Discovery of Novel Inhibitors of Uridine Diphosphate-N-Acetylenolpyruvylglucosamine Reductase (MurB) from Pseudomonas aeruginosa, an Opportunistic Infectious Agent Causing Death in Cystic Fibrosis Patients

Abstract

Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of K_d = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with K_d = 3.57 μM (LE 0.35).

Graphic abstract.

■. Introduction

Pseudomonas aeruginosa, a rod-shaped Gram-negative bacterium, is a frequent opportunistic agent of hospital-acquired infections.¹ In cystic fibrosis (CF), P. aeruginosa is responsible for 80% of the lung infections of CF patients by the age of 18.² Moreover, acquisition of P. aeruginosa by CF patients leads to 2.6 times higher risk of death, making chronic infection by this pathogen the major cause of death in this type of patient.^3,4 This bacterium has become resistant to current antibiotics such as β-lactams due to its low membrane permeability, abundant efflux pumps, and various antibiotic-degradative enzymes.⁵ Currently, the antibiotics used against P. aeruginosa in the clinic are limited and resistant strains are increasing in hospitals worldwide.⁶ Consequently, P. aeruginosa has been classified as one of the six pathogens in the world that most require new antibacterial drugs.^6–9 Therefore, it is necessary to design new antibiotics that act on novel targets of P. aeruginosa.

In the cell wall of most bacteria, one of the main components is peptidoglycan, a peptide cross-linked polymer of alternating N-acetyl-glucosamine and N-acetyl-muramic acid (UNAM) units.^10–12 The bacterial cell wall offers osmotic stability, and as a result, the enzymes involved in peptidoglycan biosynthesis are essential for bacterium survival. Therefore, the inhibition of any of these enzymes results in loss of bacterial cell wall followed by cell lysis.¹³ In recent decades, attention has been paid to the family of Mur enzymes,¹⁴ which synthesize the cell wall from the nucleotide sugar uridine diphosphate N-acetylglucosamine.

Antibiotics that block one of these enzymes are of interest, and some have already been developed. One example is fosfomycin, which inhibits MurA (UDP-N-acetylglucosamine-enolpyruvyl-transferase), the Mur enzyme that catalyzes the first step of the cell wall biosynthesis.¹⁵ Unfortunately, P. aeruginosa has also developed resistance to this antibiotic by enzymatic deactivation or by decreasing its cellular uptake.¹⁶

The second enzyme in the pathway, UDP-N-acetylenolpyruvoylglucosamine reductase (MurB), has also proved to be of interest as a novel target in P. aeruginosa because no MurB homologue exists in eukaryotic cells. MurB catalyzes the reduction of UDP-N-acetylglucosamine enolpyruvate (UN-AGEP), the product of MurA, to N-acetyl-muramic acid (UNAM) using FADH2 (Figure 1).^17,18

Figure 1. MurB enzymatic reaction.

P. aeruginosa MurB (Pa MurB) is a monomeric enzyme that comprised three different domains (PDB code 4JB1) (Figure 2). There are domain I (amino acids 1–75 and 336–339), domain II (amino acids 76-191) that binds the FAD cofactor, and domain III (amino acids 192–335), which has binding sites for the NADPH cofactor and substrate (UNAGEP).¹² The X-ray crystal structure of the complex of Pa MurB with FAD and NADP+ (PDB code: 4JB1) and the crystal structure of Escherichia coli MurB in complex with the substrate UNAGEP (Ec MurB, PDB code: 2MBR)¹⁹ have very similar threedimensional structures with identical residues in the active site¹² and FAD bound in the same manner. Both enzymes are type I UNAGEP reductases, similar to the Staphylococcus aureus MurB (SaMurB, type IIa) and Thermus caldophilus MurB (type IIb) structures^20,21 but lacking an α-helix and a protruding βαββ fold in the domain III (Figure 2). Extensive biochemical characterization of Ec MurB has led to the description of the reaction as a ping-pong bi-bi mechanism,²² where first the NADPH transfers an hydrogen to FAD followed by NADP⁺ dissociation from the enzyme. Successively, UNAGEP binds and the hydride is transferred from FADH₂ to the vinyl ether of UNAGEP, becoming UNAM.

Figure 2. Catalytic pocket and domain distribution of MurB, illustrating domain I in red, domain II in blue, and domain III in pink.

(a) Pa MurB in complex with NADP₊ (PDB code: 4JB1).¹² (b) Ec MurB in complex with UNAGEP (PDB code: 2MBR).¹⁹ FAD is shown in yellow, NADP⁺ in cyan, and UNAGEP in green.

Several MurB inhibitors have been designed using structure-based approaches²³ based on the cocrystal structures of Ec MurB and Sa MurB.^24–26 However, currently there are no inhibitors described against Pa MurB. In the present study, an in-house fragment library was screened against Pa MurB and this led to the identification ofsmall molecules that bind to Pa MurB. X-ray crystallography of one of the fragment hits was shown that it binds at the interface of FAD and the substrate binding pockets, which offers a novel strategy for the development of Pa MurB inhibitors.

■. Results

Chemical Scaffold Identification

A library of 960 rule-of-three compliant fragments was screened at a concentration of 5.0 mM against MurB using differential scanning fluorimetry (DSF) (Table 1). Fourteen fragments were shown as positive hits, and they were verified in triplicate at a 1.0 mM concentration. As a result, nine hits still had a positive thermal shift compared to the negative control. In the screening, the MurB–FAD complex was used due to the high affinity of FAD for MurB.¹²

Table 1. Hits from Differential Scanning Fluorimetry at Ligand Concentrations of 5.0 and 1.0 mM^a.

Fragment		ΔTM(°C) (±SEM) 5 mM1 mM		Fragment		ΔTM(°C) (±SEM) 5 mM 1 mM		Fragment		ΔTM(°C) (±SEM) 5 mM 1 mM
	1	+3.5	+1.0(0)		6	+1.5	+1.0(0)		11	+1.0	+0.2(0.2)
	2	+4.0	+1.0(0)		7	+1.0	+0.0(0)		12	+1.0	+0.0(0.0)
	3	+2.0	+1.5(0)		8	+1.0	+0.0(0)		13	+1.0	+0.0(0.0)
	4	+1.5	+0.5(0)		9	+1.0	+0.5(0)		14	+1.0	+0.5(0)
	5	+1.5	+1.0(0)		10	+1.0	+0.0(0)

^aFragment hits show shifts in protein melting temperatures (ΔT_M) from DMSO-d₆ at 10 μM Pa MurB concentration to the two different ligand concentrations. Each ligand is screened at concentrations of 5.0 mM (n = 1) and 1.0 mM (n = 3).

The fragment hits identified were validated by X-ray crystallography. However, only the pyrazole derivative 4 was successfully crystallized in complex with Pa MurB (Figure 3). X-ray crystallography showed that this fragment binds in the catalytic pocket in close proximity to FAD.¹⁷ The binding affinity of fragment 4 was then determined using isothermal titration calorimetry (ITC) where the affinity was measured to be K_d = 2.88 mM.

Figure 3.

X-ray structure of fragment 4 (dark blue) (a, b, PDB code: 7OR2) and NADP⁺ (cyan) (a, PDB code: 4JB1)¹² bound to the active site of Pa MurB. (a) Superimposition of Pa MurB in complex with fragment 4 (dark blue) and Pa MurB in complex with NADPH (cyan). (b) X-ray structure of fragment 4 bound to the active site of Pa MurB. FAD is depicted in yellow. Arpeggio²⁷ was used to analyze the interactions. Hydrogen bonds are shown as red dashed lines, π–π interactions are indicated in green dashed lines, and water molecules are shown as red dots. The final 2F0-Fc map around the ligand is shown in blue at 1σ.

All the analogues were screened using two different biophysical techniques, DSF (at 5 mM and/or 1 mM) and surface plasmon resonance (SPR) at 1 mM. Fragments with significant increases in ΔT_M or R higher than fragment 4 (R > R_f4) (see Figure 4) were validated by ITC. The goal was to identify potential binding analogues with higher binding affinity than fragment 4.

Figure 4.

Hit plot displaying results of analogues in SPR assay. Hits were characterized as higher RU (response units) normalized than fragment 4. Analogues in yellow showed curves that they did not reach equilibrium indicating aggregation and were ruled out as hits. Mw, molecular weight.

Modification of the Methyl Group

The methyl group on the pyrazole ring of fragment 4 points into a small pocket that the NADP⁺ does not fill (see Figure 3b). This pocket contains mainly hydrophilic residues such as Glu335, Asn243, Ser239, and Arg166. In addition, there is also a water molecule (W1, Figure 3b) in this small pocket, which is tightly bound to the hydrophilic residues by several hydrogen bonds. In order to further explore the structure–activity relationships (SAR) with this pocket, the methyl substituent was replaced by different groups that could possibly interact with the hydrophilic residues or with the water molecule (fragments 15–18) (Table 2).

Table 2. Structure and Biophysical Data for Fragments 4 and 15–18^a.

Fragment		R	ΔTM(°C) (±SEM) 1.0 mM	SPRRU>RUf4 1.0 mM	Kd (mM) ITC	LE
	4	Me	+0.5 (0)	–	2.88 ± 0.25	0.23
	15	NH2	0.0 (0)	no	–	–
	16	OH	+0.7 (0.2)	–	No binding	–
	17	CH2OH	+0.2 (0.2)	–	–	–
	18	CF3	+1.0 (0)	no	0.25 ± 0.04	0.27

^aShift in protein melting temperatures (ΔT_M) from DMSO-d₆ at 10 μM Pa MurB concentration and at 1.0 mM fragment concentrations (n = 3). SPR RU of each fragment at 1 mM in comparison with RU of fragment 4 (RU > RU_f4, RU = response units) (n = 2). Kd calculated using ITC (50 μM Pa MurB, 3.0 mM fragment). Ligand efficiencies were calculated as LE = −(RTlnK_d)/(number of heavy atoms) and are reported in kcal/mol per heavy atom. Dash entries in the table mean not measured.

The substitution at the 5-position (R in Table 2) in the pyrazole ring with a trifluromethyl group (fragment 18) was shown to give an increase in affinity where a K_d of 0.25 ± 0.04 mM was measured using ITC. This fragment was successfully crystallized in complex with Pa MurB, and the X-ray crystal structure shows a similar binding mode to the original fragment hit 4 (Figure 5a). However, the trifluromethyl group is shown to interact with the water W1 in the small pocket and another water (W3) next to it, and these interactions displace the rest of the fragment 18 closer to the α-helix 6 (see Figure 5a).

Figure 5. X-ray structures of fragment 4 and fragment 18 bound to the active site of Pa MurB.

(a) Superimposition of Pa MurB in complex with fragment 4 (dark blue) and with fragment 18 (white). (b) X-ray structure of fragment 18 bound to the active site of Pa MurB. FAD is depicted in yellow. Arpeggio²⁷ was used to analyze the interactions. Hydrogen bonds are shown as red dashed lines, π–π interactions are indicated in green dashed lines, and water molecules are shown as red dots. The final 2F0–Fc map around the ligand is shown in blue at 1σ.

Modification of the Pyrazole Ring

The CH on the 3-position of the pyrazole ring of the fragment 4 was changed for a N to observe if a possible hydrogen bond interaction could be formed with residue Tyr132 (fragment 19). This change did not give an increase in the melting temperature (Table 3). However, the X-ray crystal structure of Pa MurB in complex with fragment 19 shows a 180° flip of the five-membered ring due to the formation of an interaction of the N at the 3-position of the triazole with Arg166 (see Figure 6). In addition, this methyl group points to a small pocket that mainly contains hydrophilic residues such as Lys227, Tyr132, and Arg196 and a highly bound water molecule. These observations could suggest that an analogue ring consisting ofa pyrrole or imidazole containing two substituents at the 5- and 2-positions could also be of interest.

Table 3. Biophysical Data for Fragments 19–23^a.

Fragment		R1	R2	X	ΔTM (°C) (±SEM) 1.0 mM	SPR RU>RUf4 1.0 mM	Kd(μM) ITC	LE
	19	-			+0.5 (0)	no	877 ±154	0.28
	20	Me	Me	CH	+2.0 (0.2)	yes	110+14	0.34
	21	Me	Me	N	+0.7 (0.2)	no	–	–
	22	CFs	Me	CH	+2.0 (0)	yes	112+4.4	0.28
	23	Me	CH2CH2OH	CH	+2.0 (0)	–	280+15	0.28

^aShift in protein melting temperatures (ΔT_M) from DMSO-d₆ at a ligand concentration of 1.0 mM and 10 μM MurB concentration (n = 3). SPR RU of each fragment at 1 mM in comparison with RU of fragment 4 (RU > RU_f4, RU = response units) (n = 2). K_d calculated using ITC (50 μM MurB, 3.0 mM fragment). Ligand efficiencies were calculated as LE = –(RTlnK_d)/(number of heavy atoms) and are reported in kcal/mol per heavy atom. Dash entries in the table mean not measured.

Figure 6.

X-ray structure of fragment 4 and fragment 19 bound to the active site of Pa MurB. (a) Superimposition of Pa MurB in complex with fragment 4 (dark blue) and with fragment 19 (orange). (b) X-ray structure of fragment 19 bound to the active site of Pa MurB. FAD is depicted in yellow. Arpeggio²⁷ was used to analyze the interactions. Hydrogen bonds are shown as red dashed lines, π–π interactions are indicated in green dashed lines, and water molecules are as red dots. The final 2F0–Fc map around the ligand is shown in blue at 1σ.

Therefore, the pyrrole (fragment 20) and imidazole (fragment 21) analogues were synthesized, screened, and compared to fragment 4 using two different biophysical techniques, DSF and SPR (Table 3). Fragments with significant increase in ΔT_M or R higher than fragment 4 (R > Rf₄) at 1.0 mM were validated again by ITC. The pyrrole analogue 20 showed a greater thermal shift. Interestingly, one pyrrole derivative (fragment 3) was also identified in the initial library of hits. Subsequently, the methyl group at the 5-position was replaced with a CF₃ (fragment 22) in order to compare it with fragment 18. However, this modification showed no change in activity. Finally, the methyl at the 2-position was changed to a hydrophilic group to allow interaction with the hydrophilic residues or the water molecule of the small pocket (fragment 23). Unfortunately, a lower affinity was observed (K_d = 208 ± 15 μM). As a result, two series of compounds were taken for further optimization, the pyrazole fragment 18 and the pyrrole fragments 20 and 22.

Exploration of Substituents on the Phenyl Ring

Initially, the replacement of the phenyl group was studied (Table 4) and substitution for a benzyl or a thiophenemethyl group did not increase the affinity (fragments 24 and 25). The change of the phenyl ring for a thiophenyl or pyridinyl showed lower thermal shifts (fragments 26–28). Consequently, the phenyl ring was retained and the introduction of different substituents on the ring was explored (Table 4). An increase in the melting temperature was observed by adding large apolar groups such as halogens or methyl groups at the 2-substiuted-positions. It was observed that the larger the group, the higher the affinity (fragments 29–33). Introduction of polar and apolar groups in other positions on the phenyl ring did not improve affinity (fragments 34–40). The introduction of two apolar 3-substituted groups was shown to slightly increase the affinity (fragments 41 and 42). It was observed that some of the initial fragment hits contained phenyl groups with 3,4-dichlorophenyl substituents (fragments 5, 6, and 9). Therefore, this modification was incorporated into the developed compounds and this change showed an increase in affinity, K_d = 26.1 ± 2.7 μM (compound 43).

Table 4. Biophysical Data for Fragments 24-43_^a.

Fragment	R	ΔTM (°C) (±SEM) 1.0 mM	SPR RU>RUf4 1.0 mM	Kd(μM) ITC	LE
	24	benzyl	+1.0(0)	–	–	–
	25	thiophen-2-yl)methyl	+1.0(0)	–	–	–
	26	thiophen-2-yl	+0.3 (0.2)	–	–	–
	27	3-pyridinyl	+0.3 (0.2)	–	–	–
	28	4-pyridinyl	0.0(0)	–	–	–
	29	2-fluorophenyl	+1.0(0)	–	–	–
	30	2-chlorophenyl	+1.5(0)	yes	148+15	0.27
	31	2-bromophenyl	+2.0(0)	yes	85,.5 ± 7.7	0.29
	32	2-methylphenyl	+2.0(0)	yes	44.6 + 3.1	0.31
	33	2-trifluoromethylphenyl	+1.0(0)	–	–	–
34	4-trifluoromethylphenyl	+1.0(0)	–	–	–
35	4-methoxyphenyl	+2.0(0)	no	–	–
36	2-methyl-4-nitrophenyl	+1.7(0.7)	yes	208 ± 28	0.23
37	3-carbamoylphenyl	+0.5(0)	–	–	–
38	2,3-dimethylphenyl	+2.0(0)	yes	99.0 ± 7.2	0.27
39	2,4-dimethylphenyl	+2.3(0.2)	yes	85.5 ± 8.0	0.28
40	2,5-dimethylphenyl	+2.0(0)	yes	138 + 16	0.26
41	2,6-dimethylphenyl	+2.5(0)	–	25.8 + 5.5	0.31
42	2,6-dichlorophenyl	2.5(0)	yes	60.2 ± 4.4	0.29
43	3,4-dichlorophenyl	+2.3(0.2)	yes	26.1+2.7	0.31

^aShift in protein melting temperatures (ΔT_M) from DMSO at 1.0 mM fragment concentration and 10 μM MurB concentration (n = 3). SPR RU of each fragment at 1 mM in comparison with RU of fragment 4 (RU > RU_f4, RU = response units) (n = 2). K_d calculated using ITC (50 μM MurB, 3.0 mM fragment; except for fragment 41, where 200 μM MurB was used). Ligand efficiencies were calculated as LE = −(RTlnK_d)/(number of heavy atoms) and are reported in kcal/mol per heavy atom. Dash entries in the table mean not measured.

The introduction of the 2-methylphenyl group could orientate the pyrazole ring and the phenyl ring at around 90°, which is the conformation observed in the crystal structure of fragment 18 (Figure 5). Molecular simulations were performed to examine this (Figure 8).²⁸ In the Pa MurB crystal structure, fragment 18 shows a dihedral angle of 86.5° between the pyrazole and the phenyl ring, whereas the minimum energy structure of fragment 18 has a dihedral angle of 120°. If an ortho substituent is added in the phenyl ring, then the dihedral angle of the minimum energy structure becomes similar to the one in the crystal structure. Substituents that give closer angles to the crystal structure showed better affinity (59.8° for 2-fluorophenyl (29), 70.0° for 2-chlorophenyl (30), 69.4° for 2-bromophenyl (31), 79.6° for 2-methylphenyl (32), and 90.5° for 2,6-dimethylphenyl (41)) (Table 4). The 3,4-dichlorophenyl group could improve the binding by hydrophobic interactions with Leu300, Leu228, and Val301 (see Figure 5b). Unfortunately, attempts to obtain X-ray crystal structures of these substituted compounds with Pa MurB were not successful.

Figure 8.

(a) Comparison of the dihedral angles between the conformation of fragment 18 in complex with Pa MurB and (b) minimum energy conformations of fragments 18, 41, and 53. See the SI for minimum energy conformations of other fragments.

As a result, the 2-methylphenyl substitution was merged with the 3,4-dichlorophenyl substitution (fragments 44 and 45) (Table 5). This allowed the identification of the best substitution pattern, which was a phenyl ring substituted with a methyl group in the 2-position and two chlorines in the 4- and 5-position on the phenyl ring (fragment 44). Subsequently, some of these modifications were successfully translated into the pyrrole fragments 20 and 22 to yield fragments 46–52 and fragments 53–55, respectively. Additionally, it was observed that both chlorine atoms are important for binding as there is a decrease in thermal shift if one of the chlorines is removed (fragments 49 and 50). However, the fragment containing the pyrazole ring gave the lowest K_d of 3.57 ± 0.76 μM (fragment 44) (see Figure 7a).

Table 5. Biophysical Data for Fragments 44-55_^a.

Fragment	R	ΔTM (°C) (±SEM) 1.0 mM	SPR RU>RUf4 1.0 mM	Kd(μM) ITC	LE
44	2-methyl-4,5-dichlorophenyl	+5.0 (0)	yes	3.57 ± 0.76	0.35
45	2-methyl-3,4-dichlorophenyl	+3.7 (0.2)	yes	11.3 + 2.5	0.32
46	2-methylphenyl	+2.0 (0)	yes	64.5 + 6.1	0.34
47	2,6-dimethylphenyl	+3.0 (0)	no	62.5 ± 3.5	0.32
48	3,4-dichlorophenyl	+2.3 (0.2)	no	47.8 ± 3.8	0.33
49	3-chlorophenyl	+1.5 (0)	yes	–	–
50	4-chlorophenyl	+1.5 (0)	no	–	–
51	2-methyl-4,5-dichlorophenyl	+3.5 (0)	–	24.1+4.0	0.33
52	2-methyl-3,4-dichlorophenyl	+4.0 (0.2)	–	24.3 ± 7.4	0.33
53	2-methylphenyl	+2.5 (0)	yes	40.2+1.4	0.30
54	2-methyl-4,5-dichlorophenyl	+4.7 (0.2)	–	8.00+1.1	0.32
55	2-methyl-3,4-dichlorophenyl	+2.7 (0.2)	yes	11.4 + 3.9	0.31
NADP+	–	+3.5 (0)	–	23.6 ± 2.4	–
NADPH	–	+3.0 (0)	–	–	–

^aShift in protein melting temperatures (ΔT_M) from DMSO-d₆ at 1.0 mM fragment concentration and 10 μM Pa MurB concentration (n = 3). SPR RU of each compound at 1.0 mM in comparison with RU of fragment 4 (RU > RU_f4, RU = response units) (n = 2). K_d calculated using ITC (50 μM Pa MurB, 3.0 mM fragment; except for fragments 44, 51, and 54, which were tested at 1.0 mM, and fragments 45 and 52, which were tested at 0.5 mM). Ligand efficiencies were calculated as LE = −(RTlnK_d)/(number of heavy atoms) and are reported in kcal/mol per heavy atom. Dash entries in the table mean not measured.

