,12] Synthetic cannabinoids are the man-made type which are mainly cannabinoid (CB) receptor agonists that mimic the effects of phytocannabinoids. [11]

Synthetic cannabinoids belong to a large diverse class of lipophilic molecules which have affinity for CB receptors. [13] Two types of CB receptors are known: CB1 and CB2. These receptors are G-protein coupled receptors. CB1 receptors are expressed in the heart, liver, brain, spinal cord, central and the peripheral nervous system. [14] CB1 receptors are responsible for euphoric and anticonvulsive effects of cannabis. CB2 on the other hand are expressed in the immune system or the immune derived cells. CB2 receptors are responsible for the anti-inflammatory and therapeutic effects of cannabis. [11] Generally, synthetic cannabinoids have greater affinity for both the receptors compared to Δ9THC. Furthermore, most synthetic cannabinoids are full agonists, thus having higher response compared to Δ9THC. [10,15] Drug metabolism takes place in a variety of organs throughout the human body such as in the lungs, kidneys, heart, and blood etc. However, because of the vast amount of hepatocyte cells that make up the liver, the liver may be the organ that dominates the metabolic activities. Furthermore, the synthetic cannabinoids are metabolized and biotransformed in the liver into more hydrophilic molecules, eliminating them through urine more rapidly. [8]

1.4 Aim The aim of this project was to synthesise several synthetic cannabinoid metabolites on a multigram scale and obtain the target molecules with a purity of 95 % or above.

1.5 Targeted metabolites A total of 8 metabolites were aimed to be synthesized in this project. The structures as well as general information such as IUPAC names, formula, molecular weight, and the compound number for this project can be seen in Table 1. Out of the 8 compounds, 6 of the compounds contain an indazole core and the other 2 contain an indole core. The indazole synthetic cannabinoid metabolites include (S)-2-(1-(4-fluorobutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoic acid 1b, (S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate 2b, Methyl (S)-2-(1-(5-hydroxypentyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate 3b, 4-(3-(((2S)-1-methoxy-3,3-dimethyl-1-oxobutan-2-yl) carbamoyl)-1H-indazol-1-yl) butanoic acid 4b, (2S)-2-(((1-(3-carboxypropyl)-1H-indazol-3-yl) carbonyl) amino)-3,3-dimethylbutanoic acid 5b, and (S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3-methylbutanoate 6b. The indole synthetic cannabinoid metabolites include (S)-2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoic acid 7b and (S)-2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoic acid 8b. Every potential metabolite in this project except for metabolite 6b contains a L-tert-Leucine moiety, either with a methyl protective group or without.

3

Table 1. Chemical structures and general information of the different targeted metabolites.

NH

OH

O

N

N

O

F

IUPAC: (S)-2-(1-(4-fluorobutyl)-1H-indazole-3-carboxamido)-3,3-

dimethylbutanoic acid

Formula: C18H24FN3O3

Molecular weight: 349.3998

Compound number in this project: 1b

NH

OH

O

N

N

O

OH

IUPAC: (S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3,3-

dimethylbutanoate

Formula: C18H25N3O4



NH

O

O

N

N

O

OH

IUPAC: Methyl (S)-2-(1-(5-hydroxypentyl)-1H-indazole-3-

carboxamido)-3,3-dimethylbutanoate

Formula: C20H29N3O4



NH

O

O

N

N

O

OH

O

IUPAC: 4-(3-(((2S)-1-methoxy-3,3-dimethyl-1-oxobutan-2-yl)

carbamoyl)-1H-indazol-1-yl) butanoic acid

Formula: C19H25N3O5



4

NH

OH

O

N

N

O

OH

O

IUPAC: (2S)-2-(((1-(3-carboxypropyl)-1H-indazol-3-yl) carbonyl)

amino)-3,3-dimethylbutanoic acid

Formula: C18H23N3O5



NH

O

O

N

N

O

OH

IUPAC: Methyl (2S)-2-(((1-(4-hydroxybutyl)-1H-indazol-3-

yl)carbonyl)amino)-3-methylbutanoate

Formula: C17H23N3O4



NH

OH

O

N

O

F

IUPAC: (S)-2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-





NH

OH

O

N

O

F

IUPAC: (S)-2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-





5

2 Synthetic approach and mechanism The two main steps in the synthetic route used, are the alkylation and the amide coupling steps. The first step in the synthesis is the alkylation step followed by amide coupling. The total synthesis of the different synthetic cannabinoid metabolites is shown in Scheme 1. The targeted metabolites in this project follow a similar route with metabolite 5b being the exception which uses a chemical reaction with trifluoroacetic acid (TFA) instead of hydrolysis. Other steps such as extraction, hydrolysis and purification were also implemented. Extraction was always carried out when the alkylation and amide coupling steps were confirmed to be complete with LC-MS. Hydrolysis was done to cleave the methyl ester of the amino acid to obtain the desired compound. Purification was almost always carried out as the last step to obtain a pure compound. Purification was also carried out when the compound obtained after the alkylation step was too impure. In the sections below, the two major steps are further explained and discussed in more detail.

OH

X

NH

OOH

X

N

O

R1

NH

O

O

R3

X

N

O

R4

R2

1a-8a 1b-8b

1a: X = N R1 = CH2CH2CH2CH2F 1b: X = N R2 = CH2CH2CH2CH2F R3 = CH3 R4 = H

2a: X = N R1 = CH2CH2CH2CH2OH 2b: X = N R2 = CH2CH2CH2CH2OH R3 = CH3 R4 = H

3a: X = N R1 = CH2CH2CH2CH2CH2OH 3b: X = N R2 = CH2CH2CH2CH2CH2OH R3 = CH3 R4 = CH3

4a: X = N R1 = CH2CH2CH2C(=O)OBut 4b: X = N R2 = CH2CH2CH2COOH R3 = CH3 R4 = CH3

5a: X = N R1 = CH2CH2CH2C(=O)OBut 5b: X = N R2 = CH2CH2CH2COOH R3 = CH3 R4 = H

6a: X = N R1 = CH2CH2CH2CH2OH 6b: X = N R2 = CH2CH2CH2CH2OH R3 = H R4 = CH3

7a: X = CH R1 = CH2CH2CH2CH2F 7b: X = CH R2 = CH2CH2CH2CH2F R3 = CH3 R4 = H

8a: X = CH R1 = CH2CH2CH2CH2CH2F 8b: X = CH R2 = CH2CH2CH2CH2CH2F R3 = CH3 R4 = H Scheme 1. The total synthesis of the 8 potential synthetic cannabinoid metabolites. The notation X shows whether the core is an indazole (X = N) or an indole (X = CH); R1 is the side chain after alkylation; R2 is the side chain after amide coupling/hydrolysis/TFA treatment; R3 indicates which amino acid was used when amide coupling (L-tert-leucine: R3 = CH3, L-Valine: R3 = H); R4 shows whether hydrolysis was done or not.

