Design, Synthesis, and Pharmacological Evaluation of Second-Generation Soluble Adenylyl Cyclase (sAC, ADCY10) Inhibitors with Slow Dissociation Rates

Abstract

Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.

Graphical Abstract

INTRODUCTION

Cyclic AMP (cAMP) is an important second messenger that plays a key role in numerous signal transduction pathways. cAMP-dependent signaling cascades regulate various cellular processes such as proliferation, apoptosis, differentiation, migration, development, ion transport, pH regulation, and gene expression.^1,2 In mammals, there are two distinct types of enzyme that produce cAMP from ATP: the G-protein-regulated transmembrane adenylyl cyclases (tmACs; ADCY1–9) and a bicarbonate-regulated soluble adenylyl cyclase (sAC, ADCY10).³ Unlike tmACs, which are localized to the plasma membrane, sAC is found within the cytosol and in several compartmentalized microdomains located within cellular organelles.^4–7 sAC-generated cAMP signaling has been linked to multiple physiological functions⁸ such as insulin release from pancreatic β-cells,⁹ regulation of intraocular eye pressure,^10,11 lysosomal acidification,⁴ and, the capacitation of sperm.^12,13 Due to its varied biological roles, sAC has been identified as a potential novel drug target in a number of therapeutic areas.²

In particular, recent work has increased interest in the use of sAC as a drug target for contraception.^14,15 Safe, effective new contraceptive strategies for males, particularly nonhormonal methods, represent a profound unmet medical need as globally, approximately half of all pregnancies are unplanned.^16,17 In males, sperm are stored in the cauda epididymis in their inactivated form (“noncapacitated”) under low bicarbonate conditions (<5 mM).^18–20 Upon ejaculation, sperm are mixed with seminal fluid containing high bicarbonate levels (20–30 mM). Exposure to bicarbonate, the endogenous ligand for sAC,²¹ leads to an increase in intracellular cAMP, which stimulates sperm motility and converts sperm into their activated (“capacitated”) form able to undergo the acrosome reaction required for oocyte fertilization.¹² Consistent with these observations, two strains of mice lacking sAC are sterile due to a male-specific sperm-motility defect.^13,22 More recently, two adult human males presented as infertile with immotile sperm, a defect which was traced to an ADCY10 frameshift mutation.²³ Treatment of immotile sperm from knockout mice or the two infertile adcy10 mutant men with a cell-permeable cAMP analogue restored sperm motility, which represents key confirmatory mechanistic data that their infertility is due to the absence of sAC.²³ Importantly, despite sAC expression in other tissues,²¹ mechanism-based liabilities appear modest as both the KO mice and the humans bearing the ADCY10 frameshift mutation exhibit overall benign profiles.^13,22,23 These aggregate datasets support the hypothesis that sAC represents a highly attractive target for the development of an on-demand, nonhormonal male contraceptive.

To interrogate sAC inhibition for therapeutic utility, high-quality pharmacologic tools are required. We previously described the discovery of TDI-10229, a potent and orally bioavailable inhibitor of sAC.²⁴ Drug design efforts were aided by abundant structural information from X-ray crystallography on sAC alone and in complex with inhibitors.^25–27 While sAC is similar to transmembrane adenylyl cyclases (tmACs 1–9) in overall structure, its regulatory bicarbonate binding site has unique features, which are exploited by TDI-10229.^24,27 Briefly, TDI-10229 has an IC₅₀ of 160 nM on purified human sAC protein in an in vitro sAC biochemical assay²⁵ and a cellular IC₅₀ of 92 nM for sAC in sAC-overexpressing rat 4–4 cells;²⁵ is highly selective versus the closely related and widely expressed, tmACs 1–9; and exhibited reasonable druglike properties.²⁴ However, more recent studies suggest that TDI-10229 does not provide adequate exposure for use as a male systemic contraceptive.¹⁵ This could be due to its modest affinity (160 nM in a biochemical assay) and/or the strong dilution effect that occurs when inhibitor-containing sperm enter the inhibitor-free female body. As a potential solution, we identified fast onset and slow dissociation rates as key design goals for the development of second-generation sAC inhibitors for use as male systemic contraceptives. More generally, slow dissociation rates are often a desirable property for therapeutic use.²⁸ We now detail our optimization efforts using TDI-10229 as a starting point, leading to the identification of high-affinity sAC inhibitors with slow dissociation rates.