Figure 7.

ITC titration curves at key stages of optimization for (a) pyrazole and (b) pyrrole series. Titrations performed at 50 μM Pa MurB with 3.0 mM fragments, except for fragments 44 and M 54 where 1.0 mM was used. See the SI for titration curves of other fragments and NADP⁺.

In the pyrrole series, the two substituents at the 2- and 5-position of the pyrrole ring can also affect the dihedral angle. Consequently, molecular simulations were also performed. fragments 20 and 22, which have angles of 100 and 70.1°, respectively, have an angle more similar to the crystal structure (86.5°) than Fragment 18, which has an angle of 120°. Fragments 20 and 22 showed greater affinity (Table 3) (see Figure 7b). However, these two fragments both had similar binding affinities. This suggests that the CF₃ group is not improving the affinity as in the pyrazoles. However, if a 2-methyl group is added in the phenyl ring of these fragments, then both resulting fragments (fragments 46 and 53) have an angle more similar to the one of the crystal structure (90.0 and 91.0°, respectively). Due to the fact that fragment 53 has a CF₃ group in the 2-position instead of the CH₃, a higher affinity is now observed. Consequently, once the dihedral angle is close to what is observed in the X-ray crystal structure, better affinity can be observed by the addition of the CF₃ group. Addition of a second ortho-methyl group (fragment 47) did not change the dihedral angle (90.0°) as it did in the pyrazole series, and no change in affinity was observed.

Modifications at the Carboxylic Acid

Upon exploration of the SAR on the two rings, a further approach was explored to examine whether the compounds can be grown from the carboxylic acid moiety. NADP⁺ was shown by X-ray crystallography to form a “sandwich” π-stacking interaction with its adenine ring to the Tyr196 and Tyr264 at the entrance of the binding pocket (see Figure 3a). In the absence of NADP⁺, these two tyrosine residues are not stabilized and the α-helix and βαββ fold in domain III (see Figure 2a) are flexible. The carboxylic acid of fragment 4 forms a polar interaction with the Tyr132 and it points to the same direction as the adenine ring of NADP⁺ (see Figure 3b). Consequently, this is a good vector for developing these compounds. Therefore, the carboxylic acids of some of the previously developed fragments were grown with different functional groups (Table 6). Amide and ester derivatives did not show any activity (fragments 56–58). Only the sulfonamide derivatives were detected to be active by both DSF and ITC. If the methanesulfonyl group (fragment 59) was changed for a benzenesulfonyl group (fragment 60), then a higher ΔT_M was observed (from +0.5 to +1.0 °C, respectively). However, fragment 60 (K_d = 0.32 ± 0.06 mM) showed a similar binding affinity than the acid analogue 18 (K_d = 0.25 ± 0.04 mM). The activity was lost when the phenyl group was changed for a benzyl group (fragment 61).

Table 6. Biophysical Data for Fragments 56–64_^a.

Fragment		R	X	Y	ΔTM (°C) (±SEM) 1.0 mM	SPR RU>RUf4 1.0 mM	Kd(μM) ITC	LE
	56	NH2	CF3	H	0.0 (0)	–	–	–
	57	OEt	CF3	CL	0.0 (0)	–	–	–
	58	N-benzylamino	Me	H	0.0 (0)	–	–	–
	59	N-(methanesulfonyl)amino	CF3	H	+0.5 (0)	no	–	–
	60	N-(benzenesulfonyl)amino	CF3	H	+1.0 (0)	yes	324 ± 59	0.18
	61	N-(benzylsulfonyl)amino	Me	H	0.0 (0)	–	–	–
		R1	R2
	62	phenyl	2,6-dimethyl phenyl	+3.0 (0)	yes	25.6 + 2.9	0.22
	63	3-bromophenyl		+1.3 (0.2)	–	–	–
	64	phenyl	2-methyl-4,5-dichlorophenyl	+2.7 (0.3)	–	12.0 + 3.7	0.22

^aShift in protein melting temperatures (ΔT_M) from DMSO-d6 at 1.0 mM fragment concentration and 10 μM Pa MurB concentration (n = 3). SPR RU of each fragment at 1.0 mM in comparison with RU of fragment 4 (RU > RU_f4, RU = response units) (n = 2). K_d calculated using ITC (50 μM Pa MurB, 3.0 mM fragment). Ligand efficiencies were calculated as LE = −(RTlnK_d)/(number of heavy atoms) and are reported in kcal/mol per heavy atom. Dash entries in the table mean not measured.

Consequently, the effect in the binding affinity by the substitution of the acid moiety for a benzenesulfonyl group was investigated with the most optimized fragments such as 41 and 44. After growing fragment 41,a ΔT_M = +3.0 °C and a K_d = 25.6 ± 2.9 μΜ were observed (fragment 62). The addition of a bromine substituent on this N-benzenesulfonyl group decreased the ΔT_M to +1.3 °C (fragment 63). In the case of fragment 44, after the addition of the N-benzenesulfonyl group, a ΔT_M of +2.7 °C and a K_d = 12.0 ± 3.7μM were observed (fragment 64). Attempts to obtain X-ray crystal structures of these fragments proved unsuccessful.

Synthetic Chemistry

Different synthetic routes were employed to prepare the different types of fragments (Scheme 1). Pyrazoles (fragments 17 and 24–45) were prepared using two key steps. The first step involved the reaction of α,β-unsaturated keto esters with different hydrazines in the presence of Et₃N and EtOH at 80 °C to yield the desired 4,5-substituted pyrazoles.²⁹ The 3,4-substituted regioisomers were only observed when aliphatic hydrazines were employed as starting materials (synthesis of fragments 24 and 25). The second step involved the hydrolysis of the resulting esters with 2 M NaOH in EtOH at 80 °C. The triazole 19 was prepared from ethyl acetoacetate and phenylboronic acid in the presence of sodium azide, copper acetate, and catalytic amounts of piperidine.³⁰ No other major regioisomer was observed. The resulting product was hydrolyzed as previously. Pyrrole derivatives (20, 22, 23, and 46-55) were also prepared using two key steps. The first step involved the reaction of different α,β-keto esters with different amines using the Paal–Knorr reaction.^31,32 This reaction used acetic acid as a solvent at 120 °C when R² = CH₃. However, when R² = CF₃, para-toluenesulfonic acid in toluene at 110 °C was used instead due to the degradation ofthe α,β-keto esters in the acetic acid. Finally, the resulting ester was hydrolyzed as before. Imidazole 21 was also prepared in two main steps. In the first step, an α,β-keto ester analogue reacted with aniline in the presence of trifluoroacetic acid and butyronitrile at 120 °C.³³ The product was hydrolyzed by 6 M HCl at 100 °C. The basic conditions used previously were not used here due to the degradation of the product. Finally, the amide and sulfonamide derivatives (58 and 59–64, respectively) were prepared from the corresponding acids in a one-pot step. For the amide, the carboxylic acid was first converted into an acyl chloride by reaction with thionyl chloride at 80 °C. This intermediate was then reacted with benzyl amine in the presence of pyridine in 1,4-dioxane at room temperature to yield the corresponding amide. For the sulfonamides, the carboxylic acid reacted with the desired sulfonamide through a coupling reaction involving the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and catalytic amounts of 4-dimethylaminopyridine in dichloromethane at room temperature.

■. Discussion And Conclusions

The development of inhibitors of peptidoglycan targeting MurB enzymes has proved exceptionally challenging and currently, there are no approved antibiotics that target any of the nine subsequent steps after MurA. This study illustrates the successful application of a fragment-based approach to obtain, for the first time, a potent candidate that binds to the catalytic pocket of Pa MurB.

Screening of 960 fragment libraries by DSF and validation using X-ray crystallography allowed the identification of a pyrazole derived fragment 4, which showed a K_d of 2.8 mM by ITC. This fragment was synthetically modified to improve its binding affinity. Different substitutions were tested in comparison with the initial fragment 4 using two different biophysical techniques, DSF and SPR at 1.0 mM. Binding parameters were calculated using ITC for those fragments with significant binding increase than the initial fragment.

The substitution of the 5-methyl group on fragment 4 for a 5-trifluoromethyl group on the pyrazole ring or the substitution of the 5-methylpyrazole for a 2,5-dimethylpyrrole or 2-methyl-5-trifluoromethylpyrrole ring increased the binding affinity to values of K_d around 0.1–0.3 mM (fragments 18, 20, and 22, respectively). Subsequently, the introduction of an ortho-methyl group or a 3,4-dichloro group on the phenyl ring decreased the K_d to around 50–20 μM while maintaining the ligand efficiency. The initial library of hits contained fragments with a 2,5-dimethylpyrrole ring or 3,4-dichlolorophenyl groups, suggesting that it was important to look at these fragments for the optimization of fragment 4. The fact that the substitutions could be translated from pyrazoles to pyrroles suggests that the binding mode could be similar; however, pyrazoles showed to bind tighter. Exploration of the SAR on the carboxylic showed that a phenylsulfonamide group was tolerated, but no gain in affinity was observed.

The best fragments were obtained by merging the ortho-methyl group with the meta,para-dichloro groups and showed an LE = 0.35 (fragment 44) and an LE = 0.32 (fragment 54). As a result, fragments with higher potency to that of the cofactors of Pa MurB have been designed. Consequently, these fragments can grow into Pa MurB inhibitors that would disrupt cell wall biosynthesis. This fragment is a good candidate because the binding is mediated by π–π interactions between FAD and Tyr132; thus, there is no possibility of P. aeruginosa becoming resistant by mutations in MurB. Although Tyr132 is quite conserved in bacteria (in MurB from E. coli and S. aureus), some bacteria such as T. caldophilus have R132. However, if Tyr132 were to mutate to another amino acid, then the π-π interaction would likely be replaced with a π-polar interaction. A future work can elaborate this fragment in order to increase the interactions with MurB in the catalytic pocket.

■. Experimental Section

Cloning, Protein Expression, and Purification

MurB gen of P. aeruginosa was designed based on the sequence available in the NCBI database and synthesized using GeneArt (Invitrogen). The gene was cloned between the BamHI and HindIII sites in pET28a vector (Novagen) and modified with an N-terminal 6xHis-SUMO tag. The resulting plasmid was confirmed by DNA sequencing and transformed into the E. coli BL21(DE3) strain (Invitrogen).

Transformed cells were grown to OD₆₁₀ = 0.6 in LB media (Invitrogen) containing 30 mg L^-1 kanamycin at 37 °C. At this OD, protein expression was induced using 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at 18 °C. Cells were harvested by centrifugation and resuspended in 50 mM Tris (pH 8), 0.5 M NaCl, 5 mM MgCl₂, and 20 mM imidazole with protease inhibitor tablets (Roche) and DNAse I. Cells were lysed, sonicated, and centrifuged at 30,000g for 45 min to collect the supernatant.

Pa MurB was purified with a HiTrap IMAC Sepharose FF column (GE-Healthcare), equilibrated with 50 mM Tris (pH 7.5), 0.5 MNaCl, and 20 mM imidazole, and the elution was performed in the same buffer with 500 mM imidazole. Imidazole was removed with overnight dialysis in 50 mM Tris (pH 8) and NaCl (250 mM). Meanwhile, the SUMO tag was cleaved by adding Ulp1 Protease at a 1:100 ratio. The SUMO tag, Ulp1 protease, and Pa MurB were separated using a Superdex 200 column equilibrated with 50 mM Tris (pH 8) and NaCl (250 mM). Fraction purity was determined by SDS-PAGE, and the purest fractions were pooled and concentrated to 25.5 mg mL^-1 in the same buffer, flash frozen in liquid nitrogen, and stored at −80 °C.

Differential Scanning Fluorimetry

Differential scanning fluorimetry was performed using a Bio-Rad CFX96 Touch PCR system from 25 to 95 °C in 0.5 °C increments of 30 s duration. Samples were run in 96-well clear-bottomed plates. For these experiments, each well contained a final volume of 25 μL, consisting of 25 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5X SyproOrange, 10 μM Pa MurB, and either 5% DMSO-d₆ or ligand at 5.0 or 1.0 mMin DMSO-d₆ as specified. Controls were used for all experiments, with DMSO-d₆ (reference) and NADP⁺ (positive control) used instead of the fragment. The resulting data (fluorescence intensity vs temperature) was fitted to obtain the denaturing temperature T_M (point of sigmoidal inflection) as the maximum of each curve’s derivative. This analysis was performed using Microsoft Office Excel. The reference unfolding temperature of Pa MurB in 5% DMSO-d₆ was subtracted from the values in the presence of the fragment to obtain the thermal shift. The thermal shifts at 5.0 mM were recorded once (n = 1), and the thermal shifts at 1.0 mM were recorded three times (n = 3).

Crystallization and Data Collection

Crystallization of the complexes was carried out by seeding using Pa MurB:NADPH microcrystals as a nucleation starting point. Pa MurB:NADPH crystals were set up, manually mixing 1 μL of Pa MurB at 25.5 mg μL^-1 and 2 mM NADPH and 1 μL of crystallization condition mix (160 mM (NH₄)SO₄, 80 mM sodium acetate at pH 4.6, 20% PEG 4000, and 20% glycerol) using the hanging-drop vapor-diffusion method in 24-well VDX greased plates (Hampton Research, Aliso Viejo, California, USA). Crystallization of the fragment complexes or Apo crystal was prepared using the sitting-drop vapor-diffusion method at 25 °C, and the plates were mounted in the Mosquito Crystal robot (TPP Labtech, Hertfordshire, UK). In each crystallization drop, 0.4 μL of reservoir solution and 0.05 μL of microseeds were added to 0.2 μL of protein solution. The protein droplets were equilibrated over 70 μL of reservoir solution mix (30% glycerol and 22% PEG 1500). Suitable crystals for X-ray diffraction grew in 1 week.

Diffraction data were processed and reduced using XDS³⁰ and Aimless from the CCP4 suite.³¹ All the structures crystallized in the P6₁ space group with one protomer per asymmetric unit. Initial phases were determined using the previously published structure of Pa MurB (PDB code: 4JB1)¹² as a model with the PHASER³² program from the PHENIX software package.³³ Model building and structure validation were refined using PHENIX and Coot.³⁴

All data sets were collected at stations I03 and I04-1 at the Diamond Light Source (Oxford, UK). Data collection and refinement statistics are summarized in Table S1

General Chemistry

Commercially available starting materials and fragments 4, 15, 16, 18, and 56 were obtained from Sigma–Aldrich, Acros, Fluorochem, and Alfa Aesar. All non-aqueous reactions were performed under a nitrogen atmosphere unless otherwise stated. Watersensitive reactions were performed in anhydrous solvents in oven-dried glassware cooled under nitrogen before use. Petrol refers to petroleum spirit (b.p.: 40–60 °C), THF refers to tetrahydrofuran, and DCM refers to dichloromethane. A rotary evaporator was used to remove the solvents in vacuo.

Thin-layer chromatography was performed using Merck glass-backed silica (Kieselgel 60 F₂₅₄ 0.25 mm plates). An ultraviolet lamp (λ-_max = 254 nm) and KMnO₄ were used for visualization. Flash column chromatographywas performed using automated Isolera Spektra One/Four purification systems and an appropriately sized Biotage SNAP column containing KP-silica gel (50 μM). A Perkin-Elmer One FT-IR spectrometer was used to analyze the infrared spectra. Absorptions are reported in wavenumbers (cm⁻¹).

An SQD2 mass spectrometer detector (Waters) utilizing electrospray ionization (ESI) was used for low-resolution mass spectrometry (MS). High-resolution mass spectrometry (HRMS) was recorded using a Waters LCT Premier Time of Flight (TOF) mass spectrometer or a Micromass Quadrapole-Time of Flight (Q-TOF) spectrometer.

The purity of tested fragments was determined by high-performance liquid chromatography (HPLC). All final fragments had purity greater than 95% unless otherwise stated. HPLC was carried out using an Ultra Performance Liquid Chromatographic system (UPLC) Waters Acquity H-class. Samples were detected using a Waters Acquity TUV detector at two wavelengths (254 and 280 nm). Samples were run using an Acquity UPLC HSS column and a flow rate of 0.8 mL/min. The eluent consisted of 0.1% formic acid in water (A) and acetonitrile (B) (gradient, from 95% A to 5% A over a period of 4 min).

Proton (¹H), carbon (¹³C), and fluorine (¹⁹F) NMR data were collected on a Bruker 400 MHz spectrometer. Data were collected at 300 K. Chemical shifts (δ) are given in parts per million (ppm), and they are referenced to the residual solvent peak. ¹⁹F NMR spectra were references to TFA. Coupling constants (J) are reported in Hertz (Hz), and splitting patterns are reported in an abbreviated manner: app. (apparent), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad).

General Procedure A

A dispersion of 20% Pd(OH)₂ on carbon (0.5 equiv) was added to a solution of the benzyl derivative (1.0 eq) in ethanol (0.1 M). The mixture was stirred under hydrogen at room temperature for 24 h. Subsequently, it was filtered through celite and concentrated in vacuo to give a crude product.

General Procedure B

An aqueous solution of 2 M NaOH (6.0 eq) was added dropwise to a solution of the ester derivative (1.0 equiv) in ethanol (0.2 M), and the resulting mixture was stirred at 80 °C. After consumption of the starting material, the solvent was removed in vacuo, water (5 mL for each 1.00 mmol of the ester derivative) was added, and the mixture was washed with ethyl acetate (5 mL for each 1.00 mmol of the ester derivative). Successively, the aqueous layer was acidified to pH 4 with an aqueous solution of 1 M HCl and extracted with ethyl acetate (3 X 5 mL for each 1.00 mmol of the ester derivative). The organic phases were combined, washed with brine (5 mL for each 1.00 mmol of the ester derivative), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to yield a crude product or the corresponding acid derivative.

General Procedure C.³⁶

The amine derivative (1.0 equiv) was added to a solution of the carbonyl derivative (1.0 equiv) in acetic acid (0.27 M), and the mixture was stirred at 120 °C until consumption of the starting material. After cooling to room temperature, water (4 mL for each 1.00 mmol of amine derivative) was added and the mixture was extracted with EtOAc (3 × 8 mL for each 1.00 mmol of amine derivative). The organic phases were combined, dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product.

General Procedure D.³⁷

TsOH.H₂O (0.5 equiv) was added to a solution of the amine derivative (1.0 equiv) and the dicarbonyl derivative S9 (1.0 equiv) in toluene (concentration of the amine derivative: 0.27 M), and the mixture was stirred at 110 °C. After consumption of the starting materials, the mixture was allowed to cool to room temperature and water (3 mL for each 1.00 mmol of the amine derivative) and EtOAc (3 mL for each 1.00 mmol of the amine derivative) were added. The phases were separated, and the aqueous phase was extracted with EtOAc (3 × 3 mL for each 1.00 mmol of the amine derivative). The organic phases were combined, dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product.

General Procedure E.²⁹

Triethylamine (1.2 or 2.4 equiv if the hydrazine dihydrochloride salt derivative was used) was added dropwise to a stirred solution of ethyl-3-ethoxy-2-(2,2,2-trifluoroacetyl)acrylate (1.0 equiv) and the hydrazine hydrochloride salt derivative (1.0 equiv) in ethanol (concentration of acrylate derivative: 0.4 M), and the resulting mixture was stirred at 80 °C. After consumption ofthe starting material, the mixture was allowed to cool to room temperature and the solvent evaporated under reduced pressure. EtOAc (2 mL for each 1.00 mmol of the hydrazine derivative) and water (2 mL for each 1.00 mmol of the hydrazine derivative) were added, the phases were separated, and the aqueous phase was extracted with EtOAc (3 × 2 mL for each 1.00 mmol of the hydrazine derivative). The organic phases were combined, washed with brine (2 mL for each 1.00 mmol of the hydrazine derivative), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product.

General Procedure F

A solution of NaNO₂ (1.2 equiv) in H₂O (1.8 M) was added dropwise to a solution of the amine derivative (1.0 equiv) in concentrated HCl (0.3 M) at 0 °C. The reaction was stirred for 30 min at 0 °C, and the insolubilities were removed. Successively, a solution of SnCl₂η(H₂O)₂ (3.0 equiv) in 1:1 concentrated HCl-H₂O (1.1 M) was added. After stirring the reaction for 2.5 h at 0 °C, the precipitate was filtered, washed with a cold aqueous solution of 6.0 M HCl, washed with hexane, and dried in vacuo to give the hydrazine derivative.

General Procedure G

1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1.1 equiv), the sulfonamide derivative (1.1 equiv), and 4-dimethylaminopyridine (0.1 equiv) were added to a solution of the carboxylic acid derivative (1.0 equiv) in DCM (0.1 M). After stirring at room temperature for 18 h, water (8 mL for each 1.00 mmol of carboxylic acid derivative) was added, the phases were separated, and the aqueous phase was extracted with DCM (3 × 8 mL for each 1.00 mmol of carboxylic acid derivative). The organic phases were combined, dried under anhydrous sodium sulfate, filtered, and concentrated in vacuo to give a crude product or the sulfonamide derivative.

Ethyl 4-(Benzyloxy)-3-oxobutanoate (S1)

Benzyl alcohol (2.00 mL, 19.3 mmol) was added to a suspension of 60% NaH in mineral oil (1.16 g, 29.0 mmol) in THF (24 mL) at 0 °C. After stirring at room temperature for 2 h, ethyl 4-chloroacetoacetate (2.1 mL, 15.4 mmol) was added dropwise over 30 min and the reaction mixture was stirred at room temperature for 18 h. Successively, the mixture was acidified to pH 2 with an aqueous solution of 6.0 M HCl. Water (10 mL) and EtOAc (10 mL) were added, the phases were separated, and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to yield a crude product. The crude product was purified by flash column chromatography (0–5% EtOAc in petrol) to give the benzyl derivative S1³⁹ as yellow oil (2.50 g, 10.6 mmol, 55%). R_f 0.41 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.40-7.27 (m, 5H), 4.58 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 4.14 (s, 2H), 3.53 (s, 2H), 1.24 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 201.7, 167.0, 137.0, 128.6, 128.1, 127.9, 74.8, 73.5, 61.4,46.1, 14.1 ppm. MS (ESI): [M + H]+237.1. IR (neat): ν_max 3064–2871 (w, C–H), 1721 (s, C⩵O), 1656 (w, C⩵O), 1454 (m), 1393 (m), 1367 (m), 1317 (m), 1229 (m), 1098 (m), 1030 (m) cm^-1.