2.1 N-Alkylation The alkylation was chosen as the first step for all the metabolites synthesized in this project. The alkylation step couples the core to a side chain of choice to obtain the desired synthetic cannabinoid. This was done by dissolving the core, in this case either an indazole-3-carboxylic acid or an indole-3-carboxylic acid in dimethylformamide (DMF) due to its good solubility, followed by the addition of the base sodium hydride (NaH (60 %)). 3 equiv. of NaH (60 %) were used for optimal conversion. This was performed under 0 °C which increases the selectivity towards the first nitrogen in the indazoles but also decreases the amount of O-alkylated product. Following this, the desired side chain with a terminal bromine is added to the mixture. A proposed mechanism for the alkylation reaction can be seen in Scheme 2. The reaction is initiated by the base deprotonating the nitrogen. The bromide is now susceptible to an attack from the deprotonated nitrogen in an SN2 reaction resulting in an alkylated compound.

6

N

N

O

O–

H

Na H

DMFN

N–

O

O–

DMFN

N

O

O–

F

0 °C 0 °C

BrF

Scheme 2. Proposed mechanism for the N-alkylation of an indazole with 1-bromo-4-fluorobutane as the bromide giving an alkylated indazole core with a 4-fluorobutyl side chain.

2.2 Amide coupling Amide coupling is a reaction where the carboxylic acid is coupled to an amino acid of choice. Almost every metabolite in this project was coupled to a tert-leucine moiety. The alkylated compound was dissolved in a suitable solvent such as acetonitrile (ACN). A peptide reagent such as 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) was added together with a strong base, triethylamine (TEA) in this case. Before adding the amino acid of choice, the mixture was let to stir until an intermediate was formed confirmed by LC-MS. A proposed mechanism for the amide coupling can be seen in Scheme 3. The reaction is initiated by the carboxylate in the alkylated indazole. The carboxylate attacks the (Dimethylamino)-N,N-dimethylmethaniminium moiety. This moiety is then attacked by the resulted 1H-Benzotriazol-1-olate forming the intermediate. The amino acid is then introduced to the reaction where the nitrogen in the amino acid attacks the susceptible carbonyl kicking out 1H-Benzotriazol-1-olate. This is followed by TEA deprotonating the nitrogen giving a product which can then be purified and hydrolyzed if so desired.

N

N

O

F

O–

ACN

N+

N

N

O–

N+

N

N

N

O

F

O

NN

+

N

N

N

O–

N

N

O

F

O

N

NN

N

N

O

F

O

N

N

N

N

N

O

F

NH+

O

O

HNH2

O

O N

N

N

O

F

NH

O

O

Scheme 3. Proposed mechanism for the formation of the intermediate with TBTU as the coupling agent and amide coupling of L-tert-leucine methyl ester to an alkylated indazole.

7

3 Results and discussion 3.1 N-Alkylation The alkylations proceeded regioselectively to afford 1-substituted 1H-indazoles. The alkylation of the indole cores was much more straightforward as it only contains one nitrogen, and the only problem is the possibility of formation of an O-alkylated product. However, this was not observed in any of the syntheses.

The general synthesis method used in this project resulted in 8 possible synthetic cannabinoid metabolites. The first step of every synthesis was alkylation. All the alkylated compound and their respective yields can be seen in Table 2. The alkylated compounds used for synthesis of metabolites 3b and 6b are missing from the table as they were already synthesized beforehand. The general alkylation method was successful and resulted in good yields. However, the alkylated compound used for synthesis of metabolite 1b contained a side product, or rather an isomer shown in Scheme 4. This product is formed when the second nitrogen in the indazole core is doing the SN2 attack instead of the first nitrogen. This is most likely due to the reaction not being properly monitored and not being performed under 0 °C during the whole time as the ice bath melted and the reaction was not properly monitored. Properly monitoring and making sure that the reaction was performed in an ice bath reduced the amount of isomer formed drastically. Two of the compounds showed a yield over 100 %. As it is impossible to obtain yields over 100 %, the explanation for this is that the product might have contained impurities, unreacted reactants, solvent, and/or moisture such as DMF. The reason to not reduce the impurities in compounds with over 100 % yields is because the procedure to do so is time consuming and not worth the risk as it can result in loss of product.

Scheme 4. Mechanism for the formation of isomeric side products for compound 1a.

Table 2. Obtained compounds, which metabolite is synthesized from them and their respective yields after alkylation.

Although no plans were made to synthesize a metabolite containing an epoxide side chain, an alkylation of an epoxide was carried out in a similar manner, see compound 9a in the Appendix. However, it was most successful when the equiv bromide used was weighted in grams instead of milliliters. Furthermore, the acidification before the extraction were carried out with KHSO4 instead of the usual HCl as the epoxide was very sensitive. While the epoxide was successfully alkylated on an indazole core, it was very reactive, and it was observed that the ring was readily opened when MeOH was used as solvent to transfer the product to another flask.

Structure

N

N

O

F

OH

N

N

O

OH

OH

N

N

O

O

O

OH

N

O

F

OH

N

O

F

OH

Used for synthesis of metabolite

1b 2b 4b & 5b 7b 8b

Yield (%) 94 >100 86 96 >100

N

NH

OO

–

NH

N

OO

–

N

N

OO

–

H

Na H

DMF N

N

O

O–

F

BrF

N–

N

OO

–

DMF

1

2

8

3.2 Amide coupling The second major step in the synthesis was the amide coupling step. All the resulted metabolites from the amide couplings and their respective yields can be seen in Table 3. Although the yields were not the best, the amide coupling method was successful. All the compounds containing a side chain with a terminal hydroxy group were on the lower side in terms of yield. The reason for this is unclear but speculation can be made. For example, the low yields can potentially be due to the solvent over boiling and the product shooting up and getting trapped in the adapter of the rotary evaporator thus losing some product. Another reason could be the purification step, where tailing was present when purifying the compounds resulting in loss of some product.