RESULTS AND DISCUSSION

Design and Optimization.

The crystal structure of TDI-10229 in complex with sAC provided opportunities for structure-based drug design to fulfill our goals related to the optimization of sAC binding affinity and kinetics to improve downstream activity (Figure 1).

Figure 1.

Crystal structure of sAC complex with TDI-10229. The crystal structure of sAC in complex with TDI-10229 (2.20 Å) from PDB ID 7OVD. The protein is displayed as a green cartoon with green wires and a transparent gray surface. TDI-10229 is displayed as atom-colored balls, and sticks with magenta carbons.

TDI-10229 is substituted with methyl groups at both the 1 and 5 positions of the pyrazole and we noted the presence of a promising growth vector at the 5 position. Table 1 presents the resultant structure–activity relationships (SAR) data for substitution at the 1 and 5 positions of the pyrazole.

Table 1.

Biochemical Potency of TDI-10229 and Related Analogues

Cpd	Structure	Biochemical IC50 (nM)a
1 (TDI-10229)		159 ± 7
2		70% inhibition at 500 μM
3		1024 ± 235
4		829 ± 166
5		469 ± 87
6		112 ± 21
7		716 ± 245

^aCompounds were tested in a minimum of three independent assays, and data are reported as the mean ± standard error of the mean. The 1 and 5 position numbering on the pyrazole ring is shown for TDI-10229 (1).

Removal of the methyl group at the 5 position leads to a small 6-fold loss of affinity (3) whilst removal of the methyl groups at both the 1 and 5 positions leads to a dramatic loss of affinity (2). Interestingly, some affinity can be regained by the replacement of the 1-methyl group with a difluoromethyl group (4) or trifluoromethyl group (5). Cyclization between the 1 and 5 positions (6) can improve binding affinity, highlighting the opportunities available for growth at these positions, though an alternate cyclization (7) reduces binding affinity. Previously reported SAR highlighted some limited ability to improve binding affinity through substitution at the 5 position of TDI-10229.²⁴ However, further study of the crystal structure (Figure 1) and exploitation of structure-based design tools such as free-energy perturbation^29,30 suggested that the space filled by the methyl group at the 5 position of TDI-10229 could also be filled by substitution of the phenyl ring at the ortho or meta position. For this reason, we explored substitution around the phenyl ring of 3. The SAR data are presented in Table 2.

Table 2.

Biochemical Potency of Ortho-, Meta-, and Para-Substituted Phenyl Analogues

Cpd	R1 (SAR)	R2 (SAR)	R3 (SAR)	Biochemical IC50 (nM)a
3	H	H	H	1024 ± 235
8	Cl	H	H	64 ± 9
9	H	Cl	H	389 ± 80
10	H	H	Cl	2577 ± 300
11	OMe	H	H	67 ± 8
12	CO2Me	H	H	21 ± 3
13	C(O)NHMe	H	H	1245 ± 123
14	C(O)N(Me)2	H	H	75% inhibition at 500 μM
15	H	OMe	H	351 ± 43
16	H	CO2Me	H	436 ± 39
17	H	C(O)NHMe	H	1876 ± 208

^aCompounds were tested in a minimum of three independent assays, and data are reported as mean ± SEM.

Results for the chlorinated analogues indicate that substitution at the ortho (8) or meta (9) position leads to increases in affinity whilst substitution at the para position is not favored (10). This is in good agreement with structural data and modeling predictions, which show that the growth vector at the para position is directed toward the protein surface. Also consistent with structural data and modeling predictions, the ortho position provides the best vector for growth, with methoxy (11) and methyl ester (12) groups yielding improved IC₅₀ values of 67 and 21 nM, respectively. However, not all substitutions are favored, with methyl amide (13) having an IC₅₀ of 1245 nM and dimethyl amide (14) being almost inactive. The meta position is also amenable to substitution, with methoxy (15) and methyl ester (16) groups yielding improved IC₅₀ values but methyl amide (17) having an IC₅₀ greater than 1 μM. Considering both the SAR and the structural information, we selected alkoxy substitution at the ortho position to explore further. The SAR data are presented in Table 3.