Ethyl 5-[(Benzyloxy)methyl]-1-phenyl-1H-pyrazole-4-carboxylate(S2)

The benzyl derivative S1 (0.50 g, 2.11 mmol) and N,N-dimethylformamide dimethyl acetal (0.35 mL, 2.63 mmol) were stirred at room temperature for 18 h and concentrated in vacuo to yield the crude product. This was dissolved in EtOH (4 mL), phenylhydrazine hydrochloride (0.30 g, 2.11 mmol) and Et₃N (0.35 mL, 2.53 mmol) were added, and the reaction mixture was stirred at 80 °C for 18 h. After allowing it to cool to room temperature, the solvent was evaporated under reduced pressure and the residue was taken up in ethyl acetate (4 mL) and water (4 mL). The phases were separated, and the aqueous phase was extracted with EtOAc (3 × 4 mL). The organic phases were combined, washed with brine (4 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product. The crude product was purified using flash column chromatography (7% EtOAc in petrol) to give the benzyl derivative S2 as colorless oil (0.38 g, 1.13 mmol, 54%). R_f: 0.50 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.09 (s, 1H), 7.65 (d, J = 7.7 Hz, 2H), 7.50-7.40 (m, 3H), 7.36-7.25 (m, 5H), 4.81 (s, 2H), 4.63 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 163.3, 142.0, 138.9, 137.6, 129.2, 128.7, 128.5.128.0. 127.9.125.1.114.9, 73.1, 60.4,60.3, 14.5 ppm. MS (ESI): [M + H]+ 337.3. IR (neat): ν_max 3113–2868 (w, C–H), 1708 (s, C = O), 1549 (m), 1501 (m), 1379 (m), 1265 (s), 1241 (s), 1189 (m), 1093 (s), 1063 (s), 1022 (s) cm^-1.

Ethyl 5-(Hydroxymethyl)-1-phenyl-1H-pyrazole-4-carboxylate (S3)

According to General Procedure A, the benzyl derivative S2 (0.32 g, 0.95 mmol) gave a crude product. The crude product was purified using flash column chromatography (5% EtOAc in petrol) to give the alcohol derivative S3 as colorless oil (0.22 g, 0.89 mmol, 94%). R_f. 0.50 (50:50 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.02 (s, 1H), 7.53-7.42 (m, 5H), 4.76 (s, 2H), 4.37 (q, J =7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDQ3): δ 164.9, 146.6, 141.8, 138.3, 129.4, 129.1, 125.4, 114.0, 61.0, 55.1, 14.4 ppm. MS (ESI): [M +H]+247.1. IR (neat): ν_max3272 (s, O–H), 3057-2851 (w, C–H), 1698 (s, C⩵O), 1558 (m), 1497 (m), 1460 (m), 1410 (m), 1381(m), 1244 (s), 1207 (s), 1090 (s), 1026 (s) cm^-1.

5-(Hydroxymethyl)-1-phenyl-1H-pyrazole-4-carboxylic Acid (17)

According to General Procedure B, the alcohol derivative S3 (56 mg, 0.23 mmol) was stirred for 4 h to give the acid derivative 17 as a white amorphous solid (47 mg, 0.22 mmol, 94%). R_f: 0.16 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CD₃OD): δ 8.05 (s, 1H), 7.65-7.47 (m, 5 H), 4.80 (s, 2H) ppm. ¹³C NMR (100 MHz, CD₃OD): δ 166.5, 146.9, 143.0, 139.8, 130.3, 130.2, 126.5, 115.2, 53.8 ppm. MS (ESI): [M + H]+ 219.0. HPLC: retention time: 1.31 min (>99%). IR (neat): ν_max 3295 (s, O–H), 3063-2548 (s, C-H), 1673 (s, C⩵O), 1560 (s), 1458 (m), 1416 (m), 1282 (s), 1262 (s), 1226 (m), 1096 (w), 1019 (s), 936 (s) cm⁻¹. HRMS: calculated for C₁₁H₁₀N₂O₃ [M + H]+ = 219.0770, observed = 219.0765.

Ethyl 5-Methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (S4)

According to a procedure,³⁵ sodium azide (0.26 g, 4.10 mmol) and copper(II) acetate (37.2 mg, 0.20 mmol) were added to a solution of phenylboronic acid (0.25 g, 2.05 mmol) in DMSO (10 mL) and water (1 mL). After stirring for 4 h at room temperature, ethyl acetoacetate (0.28 mL, 2.25 mmol) and piperidine (41 μL, 0.41 mmol) were added and the reaction mixture was stirred at 80 °C for 18 h. Successively, the reaction was allowed to cool to room temperature and an aqueous solution of 5% ammonium hydroxide (20 mL) and EtOAc (20 mL) were added. The phases were separated, and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, washed with brine (3 × 30 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product, which was purified by flash column chromatography (10-20% EtOAc in petrol) to give the ester derivative S4⁴⁰ as yellow oil (0.32 g, 1.38 mmol, 67%). R_f: 0.43 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.58-7.48 (m,3H), 7.45-7.38 (m, 2H), 4.43 (q, J = 7.1 Hz, 2H), 2.55 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 161.8, 138.9, 136.7, 135.5, 130.1, 129.7, 125.4, 61.1, 14.4, 10.0 ppm. MS (ESI): [M + H]⁺ 232.1. IR (neat): ν_max 3061-2871 (w, C–H), 1712 (s, C⩵O), 1597 (w), 1566 (w), 1504 (m), 1423 (m), 1374 (m), 1350 (w), 1278 (w), 1244 (s), 1228 (s), 1207 (s), 1107 (s), 1010 (w), 980 (w) cm^-1.

5-Methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylic Add (19)

According to General Procedure B, the ester derivative S4 (83 mg, 0.36 mmol) was stirred for 3 h to give the acid derivative 19⁴¹ as a white amorphous solid (70 mg, 0.34 mmol, 94%). R_f: 0.29 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 10.83 (br. s, 1H), 7.68-7.36 (m, 5H), 2.62 (s, 3H) ppm. ¹³CNMR (100 MHz, CDCl³): δ 165.5.140.0. 136.1.135.3.130.4.129.8.125.4.10.2ppm.MS (ESI): [M + H]+ 204.1. HPLC: retention time: 1.42 min (>99%). IR (neat): vmax 3067 (s, O–H), 2928-2580 (w, C–H), 1681 (s, C⩵O), 1566 (m), 1494 (m), 1452 (m), 1270 (m), 1241 (m), 1229 (m), 1117 (m), 1090 (m) cm^-1.

Ethyl 2-Acetyl-4-oxopentanoate (S5)

According to a procedure,⁴² chloroacetone (1.26 mL, 15.8 mmol) was added to a solution of ethyl acetoacetate (2.00 mL, 15.8 mmol) in triethylamine (15 mL) and the reaction mixture was stirred at 90 °C for 18 h. After allowing the mixture to cool to room temperature, the mixture was concentrated in vacuo. Water (10 mL) and DCM (10 mL) were added, the phases were separated, and the aqueous phase was extracted with DCM (3 × 10 mL). The organic phases were combined, washed with brine (10 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product, which was purified by flash column chromatography (0–10% EtOAc in petrol) to give the carbonyl derivative S5³⁷ as yellow liquid (1.20 g, 6.44 mmol, 41%). R_f: 0.51 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 4.16 (q, J =7.1 Hz, 2H), 3.98 (dd, J =8.2, 5.7 Hz, 1H), 3.11 (dd, J = 18.5, 8.2 Hz, 1H), 2.92 (dd, J = 18.5, 5.7 Hz, 1H), 2.32 (s, 3H), 2.16 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 205.7, 202.2, 168.8, 61.8, 53.8, 41.6, 30.1, 29.7, 14.1 ppm. MS (ESI): [M + H]+ 187.1. IR (neat): ν_max 2984–2927 (w, C–H), 1739 (m, C⩵O), 1711 (s, C⩵O), 1359 (m), 1259 (m), 1229 (m), 1157 (m) cm⁻¹.

Ethyl 2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylate (S6)

According to General Procedure C, aniline (0.12 mL, 1.34 mmol) and the carbonyl derivative S5 were stirred for 18 h to give a crude product, which was purified by flash column chromatography (5% EtOAc in petrol) to give the ester derivative S6³⁶ as yellow oil (0.27 g, 1.11 mmol, 83%). R_f 0.70 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.52-7.41 (m, 3H), 7.17 (d, J = 7.6 Hz, 2H), 6.37 (s, 1H), 4.28 (q, J = 7.1 Hz, 2H), 2.29 (s, 3H), 1.97 (s, 3H), 1.35 (t, J = 7.1, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 165.8, 137.8, 136.3, 129.4, 128.8, 128.6, 128.3, 111.5, 107.6, 59.3, 14.7, 12.7, 12.5 ppm. MS (ESI): [M + H]⁺ 244.2. IR (neat): ν_max 2981–2898 (w, C–H), 1738 (w), 1693 (s, C= O), 1540 (w), 1500 (w), 1408 (m), 1369 (w), 1226 (s), 1216 (s), 1079 (s), 1014 (m) cm⁻¹.

2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic Acid (20)

According to General Procedure B, the ester derivative S6 (0.10 g, 0.41 mmol) was stirred for 2 days to give the acid derivative 20⁴¹ as a white amorphous solid (70 mg, 0.32 mmol, 78%). R_f: 0.54 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 10.86 (br. s, 1H), 7.55-7.42 (m, 3H), 7.20 (d, J = 7.4 Hz, 2H), 6.44 (s, 1H), 2.32 (s, 3H), 1.99 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 171.4, 137.8, 137.7, 129.5, 129.2, 128.7, 128.3, 110.8, 108.2, 12.8, 12.7 ppm. MS (ESI): [M + H]⁺ 216.1. HPLC: retention time: 1.72 min (>99%). IR (neat): νmax 3056 (s, O–H), 2917-2584 (s, C–H), 1651 (s, C⩵O), 1579 (m), 1531 (m), 1494 (m), 1453 (m), 1400 (m), 1261 (s), 1084 (m) cm⁻¹.

Methyl 2-Acetamido-3-oxobutanoate (S7)

According to a procedure,⁴³ a solution of sodium nitrite (3.30 g, 48.0 mmol) in water (4 mL) was added dropwise to a solution of methyl acetoacetate (4.00 mL, 36.8 mmol) in acetic acid (10 mL). After stirring the solution at room temperature for 2 h, water (25 mL) was added and stirred for further 30 min. Successively, the solution was extracted with diethyl ether (3 × 50 mL). The organic phases were combined, washed with a saturated sodium bicarbonate aqueous solution (50 mL), dried under anhydrous sodium sulfate, filtered, and concentrated in vacuo. Acetic acid (31.5 mL) and acetic anhydride (9.20 mL) were added, and the mixture was cooled to 0 °C. Subsequently, zinc powder (12.0 g, 184 mmol) was slowly added and the reaction was stirred at room temperature for 18 h. After filtering the mixture through celite, water (30 mL) and DCM (50 mL) were added. The phases were separated, and the aqueous phase was extracted with DCM (3 × 50 mL). Successively, the organic phases were combined, washed with a saturated sodium bicarbonate aqueous solution (50 mL), filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (33% EtOAc in petrol) and triturated with Et₂O to give the carbonyl derivative S7⁴³ as a white solid (2.55 g, 14.7 mmol, 40%). R_f 0.36 (90:10 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 6.76 (app. br. s, 1H), 5.25 (d, J = 6.6 Hz, 1H), 3.78 (s, 3H), 2.35 (s, 3H), 2.04 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 198.6, 169.9, 166.7, 63.0, 53.3, 28.1, 22.7 ppm. MS (ESI): [M + H]⁺ 174.1. IR (neat): ν_max 3231 (s, N–H), 3028–2956 (w, C–H), 1741 (s, C⩵O), 1724 (s, C⩵O), 1632 (s, C⩵O), 1524 (s), 1433 (m), 1375 (m), 1290 (m), 1223 (s), 1157 (s), 1140 (s) cm⁻¹.

Methyl 2,5-Dimethyl-1-phenyl-1H-imidazole-4-carboxylate (S8)

According to a modified procedure,³⁸ trifluoroacetic acid (0.12 mL, 1.48 mmol) was added to a solution of the dicarbonyl derivative S7 (0.20 g, 1.14 mmol) and aniline (0.14 mL, 1.48 mmol) in butyronitrile (4.40 mL). After stirring at 120 °C for 4 h, DCM (10 mL) was added and the solution was washed with a saturated sodium carbonate aqueous solution (10 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (20% petrol in EtOAc) to give the ester derivative S8⁴⁴ as a white solid (0.11 g, 0.47 mmol, 41%). R_f 0.23 (80:20 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.50-7.40 (m, 3H), 7.11 (d, J = 7.1 Hz, 2H), 3.81 (s, 3H), 2.23 (s,3H),2.13 (s, 3H) ppm. ¹³C NMR(100MHz, CDCl₃): δ 164.2,144.6,136.9, 135.5,129.8, 129.4, 127.3, 51.2, 13.7, 10.7 ppm. MS (ESI): [M + H] + 231.1. IR (neat): ν_max 3049–2925 (w, C–H), 1709 (s, C⩵O), 1543 (m), 1494 (m), 1434 (m), 1403 (m), 1372 (m), 1360 (s), 1186 (s), 1176 (s), 1092 (s) cm⁻¹.

2,5-Dimethyl-1-phenyl-1H-imidazole-4-carboxylic Acid (21)

A 6 M HCl aqueous solution (0.50 mL) was added to the ester derivative S8 (10 mg, 43.4 μMol), and the mixture was stirred at 100 °C for 18 h. The solvent was removed in vacuo to yield the acid derivative 21⁴⁴ as a hydrochloride salt (9 mg, 43.4 μMol, >99%). R_f 0.11 (20:80 MeOH.DCM). ¹H NMR (400 MHz, CD₃OD): δ 7.76-7.68 (m, 3H), 7.57-7.50 (m, 2H), 2.46 (s, 3H), 2.38 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.7, 147.8, 138.7, 133.5, 132.6, 131.7, 128.5, 121.4, 11.6, 10.4 ppm. MS (ESI): [M + H]⁺ 217.1. HPLC: retention time: 0.97 min (91%). IR (neat): ν_max 2761 (s, O–H, C–H), 1721 (s, C⩵O), 1444 (m), 1331 (m), 1220 (m), 1170 (s), 1117 (s) cm⁻¹.

Ethyl 4-Oxo-2-(2,2,2-trifluoroacetyl)pentanoate (S9)

Ethyl 4,4,4-trifluoroacetoacetate (2.31 mL, 15.8 mmol) was added dropwise to a suspension of 60% NaH in mineral oil (0.63 g, 15.8 mmol) in 1,2-dimethoxyethane (8.00 mL) at 0 °C. After stirring for 30 min at 0 °C, chloroacetone (1.45 mL, 18.2 mmol) in 1,2-dimethoxyethane (2 mL) and KI (32 mg, 0.19 mmol) were added and the reaction was stirred at 85 °C for 18 h. The mixture was diluted with water (20 mL) and extracted with Et₂O (3 × 20 mL). The organic phases were combined, washed with brine (20 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product. The crude product was purified by vacuum distillation to yield the carbonyl derivative S9⁴⁵ as orange oil (2.31 g, 9.62 mmol, 61%). R_f 0.44 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 4.34 (dd, J = 9.6, 4.7 Hz, 1H), 4.20 (app. qd, J = 7.2, 3.1 Hz, 2H), 3.28 (dd, J = 18.5, 9.6 Hz, 1H), 3.12 (dd, J = 18.5, 4.7 Hz, 1H), 2.20 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 204.1, 187.2 (q, J = 36.8 Hz), 166.7, 115.3 (q, J =291.3 Hz), 62.7,47.7,42.1, 29.2, 13.9ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -78.0 ppm. MS (ESI): [M - H]^- 239.0. IR (neat): ν_max 2988–2924 (w, C–H), 1718 (s, C⩵O), 13.70 (w), 1267 (m), 1157 (s), 1096 (m), 1041 (m) cm⁻¹.

Ethyl 5-Methyl-1-phenyl-2-(trifluoromethyl)-1H-pyrrole-3-car-boxylate (S10)

According to General Procedure D, aniline (37.3 μL, 0.41 mmol) was stirred for 2.5 h to give a crude product, which was purified by flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S10 as yellow oil (18.0 mg, 60.5 μMol, 15%). R_f: 0.59 (30:70EtOAc:petrol). ¹H NMR(400MHz, CDCl₃): δ7.51-7.44 (m, 3H), 7.25-7.20 (m, 2H), 6.47 (s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 1.94 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 163.6,137.6,133.7, 129.3,129.3,127.8 (q, J = 0.7Hz), 122.2 (q, J = 38.3 Hz), 120.5 (q, J = 269.4 Hz), 118.4 (q, J = 2.1 Hz), 110.0, 60.7, 14.2, 12.5 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -54.4 ppm. MS (ESI): [M + H] +298.1. IR(neat): ν_max 2981–2853 (w, C–H), 1721 (s, C⩵O), 1511 (m), 1495 (m), 1417 (m), 1276 (m), 1219 (s), 1174 (s), 1114 (s), 1039 (s), 996 (s) cm⁻¹.

5-Methyl-1-phenyl-2-(trifluoromethyl)-1H-pyrrole-3-carboxylic acid (22)

According to General Procedure B, the ester derivative S10 (8 mg, 26.9 μMol) was stirred for 4 h to give a crude product. The crude product was purified by flash column chromatography (30% EtOAc in petrol) to give the acid derivative 22 as a colorless amorphous solid (6 mg, 20.4 μMol, 76%). R_f. 0.34 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.55-7.49 (m, 3H), 7.31-7.24 (m, 2H), 6.60 (s, 1H), 1.99 (s,3H) ppm. ¹³C NMR (100MHz, CDCl₃): δ 168.6, 137.6, 133.8 (q, J = 1.3 Hz), 129.4, 129.3, 127.7 (q, J = 0.7 Hz), 123.4 (q, J = 38.5 Hz), 120.3 (q, J = 269.6 Hz), 116.9 (q, J = 2.0 Hz), 110.9, 12.6 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -53.5 ppm. MS (ESI): [M + H]⁺ 270.0. HPLC: retention time: 1.88 min (>99%). IR (neat): ν_max 3100–2600 (s, O–H, C–H), 1665 (s, C⩵O), 1517 (m), 1497 (m), 1456 (m), 1418 (m), 1255 (s), 1138 (s), 1000 (m) cm⁻¹. HRMS: calculated for C₁₃H₁₀F₃NO₂ [M + H]⁺ = 270.0742, observed = 270.0735.

4-(Benzyloxy)butan-2-one (S11)

Benzyl bromide (4.12 mL, 34.7 mmol) was added to a mixture of 4-hydroxy-2-butanone (2.00 mL, 23.15 mmol) and N,N-diisopropylethylamine (6.4 mL, 37.0 mmol) at room temperature. The reaction was stirred at 150 °C for 2 h and allowed to cool to room temperature. EtOAc (10 mL) and an aqueous solution of 10% sodium bisulfate (10 mL) were added, the phases were separated, and the aqueous phase was extracted with EtOAc (3 × 2 mL). The organic phases were combined, dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to yield a crude product. The crude product was purified by flash column chromatography (10% EtOAc in petrol) to give the benzyl derivative S11⁴⁶ as yellow liquid (2.60 g, 14.6 mmol, 63%). R_f·. 0.67 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.42-7.20 (m, 5H), 4.51 (s, 2H), 3.74 (t, J =6.3 Hz, 2H), 2.71 (t, J = 6.3 Hz, 2H), 2.18 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 207.2,138.1,128.5,127.8,127.7,73.3,65.3,43.8, 30.5 ppm. MS (ESI): [M + H]⁺ 179.0. IR (neat): ν_max 2863 (m, C–H), 1714 (s, C⩵O), 1454 (w), 1363 (m), 1170 (w), 1104 (s), 1085 (s) _cm⁻¹.

4-(Benzyloxy)-1-bromobutan-2-one (S12)

Bromine (0.51 mL, 10.0 mmol) was added dropwise to a solution of the benzyl derivative S11 (1.78 g, 10.0 mmol) in methanol (18 mL) at0 °C, and the reaction was stirred at room temperature for 18 h. An aqueous solution of 1.0 M K₂CO₃ (20 mL) and Et₂O (20 mL) were added. The phases were separated, and the aqueous phase was extracted with Et₂O (2 × 20 mL). The organic phases were combined, dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product. The crude product was dissolved in THF (72 mL), and an aqueous solution of 1.0 M H₂SO₄ (36.0 mL) was added. After stirring for 2 h at 65 °C and concentrating the mixture in vacuo, Et₂O (20 mL) and water (20 mL) were added and the phases were separated. The organic phase was washed with an aqueous solution of 2.0 M KHCO₃ (10 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product. The crude product was purified by flash column chromatography (6% EtOAc in petrol) to give the bromine derivative S12⁴⁷ as pale yellow oil (1.30 g, 5.05 mmol, 51%). R_f: 0.44 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.40-7.25 (m, 5H), 4.51 (s, 2H), 3.94 (s, 2H), 3.77 (t, J = 6.1 Hz, 2H), 2.92 (t, J = 6.1 Hz, 2H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 200.4, 137.9, 128.5, 127.9, 127.8, 73.4, 65.4, 40.3, 35.0 ppm. MS (ESI): [M + Na]+ 279.2. IR (neat): ν_max 3087–2865 (m, C–H), 1715 (s, C⩵O), 1495 (w), 1054 (w), 1390 (m), 1365 (m), 1326 (w), 1255 (w), 1205 (w), 1178 (w), 1095 (s), 1075 (s), 1026 (m) cm⁻¹.