Table 3. Obtained compounds and their yields after amide coupling.

Compound 1b 2b 3b 4b 5b 6b 7b 8b

Yield (%) 73 23 42 65 59 25 97 54

3.3 Methyl (S)-2-(1-(5-hydroxypentyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (3b)

The alkylation and amide coupling for the metabolite 3b was performed as usual. However, some of the solvent over boiled and the product shot up and got trapped in the adapter of the rotary evaporator. While some of the product was recovered, it might have introduced impurities. Also, when purifying with silica column chromatography, a mobile phase containing 2 % formic acid was used which was an unnecessary step. After the purification, it was confirmed by LC-MS that the formic acid had reacted with the hydroxyl group in the product, introducing an unwanted product to the mixture, see Scheme 5 for the proposed mechanism for the formation of the impurity. Some of the unwanted product was let to react with TFA in MeOH. This reaction reverted most of the unwanted product to the desired one and thus the procedure was carried out on the main mixture. However, this mistake was responsible in loss of a decent amount of the product and the usage of formic acid in the purification step was unnecessary as the formic acid did not contribute much to the purification according to the thin-layer chromatography (TLC) tests to justify using it.

NH

O

O

N

N

O

OH

OH

O

H

H+

OH

O+

H

H

C+

OH

O

H

H

O+

O

H H

H

NH

O

O

N

N

O

O+

H

OH

H OH

NH

O

O

N

N

O

O+

H

OH

H OH

NH

O

O

N

N

O

O O+

H OH

H

H

NH

O

O

N

N

O

O

H

O+

H

NH

O

O

N

N

O

O

O

- H+

TFA

MeOH

NH

O

O

N

N

O

OH

Scheme 5. Proposed mechanism for the formation of the impurity due to the usage of formic acid in the mobile phase.

9

3.4 NMR data Nuclear magnetic resonance (NMR) spectroscopy is an extremely important spectroscopic technique for identification and analysis of organic compounds. 1H-NMR can give information about the different proton environments as well as abundance, and the adjacency of the protons in relation to each other. Similarly, 13C-NMR allows identification of carbon atoms as well as the abundance and adjacency. NMR was used and was the last requirement to ascertain the identity of the final compounds. A comparison of a 13C-NMR of compounds 1b and 7b can be seen in Figure 2 where the only difference between the compounds being the core of the compound (indazole v. indole), see the red elliptical circles.

Figure 2. 13C-NMR spectrum of compound 1b and 7b, showing the similarities and the difference between them. The red elliptical circles show the only difference between the compounds.

Furthermore, a typical 1H-NMR for a compound with an indazole core can be seen in Figure 3. The green elliptical circles in Figure 3 shows the protons which belong to the indazole core, whereas the blue elliptical circles show the protons in the tertiary butyl group in the amino acid part of the molecule. The remaining protons and their corresponding peaks are marked with different colors.

Figure 3. 1H-NMR spectra of compound 3b. The green elliptical circles on shows the protons which belong to the indazole core, the blue elliptical circles show the protons in the tertiary butyl group in the compound. The remaining protons and their corresponding peaks are marked in different colors.

NH

OH

O

N

O

F

N H

O H

O

N N

O

F 7b 1b

NH

O

O

N

N

O

OH

10

4 Conclusions A simple synthetic route for the synthesis of potential synthetic cannabinoid metabolites was applied in this project. A total of 8 different possible synthetic cannabinoid metabolites were successfully synthesized, indicating that the method is effective. The alkylation resulted in good yields ranging from 86 – 100 % with an overall average of 95 % yield. The amide coupling resulted in worse yields ranging from 23 – 97 % with an overall average of 51 % yield. LC-MS confirmed the molecular masses of the synthesized metabolites. The metabolites were also characterized by 1H and 13C-NMR.

The main future perspective that can be taken from this project is that further optimization can be done to improve the yields of the amide coupling step as some of the compounds, while successfully synthesized, either produced unacceptable or low yields.

11

5 Acknowledgments I would like to express my gratitude to have gotten the opportunity of doing my master thesis within the field of synthetic chemistry. I would like to thank my examiner Professor Peter Konradsson for making it possible. His stories were inspiration and the discussions we had were amusing.

I would also like to thank my supervisor Xiongyu Wu for his patience, encouragement and inspiring me to make my own decisions and learn from them.

Finally, I would like to give a special thanks to my other supervisor Tobias Rautio for his patience with me and being there since day one sharing his knowledge and helping me in everything.

12

6 Experimental General Information

HPLC-MS was performed on a Waters system (Column: Xbridge C18, 3.5 µM, 4.6 x 50 mm) with

mobile phase: water phase A: ACN:H2O 5:95, with 10 mM NH4OAc; organic phase B: ACN:H2O 90:10,

with 10 mM NH4OAc. For mass determination, positive and negative ESI was used together with

ELSD and diode array detectors. Column purification was performed using the following silica gel:

High purity grade (Merck Grade 9385), pore size 60 Å, 230–240 mesh particle size. The structures

were characterized by 1H and 13C-NMR and the spectrum were recorded by a Varian Mercury

500/126 MHz instrument at 25 °C in CDCl3.

(S)-2-(1-(4-fluorobutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoic acid (1b)

N-Alkylation

Indazole-3-carboxylic acid (800 mg, 4.9 mmol) was dissolved in DMF (10 mL) at 0 ℃. NaH (60 %) (3 equiv, 591 mg, 14.8 mmol) was added and the mixture was stirred for 5 min. 1-bromo-4-fluorobutane (1.3 equiv, 994 mg, 6.4 mmol) was added and the mixture was stirred overnight (18 h) at 0 ℃. The mixture was quenched with H2O (20 mL) and stirred in rt for 1 h. The mixture was washed with heptane (10 mL), the water phase was collected and the pH was adjusted to 1-2 with hydrochloric acid (HCl) (3M). Then extracted with EtOAc (50x3 mL), the organic phase was collected and washed with H2O (30x4 mL) and brine (40 mL), dried with MgSO4, and filtered. The solvent was removed in vacuo giving 1-(4-fluorobutyl)-1H-indazole-3-carboxylic acid 1a (1.092 g, 94 % yield).