Table 3.

Biochemical Potency of Ortho Alkoxy-Substituted Phenyl Analogues

Cpd	R1 (SAR)	Biochemical IC50 (nM)a
18		87.1 ± 11.5
19		12.3 ± 0.8
20		6.7 ± 0.5
21		7.8 ± 1.2
22		16.9 ± 3.1

^aCompounds were tested in a minimum of three independent assays, and data are reported as mean ± SEM.

Simple substitution with a phenyl group (18) does not improve biochemical IC₅₀, but modeling indicated that an alkyl chain with a hydrophilic or weakly basic group should be favorable. Correspondingly, the morpholine compound (19) showed an improved biochemical IC₅₀ of 12.2 nM. Analogues such as the bridgehead (20), 2-oxa-6-azaspiro[3.3]heptane (21), and piperazin-2-one (22) are also highly potent. We focused on compound 19 as a high-affinity inhibitor. Noting the potential metabolic liability^31,32 of the N-methyl pyrazole group, we incorporated SAR from compound 4 (Table 1) and replaced the methyl with a difluoromethyl group.³³ The resulting compound (23) showed a biochemical IC₅₀ of 4.0 nM. As an initial check on the improved potency of 23, we examined its efficacy in an sAC cellular assay. The cellular IC₅₀ of 23 was 2.0 nM, significantly improved from TDI-10229’s cellular IC₅₀ of 92 nM.

Given the sAC concentration of 5.0 nM in the biochemical assay, measurement of IC₅₀ values below 2.5 nM cannot be effectively determined, assuming all of the protein is enzymatically competent. To quantify the increased potency of compounds such as 23, we turned to biophysical measurement of compound binding using the well-established technique of surface plasmon resonance (SPR).³⁴ This approach offered the added advantage of allowing us to investigate the kinetics of binding to assess progress made on our goal of identifying compounds with extended residence times. SPR studies have additional value because, as noted above, the biochemical assay may have reached a floor where inhibition with an IC₅₀ better than 2.5 nM cannot be measured effectively. Table 4 shows the biochemical IC₅₀, cellular IC₅₀, and SPR data for 23 along with additional morpholine-modified analogues in the context of a difluoromethyl group at the pyrazole 1 position.

Table 4.

Biochemical Potency and Cell-Based Activity of Ortho Alkoxy-Substituted Phenyl Analogues

Cpd	R1 (SAR)	Biochemical IC50(nM)a	Cell IC50(nM)a	SPR KD(nM)a	SPR Residence Time(s)a
23		3.9 ± 0.3	1.9 ± 0.9	3.3 ± 1.8	1832 ± 458
24		24.2 ± 3.9	37.7 ± 9.3	4.7 ± 1.5	484 ± 54
25		2.1 ± 0.2	5.5 ± 1.7	5.5 ± 1.4	526 ± 108
26		2.4 ± 0.3	14.6 ± 1.3	2.1 ± 1.1	2103 ± 319
27		4.0 ± 0.5	16.7 ± 6.3	3.6 ± 1.9	2716 ± 893
28		2.2 ± 0.1	19.6 ± 2.6	2.5 ± 0.4	1712 ± 267
29		1.0 ± 0.1	2.5 ± 0.2	1.7 ± 0.5	3016 ± 799
30		3.3 ± 0.2	34.8 ± 8.5	1.1 ± 0.1	2717 ± 338
31		5.5 ± 0.8	22.9 ± 4.5	3.7 ± 1.2	1301 ± 239
32		4.6 ± 0.9	10.8 ± 2.1	2.3 ± 0.5	2579 ± 708
33 (TDI-11861)		3.3 ± 0.3	5.5 ± 0.9	1.4 ± 0.3	3181 ± 636

^aCompounds were tested in a minimum of three independent assays, and data are reported as mean ± SEM.

All 11 compounds in Table 4 displayed enzymatic IC₅₀, cellular IC₅₀, and SPR-derived K_D values in the nanomolar range. The variety of substituents accommodated at the ortho phenyl position offered an ability to modulate compound properties. In particular, the activity of the amide (24) indicates that the basicity of the morpholine group is not required for high-affinity binding. Table 5 presents comparative SPR traces and associated data for TDI-11861 versus TDI-10229.