Ethyl 2-Acetyl-6-(benzyloxy)-4-oxohexanoate (S13)

Ethyl acetoacetate (0.12 mL, 0.97 mmol) was added dropwise to a suspension of 60% NaH in mineral oil (39 mg, 0.97 mmol) in 1,2-dimethoxyethane (1 mL) at 0 °C. After stirring for 10 min at 0 °C, the halogen derivative S12 (0.25 g, 0.97 mmol) was added and the reaction was stirred at room temperature for 18 h. Successively, an aqueous solution of 1.0 M HCl (1 mL) and EtOAc (1 mL) were added, the phases were separated and the aqueous phase was extracted with EtOAc (3 × 1 mL). The organic phases were combined, dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product. The crude product was purified by flash column chromatography (10-20% EtOAc in petrol) to give the benzyl derivative S13⁴⁸ as colorless liquid (0.28 g, 0.91 mmol, 94%). R_f 0.50 (40:60 EtOAcpetrol). ¹H NMR (400 MHz, CDCl₃): δ 7.40-7.21 (m, 5H), 4.49 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 4.02 (app. t, J =6.9 Hz, 1H), 3.79-3.66 (m, 2H), 3.15 (dd, J = 18.5, 8.2 Hz, 1H), 2.96 (dd, J = 18.5, 5.7 Hz, 1H), 2.74 (t, J = 6.2 Hz, 2H), 2.34 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 206.3, 202.2, 168.8, 138.1, 128.5, 127.8, 127.7, 73.3, 65.1, 61.8, 53.7, 42.9, 41.3, 30.1, 14.1 ppm. MS (ESI): [M + Na]+ 329.2. IR (neat): ν_max 2981–2869 (m, C–H), 1739 (m, C⩵O), 1712 (s, C⩵O), 1454 (w), 1399 (w), 1360 (m), 1256 (m), 1205 (m), 1147 (m), 1097 (m), 1022 (m) cm⁻¹.

Ethyl 5-[2-(Benzyloxy)ethyl]-2-methyl-1-phenyl-1H-pyrrole-3-carboxylate (S14)

According to General Procedure C, aniline (63 μL, 0.68 mmol) and the carbonyl derivative S13 were stirred for 18 h to give a crude product, which was purified by flash column chromatography (5% EtOAc in petrol) to give the ester derivative S14 as a white solid (0.16 g, 0.44 mmol, 65%). R_f 0.48 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.50-7.40 (m, 3H), 7.36-7.22 (m, 5H), 7.17-7.10 (m, 2H), 6.45 (s, 1H), 4.44 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 3.54 (t, J = 7.3 Hz, 2H), 2.64 (t, J = 7.3 Hz, 2H), 2.27 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 165.7, 138.3, 137.5, 136.5, 130.0, 129.5, 128.8, 128.5, 128.4, 127.8, 127.7, 111.8, 107.6, 73.0, 69.1, 59.4, 27.3, 14.7, 12.3 ppm. MS (ESI): [M + H] + 364.3. IR(neat): ν_max 2981-2854 (m, C–H), 1685 (s, C⩵O), 1526 (w), 1494 (m), 1430 (m), 1379 (m), 1365 (m), 1221 (s), 1119 (m), 1084 (s), 1072 (s), 1024 (m), 1000 (m) cm⁻¹.

Ethyl 5-(2-Hydroxyethyl)-2-methyl-1-phenyl-1H-pyrrole-3-carboxylate (S15)

According to General Procedure A, the benzyl derivative S14 (0.13 g, 0.36 mmol) gave a crude product. The crude product was purified using flash column chromatography (30% EtOAc in petrol) to give the alcohol derivative S15 as colorless oil (86.0 mg, 0.31 mmol, 87%). R_f: 0.31 (50:50 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃). δ 7.52-7.39 (m, 3H), 7.16 (d, J = 7.2 Hz, 2H), 6.46 (s, 1H), 4.26 (q, J = 7.1 Hz, 2H), 3.60 (t, J = 6.7 Hz, 2H), 2.56 (t, J = 6.7 Hz, 2H), 2.25 (s, 3H), 2.04 (br. s, 1H), 1.33 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 165.7, 137.4, 136.9, 129.6, 129.5, 128.8, 128.4, 111.8, 107.8, 61.2, 59.4, 30.0, 14.6, 12.3 ppm. MS (ESl): [M + H]⁺ 274.2. IR (neat): ν_max 3447 (s, O–H), 2978-2875 (m, C–H), 1693 (s, C⩵O), 1674 (s), 1597 (w), 1572 (w), 1529 (m), 1498 (m), 1418 (m), 1378 (m), 1352 (w), 1219 (s), 1079 (s), 1047 (m), 1012 (m) cm⁻¹.

5-(2-Hydroxyethyl)-2-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid (23)

According to General Procedure B, the ester derivative S15 (56 mg, 0.20 mmol) was stirred for 5 h to give a crude product. The crude product was purified by flash column chromatography (50% EtOAc in petrol) to give the acid derivative 23 as a white amorphous solid (36 mg, 0.15 mmol, 75%). R_f. 0.50 (EtOAc). ¹H NMR (400MHz, CD₃OD): δ 7.60-7.46 (m, 3H), 7.25 (d, J =7.7 Hz, 2H), 6.42 (s, 1H), 3.55 (t, J = 7.2 Hz, 2H), 2.54 (t, J = 7.2 Hz, 2H), 2.23 (s, 3H) ppm. ¹³C NMR (100 MHz, CD₃OD): δ 169.4, 138.8, 137.8, 131.3, 130.6, 130.0, 129.6, 112.6, 109.2, 61.9, 31.0, 12.5 ppm. MS (ESI): [M + H]⁺ 246.1. HPLC: retention time: 1.50 min (>99%). IR (neat): ν_max 3326 (m, OH), 2958–2850 (m, C–H), 1673 (s, C⩵O), 1568 (m), 1529 (m), 1492 (m), 1430 (m), 1375 (m), 1357 (m), 1217 (s), 1054 (s), 1025 (s) cm⁻¹. HRMS: calculated for C₁₄H₁₅NO₃ [M + H]⁺ = 246.1130, observed = 246.1124.

Ethyl 1-Benzyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S16)

According to General Procedure E, benzylhydrazine dihydrochloride (0.50 g, 2.56 mmol) was stirred for 3.5 h to give a crude product. The crude product was purified by flash column chromatography (5% EtOAc in petrol) to give the ester derivative S16 as colorless oil (0.48 g, 1.60 mmol, 63%). R_f: 0.56 (20:80 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.01 (s, 1H), 7.40–7.10 (m, 5H), 5.54 (s, 2H), 4.32 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR(100 MHz, CDCl₃): δ 161.0, 146.9, 142.0, 135.3, 131.9 (q, J = 40.1 Hz), 128.9, 128.4, 127.2, 119.6 (q, J = 271.0 Hz), 61.2, 56.7 (q, J = 3.2 Hz), 14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.4 ppm. MS (ESI): [M + H]⁺ 299.2. IR (neat): ν_max 2982 (w, C–H), 1735 (s, C⩵O), 1559 (m), 1477 (m), 1294 (s), 1222 (s), 1187 (s), 1152 (s), 1042 (s) cm⁻¹.

1-Benzyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (24)

According to General Procedure B, the ester derivative S16 (0.20 g, 0.67 mmol) was stirred for 5.5 h to give the acid derivative 24 as a white amorphous solid (0.16 g, 0.60 mmol, 90%). R_f: 0.18 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.08 (s, 1H), 7.407.10 (m, 5H), 5.55 (s, 2H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.5, 146.0, 142.8, 135.1, 132.7 (q, J = 40.0 Hz), 128.9, 128.5, 127.2, 119.4 (q, J = 271.7 Hz), 56.8 (q, J = 3.2 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ - 57.4 ppm. MS (ESI): [M - H]^- 269.0. HPLC: retention time: 1.79 min (>99%). IR (neat): ν_max 3040 (m, O–H), 2928-2525 (w, C–H), 1700 (m, C⩵O), 1562 (m), 1480 (m), 1410 (m), 1300 (s), 1231 (s), 1174 (s), 1136 (s), 1037 (s), 1014 (s) cm⁻¹. HRMS: calculated for C₁₂H₉F₃N₂O₂ [M + H]⁺ = 271.0694, observed = 271.0682.

[(Thiophen-2-yl)methyl]hydrazine Dihydrochloride (S17)

tert-Butyl carbazate (1.50 g, 11.3 mmol) was added to a solution of 2-thiophenecarboxaldehyde (1.00 mL, 10.8 mmol) in MeOH (25 mL) at room temperature. After stirring the mixture at 65 °C for 1 h, the solvent was removed in vacuo, the crude product was dissolved in THF (45 mL), and sodium cyanoborohydrate (1.00 g, 16.2 mmol) was added. Subsequently, AcOH (17.0 mL) was added dropwise and the mixture was stirred at room temperature for 24 h. Successively, a saturated aqueous solution of NaHCO₃ (20 mL) was slowly added and the mixture was extracted with EtOAc (2 × 10 mL). The organic phases were combined, washed with brine (10 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product. EtOH (50 mL) and an aqueous solution of concentrated HCl (5 mL) were added. After stirring the mixture at 80 °C for 18 h, the solvent was removed in vacuo to yield the hydrazine derivative S17⁴⁹ as a white amorphous solid (1.90 g, 9.44 mmol, 87%). R_f: 0.65 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CD₃OD) δ 7.49 (dd, J = 5.1, 1.1 Hz, 1H), 7.20 (dd, J = 3.5, 1.1 Hz, 1H), 7.06 (dd, J = 5.1, 3.5 Hz), 4.37 (s, 2H) ppm. ¹³C NMR (100 MHz, CD₃OD): δ 135.5, 130.4, 128.4, 128.3, 49.9 ppm. MS (ESI): [M + H] + 128.9. IR (neat): ν_max 3205 (m, N-H), 2990-2862 (s, N-H, C–H), 1582 (m), 1501 (m), 1375 (m), 1243 (w), 1047 (w), 1022 (w) cm⁻¹.

Ethyl 1-[(Thiophen-2-yl)methyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S18)

According to General Procedure E, the hydrazine derivative S17 (0.25 g, 1.24 mmol) was stirred for 18 h to give a crude product. The crude product was purified by flash column chromatography (5-30% EtOAc in petrol) to give the ester derivative S18 as pale brown oil (0.20 g, 0.66 mmol, 53%). R_f: 0.56 (20:80 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.98 (s, 1H), 7.27 (dd, J = 5.1, 1.2 Hz, 1H), 7.04 (d, J = 3.5 Hz, 1H), 6.95 (dd, J = 5.1, 3.5 Hz, 1H), 5.67 (s, 2H), 4.31 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H) ppm. ¹³CNMR(100 MHz, CDCl₃): δ 160.9, 142.2, 136.8, 131.4 (q, J = 40.3 Hz), 127.6, 127.0, 126.8, 119.6 (q, J = 271.1 Hz), 116.4 (q, J = 1.6 Hz), 61.2, 51.4 (q, J = 3.5 Hz), 14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.1 ppm. MS (ESI): [M + H]⁺ 305.1. IR (neat): ν_max 2983 (w, C–H), 1732 (s, C⩵O), 1558 (w), 1476 (w), 1408 (w), 1373 (w), 1292 (s), 1218 (s), 1188 (s), 1148 (s), 1039 (s), 1021 (s) cm⁻¹.

1-[(Thiophen-2-yl)methyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (25)

According to General Procedure B, the ester derivative S18 (0.10 g, 0.33 mmol) was stirred for 6.5 h to give the acid derivative 25 as a white amorphous solid (72.0 mg, 0.26 mmol, 79%). R_f: 0.29 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 11.02 (br. s, 1H), 8.09 (s, 1H), 7.29 (dd, J = 5.2, 1.2 Hz, 1H), 7.07 (d, J = 3.5 Hz, 1H), 6.97 (dd, J = 5.2, 3.5 Hz, 1H), 5.71 (s, 2H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.2, 143.1, 136.4, 132.4 (q, J = 41.1 Hz), 127.8, 127.1, 127.0, 119.4 (q, J = 271.7 Hz), 115.1 (q, J = 1.4Hz), 51.6 (q, J = 3.6 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.2 ppm. MS (ESI): [M - H] ^- 275.0. HPLC: retention time: 1.65 2864-2557 (m, O–H, C–H), 1687 (s, C⩵O), 1565 (m), 1479 (m), 1422 (m), 1334 (m), 1302 (s), 1239 (s), 1129 (w), 1029 (s), 1009 (s) cm⁻¹. HRMS: calculated for C₁₀H₇F₃N₂O₂S [M - H]^- = 275.0102, observed = 275.0104.

N-(Thiophen-2-yl)(tert-butoxy)carbohydrazide (S19)

tert-Butyl carbazate (3.50 g, 26.8 mmol), Cs₂CO₃ (6.90 g, 21.2 mmol), CuI (0.25 g, 1.40 mmol), and trans-4-hydroxy-L-proline (0.35 g, 2.67 mmol) were added to a solution of 2-bromothiophene (1.00 mL, 10.3 mmol) in DMSO (50 mL), and the reaction was stirred at 80 °C for 18 h. After allowing the mixture to cool to room temperature, water (40 mL) and EtOAc (10 mL) were added. The phases were separated, and the aqueous phase was extracted with EtOAc (2 × 10 mL). The organic phases were combined, washed with brine (10 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (10% EtOAc in petrol) to give the carbamate derivative S19⁵⁰ as pale brown oil (0.52 g, 2.42 mmol, 24%). R_f: 0.60 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 6.88 (br. s, 1H), 6.84-6.78 (m, 2H), 4.56 (s, 2H), 1.56 (s, 9H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 153.2, 146.8, 125.3, 117.3, 112.6, 83.2, 28.3 ppm. MS (ESI): [M + H] + 158.9 (^tBu lost during MS). IR (neat): vmax 3324 (m, N–H), 3273 (w, N–H), 3201 (w, N–H), 2977–2930 (m, C–H), 1692 (s, C⩵O), 1621 (w), 1534 (m), 1473 (w), 1447 (m), 1368 (s), 1322 (s), 1280 (m), 1251 (m), 1224 (m), 1150 (s), 1082 (m), 1050 (m), 998 (s) cm^–1.

Ethyl 1-(Thiophen-2-yl)-5-(trifluoromethyl)-1 H-pyrazole-4-carboxylate (S20)

A solution of 4.0 M HCl in 1,4-dioxane (1.00 mL) was added to a solution of the derived carbamate S19 (0.10 g, 0.47 mmol) in DCM (1 mL). The reaction was stirred at room temperature for 3 days, and the solvent was removed in vacuo to yield a crude product. The crude product was dissolved in EtOH (1.00 mL), and ethyl-3-ethoxy-2-(2,2,2-trifluoroacetyl)acrylate (0.10 mL, 0.53 mmol) and Et3N (79 μL, 0.57 mmol) were added. The mixture was stirred at 80 °C for 5 h. After allowing the reaction to cool to room temperature, the solvent was evaporated in vacuo and EtOAc (1 mL) and water (1 mL) were added. The phases were separated, and the aqueous phase was extracted with EtOAc (3 × (1 mL)). The organic phases were combined, washed with brine (1 mL), dried under anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a crude product. The crude product was purified by flash column chromatography (5% EtOAc in petrol) to give the ester derivative S20 as yellow oil (75 mg, 0.26 mmol, 55%). R_f: 0.62 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.10 (s, 1H), 7.37 (dd, J = 5.6, 1.4 Hz, 1H), 7.17 (dd, J = 3.8,1.4 Hz, 1H), 7.01 (dd, J = 5.6, 3.8 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.7, 142.8, 139.0, 133.9 (q, J = 40.1 Hz), 126.0, 125.6, 125.5 (q, J = 1.5 Hz), 118.9 (q, J = 271.7 Hz), 117.1 (q, J =1.3 Hz),61.5, 14.2ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ –57.3 ppm. MS (ESI): [M + H] + 291.1. IR (neat): ν_max 3109–2907 (w, C–H), 1734 (s, C⩵O), 1566 (w), 1554 (w), 1466 (w), 1397 (w), 1377 (w), 1291 (s), 1230 (s),1185 (s), 1140 (s), 1035 (s) cm⁻¹.

1-(Thiophen-2-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (26)

According to General Procedure B, the ester derivative S20 (60 mg, 0.21 mmol) was stirred for 5.5 h to give the acid derivative 26 as a white amorphous solid (50 mg, 0.19 mmol, 90%). R_f: 0.33 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 11.34 (br. s, 1H), 8.21 (s, 1H), 7.40 (dd, J = 5.6, 1.4 Hz, 1H), 7.21 (dd, J = 3.8, 1.4 Hz, 1H), 7.04 (dd, J = 5.6, 3.8 Hz) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.1, 143.6, 138.7, 134.9 (q, J = 40.7Hz), 126.3, 125.8 (q, J = 1.5 Hz), 125.7, 118.7 (q, J = 272.1 Hz), 115.8 (q, J = 1.3 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ –56.3 ppm. MS (ESI): [M – H]^– 261.0. HPLC: retention time: 1.64 min (>99%). IR (neat): ν_max 2856–2583 (m, O–H, C–H), 1702 (m, C⩵O), 1568 (w), 1545 (w), 1418 (w), 1299 (m), 1254 (m), 1235 (m), 1187 (m), 1134 (s), 1026 (m) cm^–1. HRMS: calculated for C₉H₅F₃N₂O₂S [M + H]⁺ = 263.0102, observed = 263.0110.

Ethyl 1-(Pyridin-3-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxy-late (S21)

According to General Procedure E, 3-hydrazinopyridine dihydrochloride (0.30 g, 1.64 mmol) was stirred for 18 h to give a crude product. The crude product was purified by flash column chromatography (20–40% EtOAc in petrol) to give the ester derivative S21⁵¹ as yellow oil (86 mg, 0.30 mmol, 18%). R_f: 0.40 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.75 (app. d, J = 4.9 Hz, 1H), 8.71 (s, 1H), 8.14 (s, 1H), 7.77 (app. dt, J =8.2, 1.9 Hz, 1H), 7.46 (dd, J = 8.2, 4.9 Hz, 1H), 4.36 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.7, 150.9, 146.8 (q, J =1.1 Hz), 143.2, 136.2, 133.3 (q, J = 0.9 Hz), 133.1 (q, J = 40.1 Hz), 123.7, 119.0 (q, J =271.5 Hz), 117.5 (q, J = 1.5 Hz), 61.5, 14.1 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ –56.1 ppm. MS (ESI): [M + H]⁺ 286.2. IR (neat): ν_max 2988 (w, C–H), 1710 (m, C⩵O), 1554 (w), 1491 (w), 1465 (w), 1411 (w), 1384 (w), 1295 (w), 1245 (m), 1223 (m), 1190 (s), 1148 (m), 1082 (s), 1027 (m) cm^–1.

1-(Pyridin-3-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (27)

According to General Procedure B, the ester derivative S21 (66 mg, 0.23 mmol) was stirred for 18 h to give the acid derivative 2T⁴⁷ as a white amorphous solid (57 mg, 0.22 mmol, 96%). R_f: 0.16 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CD₃OD): δ 8.75 (app. d, J = 4.9 Hz, 1H), 8.72 (d, J =2.5 Hz, 1H), 8.21 (s, 1H), 8.02 (app. dt, J = 8.3,2.0 Hz, 1H), 7.65 (dd, J 8.3, 4.9 Hz, 1H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 163.3,151.7,147.6 (q, J = 1.1 Hz), 144.5, 138.1, 135.7 (q, J = 1.0 Hz), 134.0, 125.5, 120.5 (q, J =270.6 Hz), 119.1 (q, J = 1.4 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ – 57.5 ppm. MS (ESI): [M + H]⁺ 258.1. HPLC: retention time: 1.36 min (>99%). IR (neat): ν_max 3118–2413 (m, O–H, C–H), 1878 (w), 1718 (m, C⩵O), 1562 (m), 1489 (w), 1433 (m), 1378 (w), 1366 (w), 1299 (m), 1256 (m), 1225 (m), 1184 (s), 1144 (s), 1082 (m), 1045 (s), 1027 (s) cm^–1.

Ethyl l-(Pyridin-4-yl)-5-(trifluoromethyl)-lH-pyrazole-4-carboxylate (S22)

According to General Procedure E, 4-hydrazinopyridine hydrochloride (20 mg, 0.14 mmol) was stirred overnight to give a crude product. The crude product was purified by flash column chromatography (30% EtOAc in petrol) to give the ester derivative S22⁵¹ as colorless oil (18 mg, 63.1 μMol, 45%). R_f: 0.38 (60:40 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.80 (d, J = 5.3 Hz, 2H), 8.15 (s, 1H), 7.43 (d, J = 5.3 Hz, 2H), 4.38 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.6, 151.1, 146.4, 143.4, 132.6 (q, J = 40.7 Hz), 120.0, 119.0 (q, J = 271.6 Hz), 118.2 (q, J = 1.4 Hz), 61.7, 14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ – 55.9 ppm. MS (ESI): [M + H]⁺286.1. IR (neat): ν_max 1737 (s, C⩵O), 1590 (s), 1501 (w), 1299 (m), 1245 (s), 1148 (s), 1040 (m) cm^–1. HRMS: calculated for C₁₂H₁₀F₃N₃O₂ [M + H]⁺ = 286.0803, observed = 286.0800.

l-(Pyridin-4-yl)-5-(trifluoromethyl)-lH-pyrazole-4-carboxylic Acid (28)

According to General Procedure B, the ester derivative S22 (18 mg, 63.1 μMol) was stirred for 2 h to give the acid derivative 28⁵¹ as a white amorphous solid (15 mg, 59.3 μMol, 94%). R_f 0.13 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 13.49 (br. s, 1H), 8.82 (d, J = 6.2 Hz, 2H), 8.32 (s, 1H), 7.64 (d, J = 6.2 Hz, 2H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): δ 162.1, 151.6, 146.4, 143.7, 132.0, 131.7, 121.0, 118.4 (q, J = 1.4 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ –54.4 ppm. MS (ESI): [M + H]⁺ 258.1. HPLC: retention time: 1.34 min (>99%). IR (neat): ν_max 2447 (w, O–H), 1690 (s, C⩵O), 1601 (m), 1555 (m), 1400 (m), 1239 (m), 1209 (m), 1187 (m), 1126 (s), 1029 (m), 970 (m). HRMS: calculated for C₁₀H₆F₃N₃O₂ [M + H]⁺ = 258.0490, observed = 258.0486.