Amide coupling and hydrolysis

1-(4-fluorobutyl)-1H-indazole-3-carboxylic acid 1a (1.092 g, 4.6 mmol) was dissolved in ACN (100 mL). TBTU (1.5 equiv, 2.22 g, 6.934 mmol) and TEA (3 equiv, 1.93 mL, 13.86 mmol) was added and the mixture was stirred for 30 min. L-Tert-Leucine methyl ester hydrochloride (1.5 equiv, 1.26 g, 6.934 mmol) was added and the mixture was stirred overnight (18 h). The solvents were removed in vacuo. Then redissolved in EtOAc (100 mL) and washed with H2O (20x2 mL), the organic phase was collected and dried with MgSO4 and filtered. The solvents were removed in vacuo. The obtained product was dissolved in DCM and purified with a silica gel column using pre-absorption and DCM/MeOH (95:5) as a mobile phase. The obtained fractions were analysed with TLC (DCM/MeOH (95:5)). The fractions containing the compound were collected and the solvents were removed in vacuo. Hydrolysis was done by adding MeOH (20 mL) and NaOH (3 equiv, 1.65

mL, 6M) and the mixture was let to stir. The reaction was monitored with LC-MS. When the reaction was done, the pH was adjusted to 1-2 with HCl (3M), and extracted with EtOAc (50x3 mL), the organic phase was collected and washed with H2O (30x4 mL), dried with MgSO4, and filtered. The solvent was removed in vacuo giving (S)-2-(1-(4-fluorobutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoic acid 1b (1.176 g, 73 % yield). 1H-NMR (500 MHz, CDCl3) δ 8.30 (d, J = 8 Hz, 1H), 7.52 (d, J = 9.5 Hz, 1H), 7.37 – 7.35 (m, 2H), 7.24 – 7.21 (m, 1H), 4.71 (d, J = 10 Hz, 1H), 4.48-4.38 (m, 4H), 2.07 (p, J = 7 Hz, 2H), 1.74 – 1.63 (m, 2H), 1.09 (s, 9H). 13C-NMR (126 MHz, CDCl3) δ 176.02, 162.61, 140.98, 136.82, 127.01, 123.00, 122.93, 122.91, 109.26, 83.57 (d, JCF = 165 Hz), 59.79, 49.00, 35.02, 27.74 (d, JCF = 20 Hz), 26.83, 25.98 (d, JCF = 3.8 Hz).

NH

OH

O

N

N

O

F

N

N

O

F

OH

13

(S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (2b)

N-Alkylation

Indazole-3-carboxylic acid (2 g, 12.33 mmol) was dissolved in DMF (10 mL) at 0 ℃. NaH (60 %) (3 equiv, 1.48 g, 37 mmol) was added and the mixture was stirred for 5 min. 4-bromobutyl acetate (1.3 equiv, 2.32 mL, 16.03 mmol) was added and the mixture was stirred overnight (18 h) at 0 ℃. with the reaction was not completed according to LC-MS. Additional DMF (10 mL), 4-Bromobutyl acetate (1.2 equiv, 2.14 mL, 14.8 mmol) and NaH (60 %) (1 equiv, 493 mg, 12.33 mmol) were added, and the resulting mixture was stirred overnight (18 h). The mixture was quenched with H2O (30 mL) and stirred in rt for 1 h. The hydrolysis was not completed, then more NaOH (4 equiv, 1.97 g, 49.2 mmol) was added. The mixture was stirred at rt. After 30 min,

additional NaOH (4 equiv, 5.7 mL, 6M) was added and the mixture was stirred further for 1 h until the full conversion was achieved. The reaction was monitored with LC-MS. The mixture was washed with heptane (10 mL), the water phase was collected, and the pH was adjusted to 1-2 with HCl (3M), and extracted with EtOAc (50x3 mL), the organic phase was collected and washed with H2O (30x4 mL). The solvent was removed in vacuo together with small amount of toluene giving 1-(4-hydroxybutyl)-1H-indazole-3-carboxylic acid 2a (3 g, >100 % yield).

Amide coupling and hydrolysis

1-(4-hydroxybutyl)-1H-indazole-3-carboxylic acid 2a (1.25 g, 5.3 mmol) was dissolved in ACN (100 mL). TBTU (1.5 equiv, 2.57 g, 7.95 mmol) and TEA (3 equiv, 2.23 mL, 15.9 mmol) was added and the mixture was stirred for 30 min. L-Tert-Leucine methyl ester hydrochloride (1.5 equiv, 1.45 g, 6.934 mmol) was added and the mixture was stirred overnight (18 h). The solvents were removed in vacuo. Then redissolved in EtOAc (100 mL) and washed with H2O (20x2 mL), the organic phase was collected and dried with MgSO4 and filtered. The solvents were removed in vacuo. The obtained product was dissolved in DCM and purified with a silica gel column using pre-absorption and DCM/MeOH (95:5) as a mobile phase. The obtained fractions were analysed with TLC (DCM/MeOH (95:5)). The fractions containing the compound were collected and the solvents were removed in vacuo. Hydrolysis was done by adding MeOH (20 mL) and NaOH (3 equiv, 415

L, 6M) and the mixture was let to stir. The reaction was monitored with LC-MS. When the reaction was complete, the pH was adjusted to 1-2 with HCl (3M), and extracted with EtOAc (50x2 mL), the organic phase was collected and washed with H2O (30x4 mL), dried with MgSO4, and filtered. The solvent was removed in vacuo. The obtained product was preabsorbed into a silica gel and column separation with DCM/MeOH (9:1) as the mobile phase was carried out. The obtained fractions were analysed with TLC (DCM/MeOH (9:1)). The fractions containing the compound were collected and the solvents were removed in vacuo giving (S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate 2b (527 mg, 23 % yield). 1H-NMR (500 MHz, CDCl3) δ 8.27 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 9.5 Hz, 1H), 7.40 – 7.30 (m, 2H), 7.23 – 7.16 (m, 1H), 4.67 (d, J = 9.5 Hz, 1H), 4.42 – 4.25 (m, 2H), 3.64 (q, J = 8.7 Hz, 2H), 2.09 – 1.90 (m, 2H), 1.54 (q, J = 6.9 Hz, 2H), 1.09 (s, 9H). 13C-NMR (126 MHz, CDCl3) δ 175.26, 162.69, 140.92, 136.76, 126.93, 122.92, 122.84, 122.78, 109.36, 62.10, 60.21, 49.13, 34.99, 29.61, 26.90, 26.22.

N

N

O

OH

OH

NH

OH

O

N

N

O

OH

14

Methyl (S)-2-(1-(5-hydroxypentyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (3b)

Amide coupling

1-(5-hydroxypentyl)-1H-indazole-3-carboxylic acid 3a (1.191 g, 4.80 mmol) was dissolved in ACN (100 mL). TBTU (1.1 equiv, 1.69 g. 5.28 mmol) and TEA (3 equiv, 2 mL, 14.39 mmol) was added and the mixture was stirred for 30 min. L-Tert-Leucine methyl ester hydrochloride (1.1 equiv, 958 mg, 5.28 mmol) was added and the mixture was stirred overnight (18 h). The solvents were removed in vacuo. Then redissolved in EtOAc (100 mL) and washed with H2O (20x2 mL), the organic phase was collected and dried with MgSO4 and filtered. The solvents were removed in vacuo. The obtained product was dissolved in DCM and was purified with a silica gel column using pre-absorption and DCM/MeOH (95:5) as a mobile phase. The obtained fractions were analysed with TLC (DCM/MeOH (95:5). The fractions containing the compound were collected and the solvents were removed in vacuo giving an impure compound. The obtained product was again dissolved in

DCM and was purified with a silica gel column using pre-absorption and Heptane/EtOAc (5:5) + 2 % formic acid as a mobile phase. The obtained fractions were analysed with TLC (Heptane/EtOAc (5:5)

+ 2 % formic acid). The obtained product was dissolved in MeOH (10 mL) and TFA (1 equiv, 198 L, 2,59 mmol) was added and the mixture was stirred for 20 min. The pH was then adjusted to 1-2 with HCl (3M), and extracted with EtOAc (20x2 mL), the organic phase was collected and washed with H2O (20x2 mL) dried with MgSO4 and filtered. The solvent was removed in vacuo giving methyl (S)-2-(1-(5-hydroxypentyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate 3b (0.762 g, 42 % yield). 1H-NMR (500 MHz, CDCl3) δ 8.37 (dt, J = 8.2, 1.0 Hz, 1H), 7.60 (d, J = 9.8 Hz, 1H), 7.47 – 7.39 (m, 2H), 7.32 – 7.26 (m, 1H), 4.76 (d, J = 9.7 Hz, 1H), 4.44 (t, J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.66 (t, J = 6.3 Hz, 2H), 2.02 (p, J = 7.3 Hz, 2H), 1.70 – 1.60 (m, 2H), 1.50 – 1.40 (m, 2H), 1.12 (s, 9H). 13C-NMR (126 MHz, CDCl3) δ 172.39, 162.47, 140.93, 136.77, 126.81, 123.01, 122.89, 122.72, 109.31, 62.63, 59.62, 51.95, 49.34, 35.17, 32.19, 29.52, 26.81, 23.14.

NH

O

O

N

N

O

OH

15

4-(3-(((2S)-1-methoxy-3,3-dimethyl-1-oxobutan-2-yl) carbamoyl)-1H-indazol-1-yl) butanoic acid (4b) & (2S)-2-(((1-(3-carboxypropyl)-1H-indazol-3-yl) carbonyl) amino)-3,3-dimethylbutanoic acid (5b)

N-Alkylation

Indazole-3-carboxylic acid (2 g, 12.33 mmol) was dissolved in DMF (10 mL) at 0 ℃. NaH (60 %) (3 equiv, 1.48 g, 37 mmol) was added and the mixture was stirred for 5 min. tert-butyl 4-bromobutanoate (1.05 equiv, 2.29 mL, 12.95 mmol) was added and the mixture was stirred overnight (18 h) at 0 ℃. Additional DMF (10 mL) and more

tert-butyl 4-bromobutanoate (0.2 equiv, 437 L, 2.47 mmol) was added. After conformation from LC-MS that the reaction was complete, the mixture was quenched with H2O (30 mL) and stirred in rt for 5 min. The mixture was washed with heptane (10 mL), the water phase was collected, and the pH was adjusted to 1-2 with HCl (3M), then extracted with EtOAc (50x3 mL), the organic phase was collected and washed with H2O (30x3 mL) and brine (40 mL), dried

with MgSO4, and filtered. The solvent was removed in vacuo giving 1-(4-tert-butoxy-4-oxobutyl)-1H-indazole-3-carboxylic acid 4a & 5a (3.241 g, 86 % yield).

Amide coupling

1-(4-tert-butoxy-4-oxobutyl)-1H-indazole-3-carboxylic acid 4a & 5a (2.5 g, 8.21 mmol) was dissolved in ACN (100 mL). TBTU (1.1 equiv, 2.9 g, 9.04 mmol) and TEA (3 equiv, 3.43 mL, 24.64 mmol) was added and the mixture was stirred for 30 min. L-Tert-Leucine methyl ester hydrochloride (1.1 equiv, 1.64 g, 9.04 mmol) was added and the mixture was stirred overnight (18 h). The solvents were removed in vacuo. Then redissolved in EtOAc (100 mL) and washed with H2O (20x2 mL), the organic phase was collected and dried with MgSO4 and filtered. The solvents were removed in vacuo. The obtained product was dissolved in DCM then purified with a silica gel column using pre-absorption and DCM/MeOH (9:1) as a mobile phase. The obtained fractions were analysed with TLC (DCM/MeOH (9:1)). The fractions containing the compound were collected and the solvents were removed in vacuo giving a compound that still contained impurities. The obtained product

was again dissolved in DCM (x3 Pasteur pipettes) and was purified with a silica gel column using 100 % DCM as a mobile phase. The obtained fractions were analysed with TLC. The fractions containing the compound were collected and the solvents were removed in vacuo giving methyl 2-(((1-(4-tert-butoxy-4-oxobutyl)-1H-indazol-3-yl)carbonyl)amino)-3,3-dimethylbutanoate (2.366 g, 67 % yield).