Ethyl 1-(2-Fluorophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S23)

According to General Procedure E, 2-fluorophenylhy-drazine hydrochloride (0.34 g, 2.10 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (30% EtOAc in petrol) to give the ester derivative S23⁵³ as a white amorphous solid (0.46 g, 1.53 mmol, 73%). R_f: 0.56 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.20 (s, 1H), 7.56–7.51 (m, 1H), 7.48 (td, J = 7.6, 1.7 Hz, 1H), 7.31 (tt, J = 7.6, 1.1 Hz, 1H), 7.28 (dt, J =8.4, 1.1 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 159.9, 155.9 (d, J = 253.4 Hz), 133.3 (q, J = 40.4Hz), 131.1 (d, J = 7.9 Hz), 127.6, 126.5 (d, J =12.7 Hz), 123.8 (d, J = 4.0 Hz), 118.0 (q, J = 271.7 Hz), 115.7, 115.5 ppm. ¹⁹F NMR (376 MHz, DMSO-d₆): δ – 54.2, –60.4 ppm. MS (ESI): [M + H]⁺ 303.1. IR (neat): ν_max 1733 (s, C⩵O), 1599 (w), 1567 (m), 1512 (s), 1302 (s), 1246 (s), 1147 (s), 1039 (s) cm^–1. HRMS: calcd for C₁₃H₁₀F₄N₂O₂ [M + H]⁺ = 303.0756, observed = 303.0751.

1-(2-Fluorophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (29)

According to General Procedure B, the ester derivative S23 (0.43 g, 1.41 mmol) was stirred for 2 h to give the acid derivative 29⁵³ as a white amorphous solid (0.36 g, 1.30 mmol, 92%). R_f: 0.32 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 8.25 (s, 1H), 7.58–7.50 (m, 1H), 7.47 (dd, J = 7.8, 1.7 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.25 (t, J = 5.3 Hz, 1H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 165.6, 156.8 (d, J = 253.8 Hz), 143.9,135.1 (q, J = 40.4Hz), 132.1 (d, J = 7.8 Hz), 128.4,127.3 (d, J = 12.6 Hz), 124.7 (d, J = 4.1 Hz), 120.1 (q, J = 271.8 Hz), 116.6, 115.4 (d, J =1.3 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ –57.7, –62.2 ppm. MS (ESI): [M – H]^- 273.1. HPLC: retention time: 1.76 min (>99%). IR (neat): ν_max 2844 (w, O–H), 1704 (s, C⩵O), 1600 (w), 1572 (m), 1509 (m), 1419 (w), 1299 (s), 1261 (s), 1189 (m), 1135 (s), 1032 (s), 970 (m) cm⁻¹.

Ethyl 1-(2-Chlorophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (57)

According to General Procedure E, 2-chlorophenylhy-razine hydrochloride (0.11 g, 0.63 mmol) was stirred for 2 h to give a crude product. The crude product was purified using flash column chromatography (5% EtOAc in petrol) to give the ester derivative 57⁵³ 9 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.18 (s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.51-7.45 (m, 1H), 7.44-7.39 (m, 2H), 4.38 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.8, 143.1, 137.3, 134.1 (q, J = 40.1 Hz), 132.2, 131.6, 130.4, 128.8 (q, J = 0.6 Hz), 127.6, 119.0 (q, J = 271.6 Hz), 116.3 (q, J = 1.4 Hz), 61.4.14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -58.2 ppm. MS (ESI): [M + H]⁺ 319.1. HPLC: retention time: 2.20 min (>99%). IR (neat): ν_max 3071-2850 (w, C–H), 1718 (s, C⩵O), 1562 (m), 1497 (m), 1444 (w), 1386 (w), 1299 (m), 1245 (s), 1221 (m), 1147 (s), 1097 (m), 1049 (m), 1017 (m) cm⁻¹.

1-(2-Chlorophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (30)

According to General Procedure B, the ester derivative 57 (45 mg, 0.14 mmol) was stirred for 1 h to give the acid derivative 30⁵³ as a yellow amorphous solid (41 mg, 0.14 mmol, >99%). R_f. 0.32 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 11.62 (br. s, 1H), 8.28 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.51 (td, J = 8.0, 7.0, 2.8 Hz, 1H),7.48-7.40 (m, 2H) ppm. ¹³C NMR (125 MHz, CD₃OD): δ 163.4, 144.2, 138.5, 135.1 (q, J = 40.0 Hz), 133.1, 133.0, 131.2, 130.2, 129.0, 120.4 (q, J = 270.8 Hz), 117.9 (q, J = 1.5 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -58.3 ppm. MS (ESI): [M + H]⁺ 291.1. HPLC: retention time: 1.91 min (>99%). IR (neat): ν_max 3000-2500 (w, O–H, C–H), 1702 (m, C⩵O), 1571 (m), 1495 (m), 1461 (w), 1424 (w), 1303 (m), 1268 (m), 1182 (s), 1143 (s), 1101 (m), 1050 (m), 1034 (s) cm⁻¹.

Ethyl 1-(2-Bromophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-car-boxylate (S24)

According to General Procedure E, 2-bromophenylhydrazine hydrochloride (0.37 g, 1.66 mmol) was stirred for 2 h to give a crude product. The crude product was purified using flash column chromatography (0-10% EtOAc in petrol) to give the ester derivative S24 as orange oil (65 mg, 0.18 mmol, 11%). R_f 0.28 (25:75 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.18 (s, 1H), 7.73 (dd, J =8.0, 1.6 Hz, 1H), 7.50-7.38 (m, 3H), 4.38 (q, J =7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.9, 143.1, 138.9, 134.0 (q, J = 40.0 Hz), 133.5, 131.8, 128.9 (q, J = 0.7 Hz), 128.2, 121.9 (q, J = 0.8 Hz), 119.0 (q, J = 271.7 Hz), 116.3 (q, J = 1.5 Hz), 61.4.12.2 ppm. ¹⁹FNMR(376MHz, CDCl₃): δ -58.0 ppm. MS (ESi): [M + H]⁺ 365.1. IR (neat): ν_max 3070-2908 (w, C–H), 1716 (s, C⩵O), 1561 (m), 1494 (m), 1385 (w), 1298 (m), 1244 (s), 1221 (s), 1146 (s), 1092 (s), 1040 (s), 970 (s) cm⁻¹.

1-(2-Bromophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (31)

According to General Procedure B, the ester derivative S24 (44 mg, 0.12 mmol) was stirred for 1 h to give the acid derivative 31⁵⁴ as a yellow amorphous solid (37 mg, 0.11 mmol, 92%). R_f. 0.30 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 8.29 (s, 1H), 7.77 (m, 1H), 7.55-7.40 (m, 3H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): δ 162.1, 143.4, 138.8, 133.6, 132.9 (q, J = 39.5 Hz), 132.9, 129.9, 129.3, 121.3, 119.3 (q, J = 270.9 Hz), 117.3 (q, J = 2.9 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.2 ppm. MS (ESI): [M - H]^- 334.9. HPLC: retention time: 1.80 min (95%). IR (neat): ν_max 2852 (w, O–H), 1702 (s, C⩵O), 1571 (m), 1493 (m), 1464 (m), 1447 (m), 1427 (m), 1144 (s) cm⁻¹. HRMS: calculated for C_uH₆BR_f3N₂O₂ [M - H]^- = 334.9643, observed = 334.9635.

Ethyl 1-(o-Tolyl)-5-(trifluoromethyl)-1 H-pyrazole-4-carboxylate (S25)

According to General Procedure E, o-tolylhydrazine hydrochloride (0.10 g, 0.63 mmol) was stirred for 3 h to give a crude product. The crude product was purified using flash column chromatography (0-10% EtOAc in petrol) to give the ester derivative S25⁵³ as yellow oil (0.11 g, 0.37 mmol, 59%). R_f. 0.60 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.15 (s, 1H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.33 (d, J = 7.1 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.24 (d, J = 7.5 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 2.04 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 161.1, 142.6, 138.6, 135.4, 133.4 (q, J = 39.7 Hz), 131.0, 130.4, 127.1 (q, J = 0.9 Hz), 126.6, 119.1 (q, J = 271.5 Hz), 115.9 (q, J = 1.3 Hz), 61.3, 16.9, 14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.8. MS (ESI): [M + H]⁺ 299.2. IR (neat): ν_max 2984 (w, C–H), 1735 (s, C⩵O), 1561 (m), 1501 (m), 1467 (m), 1383 (m), 1298 (s), 1228 (s), 1147 (s), 1072 (m), 1040 (s) cm⁻¹.

1-(o-Tolyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid (32)

According to General Procedure B, the ester derivative S25 (0.19 g, 0.63 mmol) was stirred for 1 h to give the acid derivative 32⁵³ as yellow oil (0.16 g, 0.58 mmol, 92%). R_f: 0.20 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 8.25 (s, 1H), 7.45 (app. td, J = 7.5, 1.4 Hz, 1H), 7.36 (d, J = 7.0 Hz, 1H), 7.33 (app. t, J = 7.5 Hz, 1H), 7.26 (d, J = 7.2 Hz, 1H), 2.07 (s, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 163.6, 143.8, 139.9, 136.6, 134.3, 132.0, 131.6, 128.2 (q, J = 0.9 Hz), 127.7, 120.5 (q, J = 270.8 Hz), 117.6 (q, J = 1.3 Hz), 16.8 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.9 ppm. MS (ESI): [M + H]⁺ 271.2. HPLC: retention time: 1.92 min (>99%). IR (neat): ν_max 2927-2588 (m, O–H, C–H), 1701 (s, C⩵O), 1566 (m), 1500 (w), 1466 (w), 1421 (w), 1301 (m), 1255 (m), 1223 (m), 1185 (s), 1128 (s), 1068 (w), 1033 (s) cm⁻¹.

Ethyl 5-(Trifluoromethyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyra-zole-4-carboxylate (S26)

According to General Procedure E, 2-(trifluoromethyl)phenylhydrazine hydrochloride (0.35 g, 1.66 mmol) was stirred for 18 h to give a crude product. The crude product was purified by flash column chromatography (10-20% EtOAc in petrol) to give the ester derivative S26⁵² as yellow oil (49 mg, 0.14 mmol, 8%). R_f: 0.45 (30:70EtOAc:petrol). ¹HNMR(400MHz, CDCl₃): δ 8.19 (s, 1H), 7.91-7.82 (m, 1H), 7.79-7.67(m, 2H), 7.49-7.39 (m, 1H),4.40 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 160.7,142.7,136.8,134.5 (q, J = 40Hz), 132.7,130.8,129.4, 127.9 (q, J = 32Hz), 127.4(q, J =5.0Hz), 122.5 (q, J = 274Hz), 118.9 (q, J = 272 Hz), 116.4, 61.3, 14.1 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -56.5, -60.6 ppm. MS (ESI): [M + H]⁺ 353.2. IR (neat): ν_max 1724 (s, C⩵O), 1563 (m), 1507 (m), 1319 (s), 1249 (s), 1161 (s), 968 (s) cm⁻¹. HRMS: calculated for C₁₄H₁₀F₆N₂O₂ [M + H]⁺ = 353.0724, observed = 353.0721.

5-(Trifluoromethyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrazole-4-carboxylic Acid (33)

According to General Procedure B, the ester derivative S26 (48.0 mg, 0.14 mmol) was stirred for 2 h to give the acid derivative 3 3⁵² as a yellow amorphous solid (29.1 mg, 90.0 μMol, 64%). R_f: 0.30 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 10.05 (br. s, 1H), 8.26 (s, 1H), 7.87 (dd, J = 6.9, 2.5 Hz, 1H), 7.77-7.70 (m, 2H), 7.45 (dd, J = 6.9,2.5 Hz, 1H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 177.7, 165.9, 143.4, 136.5, 135.4 (q, J = 41 Hz), 132.8, 131.0, 129.3, 127.5 (q, J = 32 Hz), 127.5 (q, J =5Hz), 122.4(q, J = 274Hz),118.6(q, J = 272 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -56.6, -60.6 ppm. MS (ESI): [M - H]^- 323.0. HPLC: retention time: 1.80 min (>99%). IR (neat): ν_max 2924 (w, O–H), 1703 (s, C⩵O), 1573 (m), 1320 (s), 1258 (m), 1137 (s), 1034 (m) cm⁻¹. HRMS: calculated for C₁₂H₆F₆N₂O₂ [M + H]⁺ = 325.0411, observed = 325.0400.

Ethyl 5-(Trifìuoromethyl)-1-[4-(trifìuoromethyl)phenyl]-1H-pyra-zole-4-carboxylate (S27)

According to General Procedure E, 4-(trifluoromethyl)phenylhydrazine hydrochloride (0.25 g, 1.17 mmol) was stirred for 4 h to give a crude product. The crude product was purified by flash column chromatography (5% EtOAc in petrol) to give the ester derivative S27⁵² as yellow oil (0.27 g, 0.77 mmol, 66%). R_f 0.68 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.13 (s, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.3 Hz, 2H), 4.37 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.8, 143.0,142.2, 132.8 (q, J = 40.4 Hz), 132.1 (q, J = 33.1 Hz), 126.5 (q, J = 3.7 Hz), 126.4 (q, J = 1.1 Hz), 123.5 (q, J = 272.5 Hz), 119.1 (q, J = 271.5 Hz), 117.5 (q, J = 1.3 Hz), 61.5, 14.1 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -56.0, -63.7 ppm. MS (ESI): [M + H]⁺ 353.2. IR (neat): ν_max 2991-2909 (w, C–H), 1737 (s, C⩵O), 1562 (w), 1466 (w), 1323 (m), 1300 (m), 1223 (s), 1175 (s), 1123 (s), 1062 (s), 1035 (s) cm⁻¹.

5-(Trifluoromethyl)-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-carboxylic Acid (34)

According to General Procedure B, the ester derivative S27 (0.10 g, 0.28 mmol) was stirred for 4 h to give the acid derivative 34⁵² as an amorphous white solid (88 mg, 0.27 mmol, 96%). R_f: 0.26 (20:80MeOH:DCM). ¹HNMR(400 MHz, CDCl₃): δ 8.25 (s, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 8.3 Hz, 2H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.0, 143.8, 141.9, 133.8 (q, J = 40.7 Hz), 132.4 (q, J = 33.1 Hz), 126.6 (q, J = 3.7 Hz), 126.5, (q, J = 1.1 Hz), 123.5 (q, J = 272.7 Hz), 118.9 (q, J = 271.9 Hz), 116.1 (q, J = 1.4 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -56.1, -63.7 ppm. MS (ESI): [M - H]^- 323.1. HPLC: retention time: 1.99 min (>99%). IR (neat): ν_max 3100–2587 (m, O–H, C–H), 1701 (m, C⩵O), 1570 (w), 1467 (w), 1422 (w), 1407 (w), 1327 (m), 1295 (m), 1259 (m), 1224 (m), 1179 (m), 1141 (s), 1112 (s), 1063 (m), 1031 (m) cm⁻¹.

Ethyl 1-(4-Methoxyphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S28)

According to General Procedure E, 4-methox-yphenyl hydrazine hydrochloride (0.22 g, 1.24 mmol) was stirred for 2 h to give a crude product. The crude product was purified by flash column chromatography (10% EtOAc in petrol) to give the ester derivative S28⁵⁵ as an amorphous orange solid (0.19 g, 0.60 mmol, 48%). R_f: 0.53 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CD₃OD): δ 8.08 (s, 1H), 7.33 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 4.37 (q, J = 7.1 Hz, 2H), 3.87 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CD₃OD): δ 161.2, 160.6, 142.3, 132.7 (q, J = 39.8 Hz), 132.4, 127.2, 119.2 (q, J = 271.4 Hz), 116.5 (q, J = 1.0 Hz), 114.3,61.3, 55.7, 14.2 ppm. ¹⁹F NMR (376 MHz, CD₃OD): δ -56.5 ppm. MS (ESI): [M + H]⁺ 315.1. IR (neat): ν_max 3117–2843 (w, C–H), 1735 (m, C⩵O), 1565 (w), 1516 (s), 1465 (w), 1446 (w), 1372 (w), 1302 (s), 1223 (s), 1185 (m), 1129 (s), 1080 (m), 1043 (s), 1018 (s), 971 (m) cm⁻¹.

1-(4-Methoxyphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carbox-ylic Acid (35)

According to General Procedure B, the ester derivative S28 (62 mg, 0.20 mmol) was stirred for 1 h to give the acid derivative 3 5⁵⁵ as an amorphous white solid (50 mg, 0.18 mmol, 90%). R_f 0.30 (20:80 MeOH.DCM). ¹H NMR (400 MHz, CDCl₃): δ 11.43 (br. s, 1H), 8.19 (s, 1H), 7.35 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 3.87 (s, 3H) ppm. ¹³C NMR (400 MHz, CDCl₃): δ 166.4, 160.7, 143.1, 133.6 (q, J =40.2 Hz), 132.1, 127.2, 119.0 (q, J = 271.6 Hz), 115.2 (q, J = 1.2 Hz), 114.4, 55.7 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -56.5 ppm. MS (ESI): [M - H]^- 285.0. HPLC: retention time: 1.80 min (>99%). IR (neat): ν_max 2968-2591 (m, O–H, C–H), 1700 (s, C⩵O), 1679 (m), 1563 (w), 1517 (s), 1466 (w), 1423 (w), 1302 (s), 1249 (s), 1175 (m), 1140 (s), 1075 (m), 1028 (s) cm⁻¹.

(2-Methyl-4-nitrophenyl)hydrazine (S29)

According to a proce-dure,⁵⁶ hydrazine monohydrate (2.00 mL, 40.6 mmol) was added to a solution of 1-fluoro-2-methyl-4-nitrobenzene (3.00 g, 19.3 mmol) in isopropyl alcohol (30.0 mL). After stirring the mixture at 90 °C for 2 h, additional hydrazine monohydrate (2.00 mL, 40.6 mmol) was added and the mixture was kept at 90 °C for a further 2 h. The mixture was allowed to cool to room temperature, and diethyl ether (25 mL) was added. The precipitate was collected by filtration, washed with water (12 mL) and diethyl ether (12 mL), and dried in vacuo. Subsequently, a 6 M HCl aqueous solution (18 mL) was added. After stirring the mixture at room temperature for 1 h, the precipitate was collected by filtration, washed with a 6 M HCl aqueous solution, and dried in vacuo to yield the hydrochloride salt of the hydrazine derivative S29⁵⁶ as a white-yellow amorphous solid (2.00 g, 9.86 mmol, 51%). R_f: 0.75 (20:80 MeOH:DCM). ¹H NMR (400MHz, DMSO-d₆): δ 10.56 (br. s, 2H), 8.88 (s, 1H), 8.09 (dd, J = 9.0, 2.7 Hz, 1H), 8.02 (d, J = 2.7 Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H), 2.27 (s, 3H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): δ 149.4, 140.2, 125.4, 124.4, 123.0, 111.2, 17.2 ppm. MS (ESI): [M + H] + 168.0. IR (neat): ν_max 3280 (s, N-H), 3074 (m, NH), 2817-2607 (m, N-H, C–H), 1593 (m), 1584 (m), 1495 (s), 1335 (s), 1298 (m), 1253 (m), 1216 (m), 1102 (m) cm⁻¹.

Ethyl 1-(2-Methyl-4-nitrophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S30)

According to General Procedure E, the hydrazine derivative hydrochloride S29 (0.25 g, 1.22 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (0-10% EtOAc in petrol) to give the ester derivative S30 as yellow oil (0.24 g, 0.70 mmol, 57%). R_f 0.40 (20:80 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.22 (d, J = 2.5 Hz, 1H), 8.18 (s, 1H), 8.17 (dd, J = 8.6,2.5 Hz, 1H), 7.44 (d, J =8.6 Hz, 1H),4.37(q, J = 7.1Hz, 2H),2.16(S, 3H),1.37(t, J = 7.1Hz,3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.6, 148.6, 143.4, 138.0, 133.6 (q, J = 40.0 Hz), 128.6,128.5,126.1,121.9,118.9 (q, J = 271.6 Hz), 116.8 (q, J = 1.4 Hz), 61.6, 17.3, 14.1 ppm. ¹⁹F NMR (396 MHz, CDCl₃): δ - 57.5 ppm.MS(ESI):[M + H]⁺ 344.2. IR (neat): ν_max 2984-2873 (w, C–H), 1726 (s, C⩵O), 1566 (w), 1534 (m), 1499 (w), 1354 (m), 1300 (m), 1229 (s), 1180 (s), 1139 (s), 1101 (s), 1039 (s) cm⁻¹.