N

N

O

O

O

OH

N

N

O

O

NH

O

OO

16

TFA treatment

A solution of TFA and DCM (1 mL: 8 mL) was mixed with half of the obtained compound (1.183 g, 2.74 mmol) and was let to stir for 30 min. Additional 1 mL TFA was added, and the mixture was let to stir until the reaction was confirmed to be complete with LC-MS giving 4-(3-(((2S)-1-methoxy-3,3-dimethyl-1-oxobutan-2-yl)carbamoyl)-1H-indazol-1-yl)butanoic acid 4b (1.167 g, 98% yield). 1H-NMR (500 MHz, CDCl3) δ 8.32 (d, J = 8.5, 1H), 7.59 (d, J = 9.5, 1H), 7.42 (q, J = 8.5, 2H), 7.28 – 7.25 (m, 1H), 4.72 (d, J = 10, 1H), 4.50 (t, J = 6.5, 2H), 3.75 (s, 3H), 2.42 (t, J = 7, 2H), 2.29 (p, J = 6.5, 2H), 1,08 (s, 9H). 13C-NMR (126 MHz, CDCl3) δ 177.67, 172.31, 162.52, 141.06, 137.00, 127.12, 123.00, 122.98, 122.86, 109.25, 59.75, 52.01, 48.29, 35.13, 30.90, 26.77, 24.65.

Hydrolysis

MeOH (10 mL), H2O (7 mL) and NaOH (5 equiv, 548 mg) was mixed with half of the obtained compound (1.183 g, 2.74 mmol) and the mixture was let to stir for 30 min. The solvent was vacuo removed and the pH was adjusted to 1-2 with HCl (3M), and extracted with EtOAc (20x2 mL), the organic phase was collected and washed with H2O (20x2 mL), dried with MgSO4, and filtered. The solvent was removed in vacuo giving (2S)-2-(((1-(3-carboxypropyl)-1H-indazol-3-yl)carbonyl)amino)-3,3-dimethylbutanoic acid 5b (1.048 g, 88 % yield). 1H-NMR (500 MHz, CDCl3) δ 8.35 (dt, J = 8.3, 1.1 Hz, 1H), 7.67 (d, J = 9.7 Hz, 1H), 7.45 – 7.37 (m, 2H), 7.31 – 7.26 (m, 1H), 4.77 (d, J = 9.7 Hz, 1H), 4.56 – 4.48 (m, 2H), 2.47 – 2.37 (m, 1H), 2.31 – 2.16 (m, 3H), 1.12 (s, 9H). 13C-NMR (126 MHz, CDCl3) δ 178.73, 176.94, 162.46, 140.93, 136.98, 127.16, 123.06, 123.03, 122.97, 109.19, 52.76, 47.92, 35.35, 30.86, 26.84, 24.47.

NH

O

O

N

N

O

OH

O

NH

OH

O

N

N

O

OH

O

17

(S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3-methylbutanoate (6b)

Amide coupling

1-(4-Hydroxybutyl)-1H-indazole-3-carboxylic acid 6a (1.435 g, 6.13 mmol) was dissolved in ACN (100 mL). TBTU (1.1 equiv, 2.16 g, 6.73 mmol) and TEA (3 equiv, 2.56 mL, 18.38 mmol) was added and the mixture was stirred for 30 min. L-Valine methyl ester hydrochloride (1.1 equiv, 1.13 g, 6.73 mmol) was added and the mixture was stirred overnight (18 h). The solvents were removed in vacuo. Then redissolved in EtOAc (100 mL) and washed with H2O (20x2 mL), the organic phase was collected and dried with MgSO4 and filtered. The solvents were removed in vacuo. The obtained product was dissolved in DCM then purified with a silica gel column using pre-absorption and DCM/MeOH (95:5 → 9:1) as a mobile phase The obtained fractions were analysed with TLC, fractions containing the compound were collected and the solvents were removed in vacuo giving (S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3-methylbutanoate 6b (544 mg, 25 % yield). 1H-NMR (500 MHz, CDCl3)

δ 8.20 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 9 Hz, 1H), 7.29 – 7.22 (m, 2H), 7.13 – 7.10 (m, 1H), 4.67 (m, 1H) 4.29 (t, J = 7 Hz, 2H), 3.65 (s, 3H), 3.52 (t, J = 6 Hz, 2H), 2.19 (m, 1H) 1.90 (qui, J = 7.5 Hz, 2H), 1.45 (q, J = 6.5 Hz, 2H), 0.92 (s, 6H). 13C-NMR (126 MHz, CDCl3) δ 172.51, 162.55, 140.67, 136.35, 126.61, 122.64, 122.54, 122.37, 109.27, 61.60, 56.75, 52.08, 49.08, 31.33, 29.52, 26.16, 19.00, 17.93.

NH

O

O

N

N

O

OH

18

(S)-2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoic acid (7)

N-Alkylation

Indole-3-carboxylic acid (1 g, 6.2 mmol) was dissolved in DMF (10 mL) at 0 ℃. NaH (60 %) (3 equiv, 744 mg, 18.61 mmol) was added and the mixture was stirred for 5 min. 1-bromo-4-fluorobutane (1.05 equiv, 699

L, 6.52 mmol) was added and the mixture was stirred for 1h at 0 ℃. The mixture was quenched with H2O (20 mL) and stirred in rt for 5 min. The mixture was washed with heptane (10 mL), the water phase was collected, and the pH was adjusted to 1-2 with HCl (3M), and extracted with EtOAc (50x3 mL), the organic phase was collected and washed with H2O (30x3 mL) and brine (40 mL), dried with MgSO4, and filtered. The solvent was removed in vacuo giving 1-(4-fluorobutyl)-1H-indole-3-carboxylic acid 7a (1.406 g, 96 % yield).