1-(2-Methyl-4-nitrophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (36)

According to General Procedure B, the ester derivative S30 (0.10 g, 0.29 mmol) was stirred for 6 h to give the acid derivative 36 as a white amorphous solid (76.0 mg, 0.24 mmol, 83%). R_f: 0.33 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 8.32 (d, J = 2.5 Hz, 1H), 8.24 (s, 1H), 8.23 (dd, J =8.6, 2.5 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 2.16 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 163.3, 150.1, 144.6, 139.3, 134.6 (q, J = 40.1 Hz), 129.9, 126.9, 122.9, 120.4 (q, J = 270.9 Hz), 118.4 (d, J = 1.0 Hz), 17.1 ppm. ¹⁹F NMR (396 MHz, CDCl₃): δ -58.8 ppm. MS (ESI): [M - H]^- 314.0. HPLC: retention time: 1.84 min (>99%). IR (neat): ν_max 3100-2580 (w, O–H, C–H), 1705 (m, C⩵O), 1686 (m), 1572 (m), 1527 (m), 1496 (m), 1347 (m), 1293 (m), 1263 (m), 1228 (m), 1179 (m), 1140 (s), 1098 (m), 1034 (m) cm⁻¹. HRMS: calculated for C₁₂H₈F₃N₃O₄ [M + H]⁺ = 316.0545, observed = 316.0542.

3-Hydrazinylbenzamide Hydrochloride (S31)

According to General Procedure F, 3-aminobenzamide (1.00 g, 7.34 mmol) gave the hydrazine derivative S31⁵⁷ as a pale brown amorphous solid (1.36 g, 7.25 mmol, 99%). R_f: 0.20 (20:80 MeOH:DCM). ¹H NMR (400 MHz, DMSO-d₆): δ 10.3 (br. s, 3H), 7.93 (br. s, 1H), 7.53 (s, 1H), 7.44 (app. d, J = 7.7 Hz, 1H), 7.34 (app. t, J = 8.0,1H), 7.13 (dd, J =8.0 and 2.4 Hz, 1H), 4.83 (br. s, 2H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): δ 167.7, 145.6, 135.1, 128.8, 120.1, 117.1, 113.7 ppm. MS (ESI): [M + H] + 152.0. IR (neat): ν_max 3458 (w, N-H), 3358 (w, N-H), 3271 (m, NH), 3176-2700 (s, N-H, C–H), 1646 (s, C⩵O), 1561 (s), 1540 (s), 1456 (s) cm⁻¹.

Ethyl 1-(3-Carbamoylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S32)

According to General Procedure E, the hydrazine derivative S31 (0.25 g, 1.33 mmol) was stirred for 6 h to give a crude product. The crude product was purified using flash column chromatography (40-60% EtOAc in petrol) to give the ester derivative S32 as a white amorphous solid (0.20 g, 0.61 mmol, 46%). R_f 0.53 (90:10 EtOAc:petrol). ¹H NMR (400 MHz, DMSO-d₆): δ 8.33 (s, 1H), 8.16 (br. s, 1H), 8.11 (app. dt, J =7.5, 1.6 Hz, 1H), 8.02 (s, 1H), 7.75-7.65 (m, 2H), 7.61 (br. s, 1H), 4.32 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): δ 166.2, 160.2, 142.3, 138.9, 135.3, 131.4, 129.4, 129.11, 128.8, 125.1, 118.9 (q, J = 271.1 Hz), 116.3 (q, J = 1.5 Hz), 61.1, 13.9 ppm. ¹⁹F NMR (376 MHz, DMSO-d₆): δ -55.5 ppm. MS (ESI): [M + H]⁺ 328.1. IR (neat): ν_max 3442 (m, N-H), 3199 (m, N-H), 2984 (w, C–H), 1729 (s, C⩵O), 1697 (s, C⩵O), 1623 (w), 1566 (w), 1449 (w), 1374 (m), 1299 (s), 1248 (s), 1224 (s), 1190 (s), 1133 (s), 1079 (m), 1038 (s), 986 (m) _cm⁻¹_.

1-(3-Carbamoylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-car-boxylic Acid (37)

According to General Procedure B, the ester derivative S32 (85 mg, 0.26 mmol) was stirred for 6 h to give the acid derivative 37 as a white amorphous solid (70 mg, 0.23 mmol, 88%). R_f 0.10 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 8.22 (app. dt, J = 7.4, 1.6 Hz, 1H), 8.18 (s, 1H), 8.08 (s, 1H), 7.75-7.64 (m, 2H), 5.05 (br. s, 2H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 167.9, 163.5, 144.0, 140.9, 133.4, 132.1, 131.4, 130.6, 128.2 (q, J = 0.8 Hz), 124.6, 120.5 (q, J = 270.7 Hz), 118.6 (q, J = 1.4 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -56.6 ppm. MS (ESI): [M - H]^- 298.0. HPLC: retention time: 1.53 min (94%). IR (neat): ν_max 3100-2555 (s, N-H, O–H, C–H), 1689 (s, C⩵O), 1571 (m), 1472 (w), 1448 (w), 1418 (m), 1398 (w), 1298 (s), 1265 (s), 1220 (m), 1196 (m), 1117 (s), 1135 (s), 1125 (s), 1069 (m), 1032 (s) cm⁻¹. HRMS: calculated for C₁₂H₈F₃N₃O₃ [M + H]⁺ = 300.0596, observed = 300.0592.

Ethyl 1-(2,3-Dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S33)

According to General Procedure E, 2,3-dimethylphenylhydrazine hydrochloride (0.25 g, 1.44 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (5% EtOAc in petrol) to give the ester derivative S33 as yellow oil (0.30 g, 0.96 mmol, 67%). R_f: 0.66 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.14 (s, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 7.08 (d, J = 7.7, 1H), 4.37 (q, J = 7.2, 2H), 2.33 (s, 3H), 1.87 (s, 3H), 1.38 (t, J = 7.2, 3H) ppm. ¹³C NMR(100MHz, CDCl₃): δ 161.1,142.5,138.6,138.4,134.0, 133.5 (q, J = 39.9 Hz), 131.7, 125.9, 124.7, 119.1 (q, J = 271.5 Hz), 115.8 (d, J = 1.3 Hz), 61.2,20.2, 14.1, 13.8 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ - 57.8 ppm. MS (ESI): [M + H]⁺ 313.1. IR (neat): ν_max 2995-2924 (w, C–H), 1713 (s, C⩵O), 1554 (m), 1481 (m), 1384 (m), 1293 (m), 1244 (s), 1230 (s), 1176 (s), 1144 (s), 1038 (s), 1017 (m) cm⁻¹.

1-(2,3-Dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (38)

According to General Procedure B, the ester derivative S33 (0.12 g, 0.38 mmol) was stirred for 4.5 h to give the acid derivative 38 as a yellow amorphous solid (95 mg, 0.33 mmol, 88%). R_f 0.22 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 11.44 (br. s, 1H), 8.25 (s, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.22 (t, J =7.7 Hz, 1H), 7.12 (d, J = 7.7 Hz, 1H), 2.36 (s, 3H), 1.91 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 166.5, 143.4, 138.6, 138.5, 134.5 (q, J = 40.2 Hz), 134.0, 131.9, 126.0, 124.7, 118.9 (q, J =271.8 Hz), 114.6 (d, J = 1.1 Hz), 20.3, 13.8 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -56.9 ppm. MS (ESI): [M + H]⁺ 285.1. HPLC: retention time: 1.88 min (>99%). IR (neat): 2926-2580 (m, O–H, C–H), 1698 (s, C⩵O), 1681 (s), 1564 (m), 1482 (w), 1456 (w), 1417 (w), 1299 (s), 1258 (s), 1225 (s), 1186 (s), 1138 (s), 1033 (s), 968 (m) cm⁻¹. HRMS: calculated for C₁₃H₁₁F₃N₂O₂ [M + H]⁺ = 285.0851, observed = 285.0851.

Ethyl 1-(2,4-Dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S34)

According to General Procedure E, 2,4-dimethyphenylhydrazine hydrochloride (0.21 g, 1.22 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (15% EtOAc in petrol) to give the ester derivative S34 as a pale red amorphous solid (0.25 g, 0.81 mmol, 66%). R_f: 0.44 (15:85 EtOAc.petrol). ¹H NMR (600 MHz, CDCl₃): δ 8.14 (s, 1H), 7.18-7.06 (m, 3H), 4.38 (q, J = 7.2 Hz, 2H), 2.39 (s, 3H), 2.00 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 161.1, 142.4, 140.4, 136.0, 134.9, 133.4 (q, J = 39.7 Hz), 131.5, 127.1, 126.7, 119.1 (q, J = 271.4Hz), 115.7, 61.2, 21.2, 16.7, 14.1 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -56.9 ppm. MS (ESI): [M + H]⁺ 313.2. IR (neat): 1715 (s, C⩵O), 1553 (m), 1550 (w), 1389 (m), 1255 (m), 1241 (s), 1149 (s), 1144 (s), 1041 (w), 1038 (m), 972 (m) cm⁻¹. HRMS: calculated for CˆH₁₅F3N₂O₂ [M + H]⁺ = 313.1163, observed = 313.1153.

1-(2,4-Dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-car-boxylic Acid (39)

According to General Procedure B, the ester derivative S34 (0.22 g, 0.70 mmol) was stirred for 2 h to give the acid derivative 39 as a red amorphous solid (0.19 g, 0.67 mmol, 96%). R_f. 0.22 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 11.74 (s, 1H), 8.25 (s, 1H), 7.21-7.11 (m, 3H), 2.40 (s, 3H), 2.02 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.4, 143.3, 140.7, 135.8, 134.8, 134.3 (q, J = 40.2 Hz), 131.6, 127.2, 126.7 (q, J = 0.7 Hz), 118.9 (q, J = 271.8 Hz), 114.6 (d, J = 1.2 Hz), 21.2, 16.7 ppm. ¹⁹F NMR (396 MHz, CDCl₃): δ -57.0 ppm. MS (ESI): [M + H]⁺ 285.1. HPLC: retention time: 1.95 min (>99%). IR (neat): 2927 (w, O–H), 1720 (s, C⩵O), 1558 (m), 1506 (m), 1305 (s), 1233 (s), 1163 (s), 1068 (m), 1031 (s) cm⁻¹. HRMS: calculated for C₁3H₁₁F3N₂O₂ [M + H]⁺ = 285.0851, observed = 285.0841.

Ethyl 1-(2,5-Dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S35)

According to General Procedure E, 2,5-dimethylphenylhydrazine hydrochloride (0.25 g, 1.44 mmol) was stirred for 16 h to give a crude product. The crude product was purified using flash column chromatography (5% EtOAc in petrol) to give the ester derivative S35 as a yellow amorphous solid (0.28 g, 0.90 mmol, 63%). R_f. 0.70 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.14 (s, 1H), 7.24-7.16 (m, 2H), 7.05 (s, 1H), 4.36 (q, J = 7.1 Hz, 2H), 2.34 (s, 3H), 1.98 (s, 3H), 1.37 (t, J =7.1 Hz, 3H) ppm. ¹³CNMR(100 MHz, CDCl₃): δ 161.1, 142.5, 138.4, 136.6, 133.4 (q, J = 39.8 Hz), 132.1, 131.1, 130.7, 127.5, 119.1 (q, J = 271.4Hz), 115.8 (d, J = 1.4Hz), 61.2, 20.7, 13.4,14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.9 ppm. MS (ESI): [M + H]⁺ 313.1. IR (neat): ν_max 3122-2926 (w, C–H), 1717 (s, C⩵O), 1553 (m), 1468 (m), 1384 (w), 1298 (m), 1234 (s), 1174 (s), 1135 (s), 1070 (w), 1036 (m) cm⁻¹.

1-(2,5-Dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-car-boxylic Acid (40)

According to General Procedure B, the ester derivative S35 (0.11 g, 0.35 mmol) was stirred for 4.5 h to give the acid derivative 40 as yellow oil (83.0 mg, 0.29 mmol, 83%). R_f: 0.37 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 8.24 (s, 1H), 7.26-7.20 (m, 2H), 7.08 (s, 1H), 2.37 (s, 3H), 2.01 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.5, 143.4, 138.3, 136.7, 134.4 (q, J = 40.1 Hz), 132.1, 131.4, 130.8, 127.5, 118.9 (q, J = 271.8 Hz), 114.6, 20.8, 16.5 ppm. ¹⁹F NMR (376 MHz, CDCl₃). δ -57.0 ppm. MS (ESI): [M - H]^- 283.0. HPLC: retention time: 1.90 2929-2600 (m, O–H, C–H), 1708 (m C⩵O), 1679 (m), 1559 (m), 1510 (w), 1458 (m), 1421 (w), 1297 (m), 1259 (s), 1232 (m), 1182 (m), 1143 (s), 1033 (m) cm ¹. HRMS: calculated for C₁₃H₁₁F₃N₂O₂ [M + H]⁺ = 285.0851, observed = 285.0855.

Ethyl 1-(2,6-dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-arboxylate (S36)

According to General Procedure E, 2,6-dimethyl-phenylhydrazine hydrochloride (0.32 g, 1.87 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (30% EtOAc in petrol) to give the ester derivative S36 as a white amorphous solid (93.7 mg, 0.30 mmol, 16%). R_f. 0.59 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.24 (s, 1H), 7.31 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 7.7 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), 1.99 (s, 6H), 1.41 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 161.0, 143.1, 138.0, 135.5, 133.5 (q, J = 40.0 Hz), 130.0, 128.3, 119.1 (q, J =271.0 Hz), 115.6 (q, J = 1.5 Hz), 61.2, 16.9, 14.1 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -58.4ppm. MS (ESI): [M + H]⁺ 313.2. IR (neat): 1736 (s, C⩵O), 1560 (s), 1484 (s), 1297 (s), 1222 (s), 1145 (s), 1039 (s), 966 (s) cm⁻¹. HRMS: calculated for C₁₅H₁₅F₃N₂O₂ [M + H]⁺ = 313.1163, observed = 313.1155.

1-(2,6-Dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-car-boxylic Acid (41)

According to General Procedure B, the ester derivative S36 (92.0 mg, 0.29 mmol) was stirred for 2 h to give the acid derivative 41 as a white amorphous solid (59.0 mg, 0.21 mmol, 70%). R_f. 0.38 (20:80MeOH:DCM). ¹HNMR(400 MHz, CDCl₃): δ 8.31 (s, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.17 (d, J = 7.6 Hz, 2H), 1.99 (s, 6H) ppm. ¹³C NMR (150 MHz, CDCl₃). δ 166.3, 144.0, 138.0, 135.5, 134.5 (q, J = 40.2 Hz), 130.2, 128.4, 118.9(q, J = 271.8 Hz), 114.5 (q, J = 1.2 Hz), 17.0 ppm. ¹⁹F NMR (376 MHz, CDCl₃). δ -58.5 ppm. MS (ESI). [M + H]⁺ 285.2. HPLC. retention time. 1.90 min (>99%). IR (neat). 2924 (w, O–H), 1704 (s, C⩵O), 1571 (m), 1485 (m), 1303 (m), 1266 (m), 1249 (m), 1149 (s), 1035 (s), 967 (s) cm⁻¹. HRMS. calculated for C₁3H₁₁F3N₂O₂ [M + H]⁺ = 285.0851, observed = 285.0847.

Ethyl 1-(2,6-Dichlorophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-arboxylate (S37)

According to General Procedure E, 2,6-dichlor-ophenylhydrazine hydrochloride (0.13 g, 0.63 mmol) was stirred for 2 h to give a crude product. The crude product was purified using flash column chromatography (10% EtOAc in petrol) to give the ester derivative S37 as a pale yellow amorphous solid (0.14 g, 0.40 mmol, 63%). R_f. 0.47 (30.70 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃). δ 8.26 (s, 1H), 7.50-7.45 (m, 2H), 7.42 (dd, J = 9.5,6.4 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 160.7, 143.9, 135.5, 134.5 (q, J = 40.4 Hz), 134.4 (q, J = 0.7 Hz), 131.8, 128.7, 118.9 (q, J = 271.6 Hz), 116.4 (q, J = 1.6 Hz), 61.4, 14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃). δ -59.6 ppm. MS (ESI). [M + H]⁺ 353.1. IR (neat). 3090–2942 (w, C–H), 1729 (s, C⩵O), 1571 (m), 1496 (m), 1442 (m), 1294 (m), 1240 (s), 1221 (s), 1149 (s), 1084 (m), 1040 (s) cm⁻¹.

1-(2,6-Dichlorophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-car-boxylic Acid (42)

According to General Procedure B, the ester derivative S37 (88.3 mg, 0.25 mmol) was stirred for 1 h to give the acid derivative 42 as a yellow amorphous solid (78.0 mg, 0.24 mmol, 98%). R_f. 0.25 (EtOAc). ¹H NMR (400 MHz, CDCl₃). δ 11.01 (br. s, 1H), 8.35 (s, 1H), 7.53-7.41 (m,3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 166.0, 144.6, 135.5 (q, J = 41.2 Hz), 134.3, 134.3 (q, J = 0.6 Hz), 132.0, 128.7, 118.6 (q, J = 272.0 Hz), 115.2 (q, J = 1.5 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃). δ -59.7 ppm. MS (ESI). [M - H]^- 323.0. HPLC. retention time. 1.90 min (94%). IR (neat). 2873 (m, O–H), 2599–2565 (w, C–H), 1699 (s, C⩵O), 1572 (m), 1494 (m), 1444 (m), 1424 (m), 1302 (m), 1259 (m), 1223 (m), 1148 (s), 1033 (m) cm⁻¹. HRMS. calculated for C₁₁H₅Cl₂F₃N₂O₂ [M + H]⁺ = 324.9758, observed = 324.9758.

Ethyl 1-(3,4-Dichlorophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S38)

According to General Procedure E, 3,4-dichlor-ophenylhydrazine hydrochloride (0.25 g, 1.17 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (5% EtOAc in petrol) to give the ester derivative S38⁵² as a yellow-white amorphous solid (0.21 g, 0.59 mmol, 51%). R_f. 0.38 (10.90 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃). δ 8.11 (s, 1H), 7.62-7.56 (m, 2H), 7.29 (d, J = 8.7 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 160.8, 143.0, 138.4, 134.7, 133.5 (q, J = 40.3 Hz), 130.9, 128.1 (q, J = 1.1 Hz), 125.2 (q, J = 1.1 Hz), 122.2, 119.1 (q, J = 271.6 Hz), 117.5, 61.6, 14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -55.2 ppm. MS (ESI): [M + H] + 353.1. IR (neat): ν_max 3068-2906 (w, CH), 1702 (s, C⩵O), 1595 (w), 1557 (m), 1486 (m), 1417 (w), 1403 (w), 1385 (m), 1356 (w), 1295 (m), 1254 (s), 1221 (m), 1174 (m), 1149 (s), 1135 (s), 1084 (m), 1039 (m), 1013 (m), 982 (m) cm⁻¹.

1-(3,4-Dichlorophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic Acid (43)

According to General Procedure B, the ester derivative S38 (0.10 g, 0.28 mmol) was stirred for 4 h to give the acid derivative 43⁵² as a light brown amorphous solid (40 mg, 0.12 mmol, 43%). R_f: 0.30 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 9.57 (br. s, 1H), 8.22 (s, 1H), 7.65-7.55 (m, 2H), 7.31 (dd, J = 8.6, 2.5 Hz, 1H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.0, 143.7, 138.2, 135.0, 133.8 (q, J = 40.6 Hz), 133.6, 131.0, 128.1 (q, J = 1.2 Hz), 125.2 (q, J = 1.2 Hz), 118.8 (q, J = 272.0 Hz), 116.1 (q, J = 1.2 Hz) ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -55.2 ppm. MS (ESI): [M - H]^- 323.0. HPLC: retention time: 2.06 min (>99%). IR (neat): ν_max 3102-2872 (m, O–H, C–H), 1703 (s, C⩵O), 1562 (m), 1484 (m), 1424 (w), 1297 (m), 1265 (m), 1249 (m), 1216 (m), 1185 (s), 1152 (s), 1132 (s), 1076 (m), 1031 (s) cm⁻¹.

(4,5-Dichloro-2-methylphenyl)hydrazine Hydrochloride (S39)

According to General Procedure F, 4,5-dichloro-2-methylaniline (0.69 g, 3.91 mmol) gave the hydrazine derivative S39 as a pale brown amorphous solid (0.49 g, 2.15 mmol, 55%). R_f 0.47 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CD₃OD): δ 7.34 (s, 1H), 7.05 (s, 1H), 2.22 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 143.7, 133,1, 131.0, 127.5, 126.2, 115.2, 16.5 ppm. MS (ESI): [M + H]⁺ 228.0. IR (neat): ν_max 3273 (m, N-H), 2872 (s, N-H), 2693 (s, N-H, C–H), 1586 (w), 1536 (s), 1493 (s), 1422 (m), 1380 (m), 1219 (w), 1174 (m), 1149 (m), 1118 (m), 941 (m) cm⁻¹.

Ethyl 1-(4,5-Dichloro-2-methylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S40)

According to General Procedure E, the hydrazine derivative S39 (0.49 g, 2.15 mmol) was stirred for 4 h to give a crude product. The crude product was purified using flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S40 as a yellow amorphous solid (0.43 g, 1.17 mmol, 54%). R_f: 0.63 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.15 (s, 1H), 7.45 (s, 1H), 7.38 (s, 1H), 4.38 (q, J =7.1 Hz, 2H), 2.00 (s, 3H), 1.38 (t, J =7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.7, 143.1, 137.6, 135.8, 134.7, 133.6 (q, J = 40.1 Hz), 132.4, 130.4, 129.0, 119.0 (q, J = 271.6 Hz), 116.5 (q, J = 1.5 Hz), 61.5, 16.5, 14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.6 ppm. MS (ESI): [M + H]⁺ 367.1. IR (neat): ν_max 3122-2869 (w, C–H), 1716 (m, C⩵O), 1555 (m), 1488 (m), 1419 (w), 1400 (w), 1383 (m), 1352 (w), 1294 (m), 1245 (m), 1149 (s), 1132 (s), 1077 (m), 1035 (m), 1008 (m), 984 (m) cm⁻¹.