Amide coupling

1-(4-fluorobutyl)-1H-indole-3-carboxylic acid 7a (1.406 g, 5.98 mmol) was dissolved in ACN (100 mL). TBTU (1.1 equiv, 2.11 g, 6.57 mmol) and TEA (3 equiv, 2.5 g, 17.92 mmol) was added and the mixture was stirred for 30 min. L-Tert-Leucine methyl ester hydrochloride (1.1 equiv, 1.19 g, 6.57 mmol) was added and the mixture was stirred overnight (18 h). The solvents were removed in vacuo. Then redissolved in EtOAc (100 mL) and washed with H2O (20x2 mL), the organic phase was collected and dried with MgSO4 and filtered. The solvents were removed in vacuo. The obtained product was dissolved in DCM and was purified with a silica gel column using pre-absorption and DCM/MeOH (95:5) as a mobile phase. The obtained fractions were analysed with TLC (DCM/MeOH (95:5)). The fractions containing the compound were collected and the solvents were removed in vacuo. Hydrolysis was done by adding MeOH (20 mL) and NaOH (3 equiv, 2.47

mL, 6M) and the mixture was let to stir. H2O (20 mL) and additional MeOH (50 mL) and NaOH (3 equiv, 2.47 mL, 6M) was added. The reaction was monitored with LC-MS. When the reaction was done, the solvent was removed in vacuo and the pH was adjusted to 1-2 with HCl (3M), and extracted with EtOAc (50x3 mL), the organic phase was collected and washed with H2O (30x4 mL), dried with MgSO4, and filtered. The solvent was removed in vacuo giving (S)-2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoic acid 7b (1.677 g, 97 % yield). 1H-NMR (500 MHz, CDCl3) δ 8.52 (s, 1H), 8.01 – 7.94 (m, 1H), 7.87 (s, 1H), 7.44 – 7.37 (m, 1H), 7.31 (dd, J = 9.5, 5.4 Hz, 2H), 6.76 (d, J = 9.1 Hz, 1H), 4.84 (d, J = 9.1 Hz, 1H), 4.44 (dt, J = 47.2, 5.8 Hz, 2H), 4.19 (t, J = 7.1 Hz, 2H), 2.00 (p, J = 7.3 Hz, 2H), 1.77 – 1.60 (m, 2H), 1.17 (s, 9H). 13C-NMR (126 MHz, CDCl3) δ 175.11, 165.62, 136.68, 132.76, 125.026, 122.76, 121.98, 119.90, 110.52, 110.21, 83.55 (d, JCF = 165.8 Hz), 60.45, 46.52, 34.99, 27.73 (d, JCF = 20 Hz), 26.91, 26.25 (d, JCF = 3.8 Hz).

N

O

F

OH

NH

OH

O

N

O

F

19

(S)-2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoic acid (8b)

N-Alkylation

Indole-3-carboxylic acid (800 mg, 4.96 mmol) was dissolved in DMF (20 mL) at 0 ℃. NaH (60 %) (3 equiv, 595 mg, 14.89 mmol) was added and the mixture was stirred for 5 min. 1-bromo-5-

fluoropentane (1.05 equiv, 647 L, 5.21 mmol) was added and the mixture was stirred for 1h at 0 ℃. The mixture was quenched with H2O (20 mL) and stirred in rt for 5 min. The mixture was washed with heptane (10 mL), the water phase was collected, and the pH was adjusted to 1-2 with HCl (3M), and extracted with EtOAc (50x3 mL), the organic phase was collected and washed with H2O (30x3 mL) and brine (40 mL), dried with MgSO4, and filtered. The solvent was removed in vacuo giving 1-(5-fluoropentyl)-1H-indole-3-carboxylic acid 8a (1.361 g, >100 % yield).

Amide coupling

1-(5-fluoropentyl)-1H-indole-3-carboxylic acid 8a (1.361 g, 5.46 mmol) was dissolved in ACN (100 mL). TBTU (1.1 equiv, 1.92 g, 6.00 mmol) and TEA (3 equiv, 2.28 mL, 16.38 mmol) was added and the mixture was stirred for 30 min. L-Tert-Leucine methyl ester hydrochloride (1.1 equiv, 1 g, 6.00 mmol) was added and the mixture was stirred overnight (18 h). The solvents were removed in vacuo. Then redissolved in EtOAc (100 mL) and washed with H2O (20x2 mL), the organic phase was collected and dried with MgSO4 and filtered. The solvents were removed in vacuo. The obtained product was dissolved in DCM and was purified with a silica gel column using pre-absorption and DCM/MeOH (9:1) as a mobile phase. The obtained fractions were analysed with TLC (DCM/MeOH (9:1)). The fractions containing the compound were collected and the solvents were removed in vacuo. Hydrolysis was done by adding H2O (20 mL), MeOH (40 mL) and NaOH (3 equiv, 2.16 mL, 6M) and

the mixture was let to stir. The reaction was monitored with LC-MS. When the reaction was complete, the solvent was removed in vacuo and the pH was adjusted to 1-2 with HCl (3M) and extracted with EtOAc (50x3 mL). The organic phase was collected and washed with H2O (30x4 mL), dried with MgSO4, and filtered. The solvent was removed in vacuo giving (S)-2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoic acid 8b (846 mg, 54% yield). 1H-NMR (500 MHz, CDCl3) δ 7.94 (s, 1H), 7.82 (s, 1H), 7.38 – 7.36 (m, 1H), 7.30 – 7.26 (m, 2H), 6.71 (d, J = 8 Hz, 1H), 4.82 (d, J = 9.5 Hz, 1H), (dt, J = 47.5, 6 Hz, 2H), 4.12 (t, J = 7 Hz, 2H), 2.81 (s, 1H), 1.88 (p, J = 7.5 Hz, 2H), 1.72 – 1.64 (m, 2H), 1.43 (p, J = 8.5 Hz, 2H), 1.14 (s, 9H). 13C-NMR (126 MHz, CDCl3) δ 174.94, 165.51, 136.61, 132.64, 125.11, 122.60, 121.84, 119.76, 110.43, 109.99, 84.32 (d, JCF = 165 Hz), 60.27, 46.74, 34.88, 29.89 (d, JCF = 20 Hz), 29.58, 26.80, 26.76 (d, JCF = 4.8 Hz).