1-(4,5-Dichloro-2-methylphenyl)-5-(trifluoromethyl)-1H-pyra-zole-4-carboxylic Acid (44)

According to General Procedure B, the ester derivative S40 (0.43 g, 1.17 mmol) was stirred for 5 h to give a crude product. The crude product was triturated with hexane to give the acid derivative 44 as a white amorphous solid (0.32 g, 0.94 mmol, 80%). R_f: 0.38 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 11.12 (br. s, 1H), 8.26 (s, 1H), 7.47 (s, 1H), 7.40 (s, 1H), 2.03 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.0, 143.9, 137.4, 135.6, 135.0, 134.6 (q, J = 40.4 Hz), 132.5, 130.6, 129.0, 118.7 (q, J = 272.0 Hz), 115.2, 16.5 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ - 57.7 ppm. MS (ESI): [M - H]^- 337.0. HPLC: retention time: 2.15 min (>99%). IR (neat): ν_max 2924-2600 (m, O–H, C–H), 1701 (m, C⩵O), 1681 (m), 1561 (m), 1490 (m), 1452 (m), 1418 (w), 1300 (m), 1260 (m), 1223 (m), 1178 (m), 1149 (s), 1074 (w), 1032 (m) cm⁻¹. HRMS: calculated for C₁₂H₇Cl₂F₃N₂O₂ [M + H]⁺ = 338.9915, observed = 338.9915.

(3,4-Dichloro-2-methylphenyl)hydrazine Hydrochloride (S41)

According to General Procedure F, 3,4-dichloro-2-methylaniline (0.20 g, 1.13 mmol) gave the hydrazine derivative S41 as a pale brown amorphous solid (0.23 g, 0.63 mmol, 56%). R_f: 0.54 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CD₃OD): δ 7.41 (d, J = 8.7 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 2.37 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 143.6, 134.2, 128.8, 127.7, 127.5, 113.5, 14.9 ppm. MS (ESI): [M + H]⁺228.1. IR (neat): ν_max 3181-3156 (m, N-H), 2990–2653 (s, N-H, C–H), 1556 (s), 1517 (m), 1483 (w), 1456 (s), 1401 (w), 1190 (m), 1170 (s), 1062 (m) cm⁻¹.

Ethyl 1-(3,4-Dichloro-2-methylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (S42)

According to General Procedure E, the hydrazine derivative S41 (0.12 g, 0.53 mmol) was stirred for 5.5 h to give a crude product. The crude product was purified using flash column chromatography (5% EtOAc in petrol) and triturated with hexane to give the ester derivative S42 as a colorless amorphous solid (0.11 g, 0.30 mmol, 57%). R_f: 0.58 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.16 (s, 1H), 7.45 (d, J =8.5 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 4.38 (q, J = 7.1, 2H), 2.07 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 160.8, 143.1, 137.7, 136.8, 135.5, 134.1, 133.7 (q, J = 40.0 Hz), 127.9, 126.1, 119.0 (q, J = 271.6 Hz), 116.5 (q, J = 1.4 Hz), 61.5, 16.1, 14.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.6 ppm. MS (ESI): [M + H] + 367.1. IR (neat): ν_max 3120 (w, C–H), 2980 (w, C–H), 1715 (s, C⩵O), 1555 (m), 1476 (m), 1385 (m), 1295 (m), 1248 (s), 1150 (s), 1086 (m), 1039 (m), 1013 (m), 974 (m) cm⁻¹.

1-(3,4-Dichloro-2-methylphenyl)-5-(trifluoromethyl)-1H-pyra-zole-4-carboxylic Acid (45)

According to General Procedure B, the ester derivative S42 (0.11 g, 0.30 mmol) was stirred for 5 h to give a crude product. The crude product was triturated with hexane to give the acid derivative 45 as a white amorphous solid (90 mg, 0.27 mmol, 90%). R_f: 0.25 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 10.43 (br. s, 1H), 8.26 (s, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 8.5 Hz, 1H), 2.10 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.0, 143.8, 137.5, 136.7, 135.8, 134.7 (q, J = 40.4 Hz), 134.2, 127.9, 126.1, 118.8 (q, J = 272.0 Hz), 115.2, 16.1 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -57.7 ppm. MS (ESI): [M - H]^- 337.0. HPLC: retention time: 2.08 min (>99%). IR (neat): ν_max 3127-2592 (m, O–H, C–H), 1678 (m, C⩵O), 1561 (m), 1459 (m), 1293 (m), 1254 (m), 1228 (m), 1183 (m), 1147 (s), 1088 (m), 1035 (m) cm⁻¹. HRMS: calculated for C₁₂H₇Cl₂F₃N₂O₂ [M + H]⁺ = 338.9915, observed = 338.9910.

Ethyl 2,5-Dimethyl-1-(2-methylphenyl)-1H-pyrrole-3-carboxylate (S43)

According to General Procedure C, 2-methylaniline (57 μL, 0.54 mmol) and the carbonyl derivative S5 were stirred for 7 h to give a crude product. The crude product was purified by flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S43⁵⁸ as a colorless oil (0.12 g, 0.47 mmol, 87%). R_f: 0.53 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.42-7.27 (m, 3H), 7.10 (d, J = 7.6 Hz, 1H), 6.39 (s, 1H), 4.28 (q, J = 7.1 Hz, 2H), 2.19 (s, 3H), 1.93 (s, 3H), 1.87 (s,3H), 1.35 (t, J = 7.1, 3H) ppm. ¹³C NMR (100MHz, CDCl₃): δ 165.9, 136.9, 136.7, 135.8, 131.0, 129.1, 128.6, 128.2, 127.0, 111.4, 107.4, 59.3, 17.1, 14.7, 12.3, 12.0 ppm. MS (ESI): [M + H]⁺ 258.1. IR (neat): v_miD1 2979-2921 (w, C–H), 1695 (s, C⩵O), 1579 (w), 1534 (m), 1495 (m), 1411 (m), 1335 (w), 1241 (m), 1215 (s), 1198 (m), 1121 (w), 1073 (s), 1002 (w) cm⁻¹.

2,5-Dimethyl-1-(2-methylphenyl)-1H-pyrrole-3-carboxylic Acid (46)

According to General Procedure B, the ester derivative S43 (30 mg, 0.11 mmol) was stirred for 2 days to give a crude product. The crude product was purified using flash column chromatography (30% EtOAc in petrol) to give the acid derivative 46⁵⁸ as a white amorphous solid (21 mg, 92 μMol, 83%). R_f: 0.21 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.43-7.28 (m, 3H), 7.13 (d, J = 7.6 Hz, 1H), 6.46 (s, 1H), 2.23 (s, 3H), 1.95 (s, 3H), 1.89 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 171.4, 137.3, 136.8, 136.7, 131.1, 129.4, 128.7, 128.6, 127.1, 110.8, 108.1, 17.1, 12.4, 12.3 ppm. MS (ESI): [M + H] + 230.1. HPLC: retention time: 1.90 min (>99%). IR (neat): ν_max 3033–2850 (s, O–H, C–H), 2746-2509 (m, C–H), 1652 (s, C⩵O), 1575 (w), 1530 (m), 1494 (m), 1425 (m), 1401 (w), 1257 (s), 1197 (w), 1122 (w), 1078 (m), 1026 (w), 1004 (w), 931 (m) cm⁻¹.

Ethyl 1-(2,6-Dimethylphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (S44)

According to General Procedure C, 2,6-dimethylaniline (65 μL, 0.53 mmol) and the carbonyl derivative S5 were stirred for 18h to give a crude product. The crude product was purified by flash column chromatography (5% EtOAc in petrol) to give the ester derivative S44 as colorless oil (0.13 g, 0.48 mmol, 91%). R_f: 0.71 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.31-7.23 (m, 1H), 7.19 (d, J = 7.5 Hz, 2H), 6.45 (s, 1H), 4.30 (q, J = 7.1 Hz, 2H), 2.18 (s, 3H), 1.93 (s, 6H), 1.86 (s, 3H), 1.38 (t, J = 7.1, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 166.0, 136.7, 135.9, 135.0, 128.8, 128.4, 127.2, 111.6, 107.7, 59.3, 17.5, 14.7, 12.0, 11.7 ppm. MS (ESI): [M + H] + 272.5, IR (neat): ν_max 2978–2920 (w, C–H), 1696 (s, C⩵O), 1533 (w), 1478 (w), 1410 (m), 1380 (w), 1214 (s), 1098 (m), 1073 (s), 1001 (w) cm⁻¹.

1-(2,6-Dimethylphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic Acid (47)

According to General Procedure B, the ester derivative S44 (61 mg, 0.22 mmol) was stirred for 2 days to give the acid derivative 47 as a white amorphous solid (40 mg, 0.16 mmol, 73%). R_f 0.70 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 7.31-7.26 (m, 1H), 7.20 (d, J = 7.6 Hz, 2H), 6.52 (s, 1H), 2.21 (s, 3H), 1.96 (s, 6H), 1.87 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 171.5,136.7,136.5, 135.9,128.9, 128.5, 127.7, 111.0, 108.4, 17.5, 12.1, 11.9 ppm. MS (ESI): [M - H]^- 242.8. HPLC: retention time: 2.01 min (>99%). IR (neat): ν_max 3033–2573 (O–H, C–H), 1649 (s, C⩵O), 1574 (w), 1530 (m), 1475 (w), 1425 (m), 1376 (w), 1330 (w), 1265 (s), 1250 (s), 1101 (w), 1078 (m), 1025 (w), 1001 (w), 972 (w), 935 (m) cm⁻¹. HRMS: calculated for C₁₅H₁₇NO₂ [M + H]⁺ = 244.1338, observed = 244.1335.

Ethyl 1-(3,4-Dichlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (S45)

According to General Procedure C, 3,4-dichloroaniline (0.11 g, 0.67 mmol) and the carbonyl derivative S5 were stirred for 18 h to give a crude product. The crude product was purified by flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S45 as colorless oil (0.15 g, 0.48 mmol, 72%). R_f 0.57 (20:80 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.58 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.36 (s, 1H), 4.27 (q, J = 7.1 Hz, 2H), 2.29 (s, 3H), 1.98 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 165.5, 137.2, 136.0, 133.5, 133.2, 131.2, 130.3, 128.6, 127.7, 112.3, 108.2, 59.5, 14.6, 12.7, 12.4ppm. MS (ESI): [M + H]⁺ 312.1. IR (neat): ν_max 2976-2858 (w, C–H), 1690 (s, C⩵O), 1585 (w), 1533 (m), 1473 (m), 1454 (m), 1410 (m), 1352 (m), 1324 (w), 1253 (w), 1219 (s), 1129 (m), 1084 (s), 1028 (s) cm⁻¹.

1-(3,4-Dichlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic Acid (48)

According to General Procedure B, the ester derivative S45 (28.0 mg, 89.7 μMol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (30% EtOAc in petrol) to give the acid derivative 48³⁷ as a white amorphous solid (15 mg, 52.8 μMol, 59%). R_f 0.22 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, DMSO-d₆): δ 11.7 (br. s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.77 (s, 1H), 7.37 (dd, J = 8.5, 2.4 Hz, 1H), 6.24 (s, 1H), 2.22 (s, 3H), 1.95 (s, 3H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): δ 166.0, 136.9,135.0,131.8,131.5,131.2,130.2,128.7,128.0,111.8,108.0, 12.3, 12.0 ppm. MS (ESI): [M + H]⁺284.0. HPLC: retention time: 1.99 min (>99%). IR (neat): ν_max 3069-2574 (s, O–H, C–H), 1644 (s, C⩵O), 1583 (w), 1530 (m), 1465 (s), 1403 (m), 1383 (m), 1327 (w), 1255 (s), 1241 (s), 1129 (m), 1091 (m), 1030 (m), 927 (m) cm⁻¹.

Ethyl 1-(3-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (S46)

According to General Procedure C, 3-chloroaniline (57 μL, 0.54 mmol) and the carbonyl derivative S5 were stirred for 18 h to give a crude product. The crude product was purified by flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S46³⁷ as colorless oil (0.12 g, 0.43 mmol, 80%). R_f 0.54 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.47-7.40 (m, 2H), 7.21 (s, 1H), 7.12-7.05 (m, 1H), 6.37 (s, 1H), 4.28 (q, J = 7.1 Hz, 2H), 2.29 (s,3H), 1.98 (s, 3H), 1.34 (t, J =7.1 Hz, 3H) ppm. ¹C NMR (100 MHz, CDCl₃): δ 165.6, 139.0, 136.1, 135.1, 130.4, 129.0, 128.6, 126.6, 125.1, 112.0, 108.0, 59.4, 14.6, 12.7, 12.4 ppm. MS (ESI): [M + H] + 278.2. IR (neat): ν_max 2978-2850 (m, C–H), 1695 (s, C⩵O), 1592 (m), 1581 (m), 1535 (m), 1479 (m), 1409 (m), 1373 (m), 1354 (w), 1216 (s), 1098 (m), 1077 (s), 1026 (w) cm⁻¹.

1-(3-Chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic Acid (49)

According to General Procedure B, the ester derivative S46 (30 mg, 0.11 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (30% EtOAc in petrol) to give the acid derivative 49³⁷ as a colorless amorphous solid (19 mg, 76.1 μMol, 69%). R_f: 0.22 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.50-7.41 (m,2H), 7.23 (s, 1H), 7.15-7.08 (m, 1H), 6.43 (s, 1H), 2.32 (s, 3H), 1.99 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 171.2, 138.9, 137.6, 135.2, 130.5, 129.2, 129.1, 128.6, 126.6, 111.2, 108.5, 12.8, 12.6 ppm. MS (ESI): [M + H] +250.1. HPLC: retention time: 1.95 min (>99%). IR (neat): ν_max 3086–2586 (s, O–H, C–H), 1646 (s, C⩵O), 1584 (m), 1534 (m), 1466 (m), 1430 (m), 1399 (w), 1378 (w), 1330 (w), 1271 (m), 1250 (s), 1122 (w), 1079 (w), 1034 (w), 945 (w) cm⁻¹.

Ethyl 1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (S47)

According to General Procedure C, 4-chloroaniline (0.10 g, 0.54 mmol) and the carbonyl derivative S5 were stirred for 18 h to give a crude product. The crude product was purified by flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S47³⁷ as a colorless amorphous solid (0.11 g, 0.40 mmol, 74%). R_f: 0.57 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.47 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 6.37 (s, 1H),4.27 (q, J =7.1 Hz, 2H), 2.28 (s, 3H), 1.97 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 165.7, 136.3, 136.1, 134.6, 129.7, 129.6, 128.7, 111.9, 107.9, 59.4, 14.6, 12.7, 12.4ppm. MS (ESI): [M + H]⁺278.2. IR (neat): ν_max 2922 (s, C–H), 2852 (m, C–H), 1694 (s, C⩵O), 1535 (w), 1492 (m), 1415 (m), 1371 (w), 1221 (s), 1081 (s), 1000 (m) cm⁻¹.

1-(4-Chlorophenyl)-2,5-dimethyl-1 H-pyrrole-3-carboxylic Acid (50)

According to General Procedure B, the ester derivative S47 (30 mg, 0.11 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (30% EtOAc in petrol) to give the acid derivative 50³² as a white amorphous solid (18 mg, 72.1 μMol, 66%). R_f: 0.22 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.48 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 6.43 (s, 1H), 2.30 (s, 3H), 1.98 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 170.8, 137.6, 136.2, 134.8, 129.8, 129.6, 129.1, 111.1, 108.5, 12.8,12.6 ppm. MS (ESI): [M +H]+250.1. HPLC: retention time: 1.95 min (>99%). IR (neat): ν_max 3089-2508 (s, O–H, C–H), 1646 (s, C⩵O), 1580 (w), 1538 (m), 1495 (m), 1468 (w), 1428 (m), 1401 (m), 1366 (w), 1329 (w), 1258 (s), 1083 (m), 1018 (w), 1002 (w), 942 (m) cm⁻¹.

Ethyl 1-(4,5-Dichloro-2-methylphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (S48)

According to General Procedure C, 4,5-dichloro-2-methylaniline (95 mg, 0.54 mmol) and the carbonyl derivative S5 were stirred for 5 h to give a crude product. The crude product was purified by flash column chromatography (5% EtOAc in petrol) to give the ester derivative S48 as pale yellow oil (0.15 g, 0.46 mmol, 85%). R_f: 0.48 (20:80 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.45 (s, 1H), 7.25 (s, 1H), 6.39 (s, 1H), 4.27 (q, J = 7.1 Hz, 2H), 2.20 (s, 3H), 1.90 (s, 3H), 1.89 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 165.6, 137.1, 136.3, 135.5, 133.3, 132.4, 130.6, 130.4, 128.0, 112.2, 108.1, 59.5, 16.7, 14.6, 12.3, 12.0ppm. MS (ESI): [M + H]⁺ 326.1. IR (neat): ν_max 2979-2922 (w, C–H), 1695 (s, C⩵O), 1581 (w), 1535 (w), 1479 (s), 1414 (m), 1331 (w), 1216 (s), 1187 (s), 1132 (m), 1077 (s), 1028 (m) cm⁻¹.

1-(4,5-Dichloro-2-methylphenyl)-2,5-dimethyl-1H-pyrrole-3-car-boxylic Acid (51)

According to General Procedure B, the ester derivative S48 (0.12 g, 0.36 mmol) was stirred for 1 day to give a crude product. The crude product was purified using flash column chromatography (0-30% EtOAc in petrol) to give the acid derivative 51 as an amorphous white solid (81 mg, 0.27 mmol, 75%). R_f: 0.20 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, DMSO-d₆): δ 7.75 (s, 1H), 7.62 (s, 1H), 6.25 (s, 1H), 2.10 (s, 3H), 1.83 (s, 6H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): δ 166.0, 137.6, 136.2, 134.6, 132.3, 131.7, 130.4, 129.0, 127.4, 111.7, 107.9, 15.9, 11.8, 11.6 ppm. MS (ESI): [M + H]⁺ 298.0. HPLC: retention time: 2.18 min (>99%). IR (neat): ν_max 2918-2579 (m, O–H, C–H), 1646 (s, C⩵O), 1581 (w), 1536 (w), 1477 (m), 1429 (w), 1406 (w), 1383 (w), 1327 (w), 1260 (s), 1233 (m), 1187 (w), 1134 (m), 1083 (w), 1028 (m), 951 (w) cm⁻¹. HRMS: calculated for C₁₄H₁₃Cl₂NO₂ [M + H]⁺ = 298.0402, observed = 298.0399.

Ethyl 1-(3,4-Dichloro-2-methylphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (S49)

According to General Procedure C, 3,4-dichloro-2-methylaniline (0.10 g, 0.54 mmol) and the carbonyl derivative S5 were stirred for 3 h to give a crude product. The crude product was purified by flash column chromatography (5% EtOAc in petrol) to give the ester derivative S49 as an amorphous white solid (0.15 g, 0.46 mmol, 85%). R_f. 0.68 (30:70 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.44 (d, J =8.4Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.39 (s, 1H), 4.28 (q, J = 7.1 Hz, 2H), 2.19 (s,3H), 1.99 (s,3H), 1.87 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 165.6, 137.9, 136.3, 135.8, 134.2, 134.0, 128.3, 128.2, 127.6, 112.1, 108.0, 59.5, 16.0, 14.6, 12.3, 12.0 ppm. MS (ESI): [M + H]⁺ 328.0. IR (neat): ν_max 3075–2917 (w, C–H), 1696 (s, C⩵O), 1577 (w), 1535 (m), 1467 (m), 1411 (m), 1395 (m), 1372 (m), 1351 (w), 1333 (w), 1249 (m), 1219 (s), 1194 (s), 1127 (w), 1080 (s), 1046 (m), 1001 (m) cm⁻¹.

1-(3,4-Dichloro-2-methylphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic Acid (52)

According to General Procedure B, the ester derivative S49 (0.12 g, 0.36 mmol) was stirred for 1 day to give a crude product. The crude product was purified using flash column chromatography (0-30% EtOAc in petrol) to give the acid derivative 52 as an amorphous white solid (90 mg, 0.30 mmol, 83%). R_f 0.15 (30:70 EtOAc.petrol). ¹H NMR (400 MHz, DMSO-d₆). δ 7.68 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.27 (s, 1H), 2.09 (s, 3H), 1.92 (s, 3H), 1.82 (s, 3H) ppm. ¹³C NMR (100 MHz, DMSO-d₆). δ 166.0, 137.4, 136.0, 134.8, 132.6, 132.5, 128.6, 128.4, 127.7, 111.8, 107.9, 15.6, 11.8, 11.6 ppm. MS (ESI). [M + H]⁺ 298.1. HPLC. retention time. 2.20 min (>99%). IR (neat). ν_max 3067-2521 (m, O–H, C–H), 1655 (s, C⩵O), 1577 (w), 1534 (m), 1469 (m), 1427 (m), 1392 (m), 1379 (m), 1359 (w), 1331 (w), 1261 (s), 1239 (m), 1194 (m), 1126 (w), 1087 (m), 1046 (w), 999 (m) cm⁻¹. HRMS. calculated for C₁₄H₁₃Cl₂NO₂ [M + H]⁺ = 298.0402, observed = 298.0394.

Ethyl 5-Methyl-1-(2-methylphenyl)-2-(trifluoromethyl)-1H-pyr-role-3-carboxylate (S50)

According to General Procedure D, 2-methylaniline (0.10 g, 0.41 mmol) was stirred for 4.5 h to give a crude product. The crude product was purified by flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S50 as yellow oil (27 mg, 86.7 μMol, 21%). R_f. 0.57 (30.70 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃). δ 7.44-7.24 (m, 3H), 7.14 (d, J = 7.8 Hz, 1H), 6.53 (s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 1.97 (s, 3H), 1.86 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 163.5, 136.8, 136.1, 133.1 (q, J = 1.4 Hz), 130.9, 129.6, 128.1 (q, J = 0.75 Hz), 126.8, 121.8 (q, J = 38.1 Hz), 120.5 (q, J = 269.3 Hz), 118.2 (q, J = 2.1 Hz), 110.3, 60.7, 16.9, 14.2, 12.1 ppm. ¹⁹F NMR (376 MHz, CDCl₃). δ - 55.8 ppm. MS (ESI). [M + H]⁺ 312.1. IR (neat). ν_max 2925 (m, C–H), 2853 (w, C–H), 1727 (m, C⩵O), 1511 (m), 1495 (m), 1460 (w), 1422 (m), 1378 (w), 1274(m), 1227 (s), 1199 (m), 1159 (s),1116 (s), 1035 (m) cm⁻¹.

5-Methyl-l-(2-methylphenyl)-2-(trifluoromethyl)-lH-pyrrole-3-carboxylic Acid (53)

According to General Procedure B, the ester derivative S50 (27.0 mg, 86.7 μMol) was stirred for 4 h to give a crude product. The crude product was purified using flash column chromatography (30% EtOAc in petrol) to give the acid derivative 53 as an amorphous white solid (16.0 mg, 56.5 μMol, 65%). R_f. 0.67 (20.80 MeOH.DCM). ¹H NMR (400 MHz, CD₃OD). δ 7.43-7.28 (m, 3H), 7.17 (d, J = 7.7 Hz, 1H), 6.63 (s, 1H), 1.99 (s, 3H), 1.88 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 168.5, 136.8, 136.0, 133.2 (q, J = 1.4Hz), 131.0, 129.7, 128.0, 126.9, 123.0 (q, J = 38.5 Hz), 120.3 (q, J = 269.9 Hz), 116.7 (q, J = 1.5 Hz), 111.2, 16.9, 12.2 ppm. ¹⁹FNMR(376 MHz, CDCl₃). δ -54.9 ppm. MS (ESI). [M + H]⁺ 284.1. HPLC. retention time: 1.88 min (>99%). IR (neat): ν_max 2920-2850 (s, O–H, C–H), 1690 (m, C⩵O), 1666 (m), 1519 (m), 1496 (w), 1460 (w), 1413 (m), 1336 (w), 1284 (m), 1252 (m), 1206 (w), 1172 (m), 1117 (s), 1046 (w), 1018 (m), 1002 (m) cm⁻¹. HRMS. calculated for C₁₄H₁₀F₃NO₂ [M + H]⁺ = 284.0898, observed = 284.0896.

Ethyl 1-(4,5-Dichloro-2-methylphenyl)-5-methyl-2-(trifluoromethyl)-1H-pyrrole-3-carboxylate (S51)

According to General Procedure D, 4,5-dichloro-2-methylaniline (0.22 g, 1.25 mmol) was stirred for 24 h to give a crude product. The crude product was purified by flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S51 as yellow oil (0.13 g, 0.34 mmol, 27%). R_f. 0.68 (30.70 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃). δ 7.44 (s, 1H), 7.29 (s, 1H), 6.53 (s, 1H), 4.32 (q, J = 7.1 Hz, 2H), 1.93 (s, 3H), 1.89 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 163.2, 136.5, 136.1, 133.9, 132.9 (q, J = 1.5 Hz), 132.3, 130.5, 129.8 (q, J =1.1 Hz), 121.8 (q, J = 38.3 Hz), 120.3 (q, J = 269.6 Hz), 118.9 (q, J = 2.3 Hz), 110.8, 60.9, 16.5, 14.2, 12.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃). δ -55.6 ppm. MS (ESI). [M + H]⁺ 380.2. IR (neat). ν_max 2983-2929 (w, C–H), 1726 (m, C⩵O), 1513 (m), 1477 (m), 1423 (m), 1276 (m), 1234 (s), 1194 (s), 1164 (s), 1121 (s), 1039 (m), 1027 (m), 1001 (m) cm⁻¹.

1-(4,5-Dichloro-2-methylphenyl)-5-methyl-2-(trifluoromethyl)-1H-pyrrole-3-carboxylic Acid (54)

According to General Procedure B, the ester derivative S51 (0.10 g, 0.26 mmol) was stirred for 18 h to give a crude product. The crude product was purified using flash column chromatography (0-30% EtOAc in petrol) and triturated with petrol to give the acid derivative 54 as an amorphous white solid (71 mg, 0.22 mmol, 85%). R_f. 0.58 (EtOAc). ¹H NMR (400 MHz, CDCl₃). δ 7.63 (s, 1H), 7.51 (s, 1H), 6.55 (s, 1H), 1.95 (s, 3H), 1.92 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃). δ 166.2, 138.3, 137.6, 134.7, 133.4, 133.4, 131.2, 131.1, 122.6 (q, J = 38.1 Hz), 121.7 (q, J = 268.7 Hz), 120.3 (q, J = 2.1 Hz), 111.9, 16.3, 12.0 ppm. ¹⁹F NMR (376 MHz, CDCl₃). δ -56.8 ppm. MS (ESI): [M - H]^- 350.0. HPLC. retention time. 2.23 min (>99%). IR (neat). ν_max 2958-2559 (w, O–H, C–H), 1673 (m, C⩵O), 1517 (m), 1475 (m), 1420 (m), 1272 (m), 1205 (m), 1159 (m), 1130 (s), 1035 (m), 1006 (m) cm⁻¹. HRMS. calculated for C₁₄H₁₀Cl₂F₃NO₂ [M + H]⁺ = 352.0119, observed = 352.0114.

Ethyl 1-(3,4-Dichloro-2-methylphenyl)-5-methyl-2-(trifluoro-methyl)-1H-pyrrole-3-carboxylate (S52)

According to General Procedure D, 4,5-dichloro-2-methylaniline (0.22 g, 1.25 mmol) was stirred for 1 day to give a crude product. The crude product was purified by flash column chromatography (0-5% EtOAc in petrol) to give the ester derivative S52 as yellow oil (80 mg, 0.21 mmol, 17%). R_f. 0.67 (30.70 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃). δ 7.43 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H),4.32 (q, J =7.2 Hz, 2H), 2.01 (s,3H), 1.87 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H) ppm. ¹³CNMR(100 MHz, CDCl₃). δ 163.2,137.3, 136.0, 134.6, 134.1, 133.2 (q, J = 1.4 Hz), 128.0, 127.1 (q, J = 0.7 Hz), 121.9 (q, J = 38.2 Hz), 120.3 (q, J = 269.5 Hz), 118.9 (q, J = 2.1 Hz), 110.7, 60.9, 16.0, 14.2, 12.2 ppm. ¹⁹F NMR (376 MHz, CDCl₃). δ -55.6 ppm. MS (ESI). [M + H]⁺ 380.2. IR (neat). ν_max 3121-2851 (w, C–H), 1721 (m, C⩵O), 1512 (w), 1460 (w), 1420 (w), 1274 (m), 1234 (m), 1187 (m), 1124 (s), 1073 (m), 1053 (m), 1030 (m), 999 (m) cm⁻¹.

1-(3,4-Dichloro-2-methylphenyl)-5-methyl-2-(trifluoromethyl)-1H-pyrrole-3-carboxylic Acid (55)

According to General Procedure B, the ester derivative S52(80.0 mg, 0.21 mmol) was stirred for 24 h to give a crude product. The crude product was purified using flash column chromatography (0-30% EtOAc in petrol) and triturated with petrol to give the acid derivative 55 as an amorphous white solid (52.0 mg, 0.15 mmol, 71%). R_f. 0.27 (30.70 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃): δ 7.58 (d, J = 8.5 Hz, 1H), 7.25 (d, J = 8.5 Hz, 1H), 6.56 (s, 1H), 2.02 (s, 3H), 1.90 (s, 3H) ppm. ¹³C NMR (100 MHz, CD₃OD): δ 166.3, 138.5, 137.5, 135.4, 134.9 (q, J = 1.4 Hz), 134.6, 129.3, 128.8 (q, J = 1.0 Hz), 122.7 (q, J = 38.1 Hz), 121.7 (q, J = 268.7 Hz), 120.3 (q, J = 2.1 Hz), 111.9, 16.0, 12.0ppm. ¹⁹FNMR(376MHz, CDCl₃): δ -56.7 ppm. MS (ESI). [M - H]^- 350.0. HPLC. retention time. 2.26 min (>99%). IR (neat). ν_max 3084-2592 (m, O–H, C–H), 1689 (s), 1579 (w), 1519 (m), 1467 (m), 1424 (m), 1391 (w), 1334 (w), 1282 (m), 1257 (s), 1178 (m), 1158 (m), 1124 (s), 1052 (m), 1012 (m), 1000 (m) cm⁻¹. HRMS. calculated for C₁₄H₁₀Cl₂F₃NO₂ [M + H]⁺ = 352.0119, observed = 352.0112.

N-Benzyl-5-methyl-1-phenyl-1 H-pyrazole-4-carboxamide (58)

Thionyl chloride (1.40 mL) was added to the carboxylic acid derivative 4 (0.13 g, 0.63 mmol), and the solution was stirred at 80 °C for 3 h. After concentrating the solution in vacuo, 1,4-dioxane (3.1 mL), benzylamine (0.10 mL, 0.95 mmol), and pyridine (77 μL, 0.95 mmol) were added and the resulting mixture was stirred at room temperature for 18h. Successively, the mixture was concentrated in vacuo and EtOAc (6 mL) was added. The organic phase was washed with a 0.1 M HCl aqueous solution (3 × 10 mL) and brine (5 mL), dried under anhydrous magnesium sulfate, and concentrated in vacuo to yield a crude product, which was purified by flash column chromatography (EtOAc) to give the amide derivative 58⁵⁹ as a pale brown amorphous solid (76 mg, 0.26 mmol, 42%). R_f 0.49 (50.50 EtOAc.petrol). ¹H NMR (400 MHz, CDCl₃). δ 7.78 (s, 1H), 7.53-7.48 (m, 2H), 7.47 7.40 (m, 3H), 7.39-7.35 (m, 4H), 7.33-7.28 (m, 1H), 6.07 (br. s, 1H), 4.63 (d, J = 5.7 Hz, 2H), 2.61 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 163.4, 142.3, 138.8, 138.3, 137.9, 129.1, 128.7, 128.5, 127.8, 127.5, 125.4, 115.3, 43.3, 11.8 ppm.MS (ESI). [M + H]⁺292.2. HPLC: retention time: 1.84 min (>99%). IR (neat): ν_max 3315 (m, NH), 1629 (s, C⩵O), 1593 (m), 1567 (s), 1536 (m), 1503 (s), 1453 (m), 1394 (s), 1353 (w), 1287 (s), 1262 (w), 1138 (w), 938 (s) cm⁻¹.

N-Methanesulfonyl-1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (59)

According to General Procedure G, the carboxylic acid derivative 18 (44 mg, 0.17 mmol) and methanesulfonamide gave the sulfonamide derivative 59 as a white amorphous solid (40 mg, 0.12 mmol, 70%). R_f 0.29 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 9.08 (s, 1H), 8.09 (s, 1H), 7.58-7.48 (m, 3H), 7.45-7.38 (m, 2H), 3.45 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 159.0, 140.1, 138.8, 132.8 (q, J = 40.4 Hz), 130.4, 129.4, 125.8 (q, J = 1.0 Hz), 118.9 (q, J = 271.6 Hz), 117.3 (q, J =1.2 Hz),41.9ppm. ¹⁹FNMR(376 MHz, CDCl₃): δ -56.4 ppm. MS (ESI): [M + H]⁺ 334.1. HPLC: retention time: 1.80 min (>99%). IR (neat): ν_max 3276 (m, N-H), 3137-2940 (w, C–H), 1703 (m, C⩵O), 1559 (w), 1502 (w), 1433 (m), 1405 (m), 1326 (m), 1294 (m), 1227 (m), 1158 (s), 1132 (s), 1082 (m), 1073 (m), 1022 (m), 973 (m) cm⁻¹. HRMS: calculated for C₁₂H₁₀F₃N₃O₃S [M + H]⁺ = 334.0473, observed = 334.0475.

N-(Benzenesulfonyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (60)

According to General Procedure G, the carboxylic acid derivative 18 (44 mg, 0.17 mmol) and benzenesulfo-namide gave the sulfonamide derivative 60 as a white amorphous solid (38 mg, 95.2 μMol, 56%). R 0.40 (20:80 MeOH:DCM). ¹H NMR (400 MHz, CDCl₃): δ 8.79 (s, 1H), 8.16 (d, J = 7.6 Hz, 2H), 8.00 (s, 1H), 7.69 (t, J = 7.4 Hz, 1H), 7.59 (t, J = 7.6 Hz, 2H), 7.55–7.46 (m, 3H), 7.41-7.35 (m, 2 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 157.7, 140.1, 138.1, 134.5, 133.0, 130.4, 129.4, 129.2, 128.7, 126.5, 125.8 (q, J = 0.8 Hz), 118.9 (q, J = 271.7 Hz), 117.7 (q, J = 1.0 Hz) ppm. ¹⁹FNMR (376 MHz, CDCl₃): δ -56.2 ppm. MS (ESI): [M + H]⁺ 396.2. HPLC: retention time: 1.96 min (>99%). IR (neat): ν_max 3247 (m, N-H), 2921-2850 (w, C–H), 1717 (m, C⩵O), 1563 (w), 1501 (w), 1448 (w), 1425 (m), 1408 (m), 1335 (m), 1294 (m), 1219 (m), 1169 (s), 1147 (s), 1135 (s), 1084 (s), 1020 (m) cm⁻¹. HRMS: calculated for C₁₇H₁₂F₃N₃O₃S [M + H]⁺ = 396.0630, observed = 396.0636.

1-Phenyl-N-phenylmethanesulfonyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (61)

According to General Procedure G, the carboxylic acid derivative 4 (0.11 g, 0.55 mmol) and benzylsulfonamide gave a crude product. The crude product was purified by flash column chromatography (30% EtOAc in petrol) to give the ester derivative 61 as a pale yellow amorphous solid (11.7 mg, 33.0 μMol, 6%). R_f 0.31 (50:50 EtOAc:petrol). ¹H NMR (400 MHz, CDCl₃): δ 8.00 (s, 1H), 7.65-7.32 (m, 10H), 4.79 (s, 2H), 2.58 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 164.6, 146.4, 141.0, 139.6, 132.0, 130.6, 130.5, 130.4, 129.8, 129.7, 129.5, 126.9, 59.6, 12.1 ppm. MS (ESI): [M + H]⁺ 356.2. HPLC: retention time: 1.83 min (>99%). IR (neat): ν_max 3330 (w, NH), 1682 (s, C⩵O), 1598 (w), 1549 (m), 1503 (m), 1455 (m), 1403 (m), 1337 (s), 1231 (m), 1154 (s), 1135 (m), 1056 (m) cm⁻¹. HRMS: calculated for C₁₈H₁₇N₃O₃S [M + H]⁺ = 356.1068, observed = 356.1064.

N-(Benzenesulfonyl)-1-(2,6-dimethylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (62)

According to General Procedure G, the carboxylic acid derivative 41 (0.11 g, 0.39 mmol) and benzenesulfonamide gave a crude product. The crude product was purified by flash column chromatography (30-100% EtOAc in petrol) to give the sulfonamide derivative 62 as a white amorphous solid (38 mg, 89.7 μMol, 23%). R_f: 0.34 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 8.14 (s, 1H), 8.05 (d, J =7.6 Hz, 2H), 7.66-7.50 (m, 3H), 7.33 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.6 Hz, 2H), 1.93 (s, 6H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 165.6, 143.4, 142.4, 139.0, 137.2, 133.3, 133.2 (q, J = 39.8 Hz), 131.3, 129.5, 129.3, 128.6, 123.0, 120.6 (q, J = 270.5 Hz), 17.0 ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ -60.6 ppm. MS (ESI): [M + H] + 424.2. HPLC: retention time: 2.10 min (96%). IR (neat): ν_max 3443 (w, N-H), 1701 (m, C⩵O), 1605 (m), 1560 (m), 1485 (m), 1448 (m), 1375 (m), 1304 (s), 1136 (s), 1087 (m), 1048 (m), 969 (m) cm⁻¹. HRMS: calculated for C₁₉H₁₆F₃N₃O₃S [M + H]⁺ = 424.0942, observed = 424.0947.

N-(3-Bromobenzenesulfonyl)-1-(2,6-dimethylphenyl)-5-(trifluor-omethyl)-1H-pyrazole-4-carboxamide (63)

According to General Procedure G, the carboxylic acid derivative 41 (0.15 g, 0.54 mmol) and 3-bromobenzene-1-sulfonamide gave a crude product. The crude product was purified by flash column chromatography (30% petrol in EtOAc) to give the sulfonamide derivative 63 as a yellow amorphous solid (21.7 mg, 43.2ˆmol, 8%). R_f: 0.12 (EtOAc). ¹H NMR(400 MHz, acetone-d₆): δ 8.22 (s, 1H), 8.18 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 7.6 Hz, 2H), 1.91 (s, 6H) ppm. ¹³C NMR (125 MHz, acetone-d₆): δ 147.4, 143.6, 139.8, 136.9, 135.8, 134.9 (q, J =39.2 Hz), 131.7,131.0,130.9,129.3,129.0,128.9,127.1,122.8,120.8 (q, J = 270.2 Hz), 17.3 ppm. ¹⁹F NMR (376 MHz, acetone-R): δ -59.0 ppm. MS (ESI): [M - H]^- 502.1. HPLC: retention time: 2.30 min (>99%). IR (neat): ν_max 3457 (w, N-H), 1706 (s, C⩵O), 1559 (m), 1364 (s), 1294 (s), 1255 (m), 1222 (s), 1175 (s), 1139 (vs), 1098 (m), 968 (m) cm⁻¹. HRMS: calculated for C₁₉H₁₅BRF₃N₃O₃S [M + H]⁺ = 500.9969, observed = 500.9985.

N-(Benzenesulfonyl)-1-(4,5-dichloro-2-methylphenyl)-5-(trifluor-omethyl)-1H-pyrazole-4-carboxamide (64)

According to General Procedure G, the carboxylic acid derivative 44 (40 mg, 0.12 mmol) and benzenesulfonamide gave a crude product. The crude product was purified by flash column chromatography (0-50% EtOAc in petrol) to give the sulfonamide derivative 64 as a white amorphous solid (18 mg, 37.6 μMol, 31%). R_f: 0.40 (EtOAc). ¹H NMR (400 MHz, CDCl₃): δ 8.11 (d, J = 7.8 Hz, 2H), 8.07 (s, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.52 (app. t, J = 7.8 Hz, 2H), 7.42 (s, 1H), 7.29 (s, 1H), 1.93 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 159.5, 141.3, 139.0, 137.8, 136.9, 135.8, 135.0, 134.0 (q, J = 3.1 Hz), 133.0 (q, J = 39.8 Hz), 132.4, 130.4, 129.1, 129.0, 128.2, 118.8 (q, J = 271.7 Hz), 16.5 ppm. ¹⁹FNMR(376 MHz, CDCl₃): δ -57.1 ppm. MS (ESI): [M - H]^- 476.1. HPLC: retention time: 2.12 min (>99%). IR (neat): ν_max 3267 (m, N-H), 2925 (w, C–H), 1704 (m, C⩵O), 1559 (w), 1492 (w), 1432 (m), 1411 (m), 1335 (w), 1293 (m), 1236 (m), 1165 (s), 1153 (s), 1134 (s), 1080 (m), 1022 (m), 987 (m) cm⁻¹. HRMS: calculated for C₁₈H₁₂Cl₂F₃N₃O₃S [M + H]⁺ = 478.0007, observed = 478.0001.

Surface Plasmon Resonance

Experiments were performed using as a running buffer that consisted of 10 mM NaPO₄ (pH 7) 150 mM NaCl, and 2% DMSO at 25 °C in a Biacore T200 (GE Healthcare). For data analyses, bulk effects were corrected using solvent correction and were performed through the Biacore T200 evaluation software 2.0 (GE Healthcare). Pa MurB was covalently coupled to a CM5 chip (GE Healthcare) by standard amine coupling protocol.

For the single concentration experiment, all fragments were diluted to 1 mM in running buffer injected for 30 s at 30 μL s⁻¹ and the dissociation was for 320 s. All fragments were tested two times in reverse orders. Sensograms were visually inspected, and fragments with significant signal increase comparing with the original fragment were selected for affinity study by ITC.

Isothermal Titration Calorimetry

Isothermal titration calorimetry experiments to quantify ligand binding to Pa MurB were performed using a Malvern MicroCal Auto-iTC200 system at 25 °C. Titrations consisted of an initial injection of 0.4 μL, which was discarded during data processing, followed by 28 further injections of 1.5 μL separated by a 120 s interval. The Pa MurB protein was dialyzed overnight at 4 °C in 25 mM Tris-HCl (pH 8.0) and 150 mM NaCl. Sample cell and syringe solutions were prepared using the same buffer, with a final DMSO-R concentration of 5%. Pa MurB concentrations of 200-50 pM were used, with ligand concentrations of3.0-0.5 mM. The protein well had a volume of 400 μL, the ligand well a volume of 200 μL, and the blank well a volume of 400 μL. Titrations were fitted with Origin software (OriginLab, Northampton, MA, USA) using a one site binding model. All ITC titration curves are shown in the SI.

Dihedral Angle Calculations

The global ground state conformations and dihedral angle calculations were performed using Schrodinger Maestro 11.⁶⁰ The scanning of the dihedral angles was performed using a MacroModel coordinate scan (force field: OPLS-2005, solvent: water, default settings).

Scheme 1. General Synthetic Routes for the Synthesis of the Tested Fragments.

■. Abbreviations

CF

cystic fibrosis

DSF

differential scanning fluorimetry

EDC

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Ec

Escherichia coli

FAD

flavin adenine dinucleotide

ITC

isothermal titration calorimetry

LE

ligand efficiency

MIC

minimum inhibitory concentration

MurB

UDP-N-acetylenolpyruvoyl-glucosamine reductase

Mw

molecular weight

NADP⁺

nicotinamide adenine dinucleotide phosphate

Pa

Pseudomonas aeruginosa

PBPs

penicillin-binding proteins

RU

response units

Sa

Staphylococcus aureus

SAR

structure–activity relationship

SPR

surf;ace plasmon resonance

UDP

uridine diphosphate

UNAGEP

UDP-N-acetylglucosamine enolpyruvate

UNAM

N-acetyl-muramic acid