N

O

F

OH

NH

OH

O

N

O

F

20

1-[3-(oxiran-2-yl) propyl]-1H-indazole-3-carboxylic acid (9a)

N-Alkylation

Indazole-3-carboxylic acid (0.2 g, 1.23 mmol) was dissolved in DMF (10 mL) at 0 ℃. NaH (60 %) (3 equiv, 14.79 mmol) was added and the mixture was stirred for 5 min. 2-(3-Bromopropyl) oxirane (1.1 equiv, 1.35 mmol) was added and the mixture was stirred at 0 ℃. Additional 2-(3-Bromopropyl) oxirane (0.2 equiv, 0.24 mmol) was added. After conformation from LC-MS that the reaction was complete, the mixture was quenched with H2O (10 mL) and stirred in rt for 3 min. The mixture was washed with heptane (10 mL), the water phase was collected, and the pH was adjusted to 1-2 with KHSO4, and extracted with EtOAc (30x3 mL), the organic phase was collected and washed with H2O (10x5 mL) and brine (20 mL), dried with MgSO4, and filtered.

The solvent was removed in vacuo giving 1-[3-(oxiran-2-yl) propyl]-1H-indazole-3-carboxylic acid 9a.

N

N

O

O

OH

21

7 References [1] Bertol, E., Vaiano, F., Di Milia, M. G., & Mari, F. (2015). In vivo detection of the new psychoactive

substance AM-694 and its metabolites. Forensic science international, 256, 21-27.

[2] What are NPS? https://www.unodc.org/LSS/Page/NPS (accessed Mar 23, 2021).

[3] Khullar, V., Jain, A., & Sattari, M. (2014). Emergence of new classes of recreational drugs—

Synthetic cannabinoids and cathinones. Journal of general internal medicine, 29(8), 1200-1204.

[4] Arfken, C. L., Owens, D., Madeja, C., & DeAngelis, C. (2014). Exploratory comparative study on

the diffusion of synthetic cannabinoids and synthetic cathinones. Journal of Psychoactive Drugs, 46(5), 362-368.

[5] United Nations Office on Drugs and Crime (UNODC) 2020 Current NPS Threats, Volume II, January 2020. https://www.unodc.org/documents/scientific/Current_NPS_Threats_Volume_II_Web.pdf

[6] Wu, X., Bopp, D., Wallgren, J., Dahlén, J., & Konradsson, P. (2020). Synthesis of nine potential

synthetic cannabinoid metabolites with a 5F-4OH pentyl side chain from a scalable key intermediate. Synthetic Communications, 1-10.

[7] Wallgren, J., Vikingsson, S., Åstrand, A., Josefsson, M., Green, H., Dahlén, J., ... & Konradsson, P. (2018). Synthesis and identifications of potential metabolites as biomarkers of the synthetic cannabinoid AKB-48. Tetrahedron, 74(24), 2905-2913.

[8] Diao, X., & Huestis, M. A. (2019). New synthetic cannabinoids metabolism and strategies to best identify optimal marker metabolites. Frontiers in chemistry, 7, 109.

[9] Potts, A. J., Cano, C., Thomas, S. H. L., & Hill, S. L. (2020). Synthetic cannabinoid receptor agonists: classification and nomenclature. Clinical Toxicology, 58(2), 82-98.

[10] Wallgren, J. (2020). An insight into the metabolism of New Psychoactive Substances: Structural elucidation of urinary metabolites of synthetic cannabinoids and fentanyl analogues using synthesized reference standards (Vol. 2093). Linköping University Electronic Press.

[11] Chilakapati, J., & Farris, F. F. (2014). Cannabinoids.

[12] Lu, H. C., & Mackie, K. (2016). An introduction to the endogenous cannabinoid system. Biological psychiatry, 79(7), 516-525.

[13] Sedefov, R., Gallegos, A., King, L., Lopez, D., Auwärter, V., Hughes, B., & Griffiths, P. (2009). Understanding the ‘Spice’phenomenon. Thematic papers, European Monitoring Centre for Drugs and Drug Addiction.

[14] Howlett, A. C., Barth, F., Bonner, T. I., Cabral, G., Casellas, P., Devane, W. A., ... & Pertwee, R. G. (2002). International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacological reviews, 54(2), 161-202.

[15] Wiley, J. L., Lefever, T. W., Marusich, J. A., Grabenauer, M., Moore, K. N., Huffman, J. W., & Thomas, B. F. (2016). Evaluation of first generation synthetic cannabinoids on binding at non-cannabinoid receptors and in a battery of in vivo assays in mice. Neuropharmacology, 110, 143-153.

https://www.unodc.org/LSS/Page/NPS

https://www.unodc.org/documents/scientific/Current_NPS_Threats_Volume_II_Web.pdf

22

8 Appendix (S)-2-(1-(4-fluorobutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoic acid (1b)

1H-NMR

13C-NMR

NH

OH

O

N

N

O

F

NH

OH

O

N

N

O

F

23

(S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (2b)

1H-NMR

13C-NMR

NH

OH

O

N

N

O

OH

NH

OH

O

N

N

O

OH

24

Methyl (S)-2-(1-(5-hydroxypentyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (3b)

1H-NMR

13C-NMR

NH

O

O

N

N

O

OH

NH

O

O

N

N

O

OH

25

4-(3-(((2S)-1-methoxy-3,3-dimethyl-1-oxobutan-2-yl) carbamoyl)-1H-indazol-1-yl) butanoic acid (4b)

1H-NMR

13C-NMR

NH

O

O

N

N

O

OH

O

NH

O

O

N

N

O

OH

O

26

(2S)-2-(((1-(3-carboxypropyl)-1H-indazol-3-yl) carbonyl) amino)-3,3-dimethylbutanoic acid (5b)

1H-NMR

13C-NMR

NH

OH

O

N

N

O

OH

O

NH

OH

O

N

N

O

OH

O

27

(S)-2-(1-(4-hydroxybutyl)-1H-indazole-3-carboxamido)-3-methylbutanoate (6b)

1H-NMR

13C-NMR

NH

O

O

N

N

O

OH

NH

O

O

N

N

O

OH

28

(S)-2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoic acid (7b)

1H-NMR

13C-NMR

NH

OH

O

N

O

F

NH

OH

O

N

O

F

29

(S)-2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoic acid (8b)

1H-NMR

13C-NMR

NH

OH

O

N

O

F

NH

OH

O

N

O

F

Multigram scale synthesis of synthetic cannabinoid metabolites

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