Table 5.

SPR Data for TDI-10229 and TDI-11861: The SPR Data and Example Traces for TDI-10229 and TDI-11861

Cpd	1 (TDI-10229)	33 (TDI-11861)
Structure
SPR KDa	175.6 ± 23.7 nM	1.4 ± 0.3 nM
SPR kona	2.4 ± 0.4 × 105 M−1s−1	2.7 ± 0.2 × 105 M−1s−1
SPR koffa	39.8 ± 2.2 × 10−3 s−1	0.4 ± 0.1 × 10−3 s−1
SPR Residence Timea	25 ± 1 s	3181 ± 636 s
Example SPR Trace

^aCompounds were tested in a minimum of three independent assays, and data are reported as mean ± SEM.

The SPR data reveal a fast off-rate profile for TDI-10229, with a residence time of only 25 s. This supports the idea that, in the context of a male contraceptive indication, the risk of post-copulation female genital tract dilution represents a valid concern. SPR also confirms the significantly improved binding affinity of TDI-11861 relative to TDI-10229 (1.4 nM compared with 176.0 nM). The concomitant improvement in dissociation rates is approximately 100-fold (3181 s compared with 25 s). Notably, both compounds exhibit similarly fast on rates, demonstrating that the improvement in potency for TDI-11861 is due almost entirely to the decrease in its dissociation rate.

As an independent check on the slow dissociation of sAC inhibitors such as TDI-11861, we performed jump dilution recovery assays.³⁵ Briefly, in these assays, sAC protein at 5× its standard assay concentration is preincubated with an inhibitor at 10× its IC₅₀ concentration. Following a 15 min preincubation to ensure equilibration of sAC plus inhibitor, the inhibitor-protein solution is diluted 100-fold into the standard activity assay system. Cyclase activity is initiated with the addition of substrate ATP, and the rate of conversion of substrate ATP into product cAMP is measured at various time points over the next hour. Compounds with short residence times will show minimal inhibition of sAC activity after the 100× dilution step, and their rate of enzymatic conversion of ATP into cAMP will be indistinguishable from assays performed in the absence of any inhibitor. In contrast, compounds with long residence times will show a diminished enzymatic rate at early times, as inhibitor remains engaged even after dilution. The time it takes to regain the uninhibited rate of sAC activity from the initially inhibited state defines the residence time for the inhibitor tested. These jump dilution assay results illustrate that advanced analogues, such as TDI-11861, exhibit slower dissociation rates from sAC relative to TDI-10229 (Figure 2). Importantly, these experiments recapitulate the vaginal dilution effect discussed above.

Figure 2.

Jump dilution studies of sAC inhibitors. (a) Conceptual schematic illustrating jump dilution activity recovery assay protocol. (b) Comparison of residence time from SPR and jump dilution. The residence time of TDI-10229 is too short to accurately measure via jump dilution but is included for completeness. (c) Jump dilution plots of cAMP accumulation versus time.

The residence times from SPR and jump dilution are in reasonable agreement. A consistent 2- to 3-fold difference is observed and may be due to slight differences in experimental conditions (i.e., inclusion of substrate and divalent ion cofactor in jump dilution assay). Based on its biochemical potency (IC₅₀ = 3.3 nM), cellular potency (IC₅₀ = 5.5 nM), and SPR affinity (K_D = 1.4 nM), coupled to its comparatively long residence time (3181 and 1220 s in SPR and jump dilution, respectively), TDI-11861 is among the most potent sAC inhibitors synthesized to date. To confirm the binding mode of TDI-11861, we obtained crystallographic data in complex with sAC at 1.8 Å resolution (Figure 3; Table S1).

Figure 3.

Crystal structure of sAC complex with TDI-11861. The protein is displayed as a green cartoon with green wires. TDI-11861 is displayed as atom-colored balls, and sticks with cyan carbons. Hydrogen-bonding interactions are shown as dashed yellow lines.

The protein and ligand structures are very well aligned with the crystal structure of TDI-10229²⁴ with the key hydrogen-bonding contacts made by the aminochloropyrimidine head-group maintained from TDI-10229 and its parent LRE1.^24,25 An additional hydrogen-bonding contact is seen between the ligand hydroxyl group and the sidechain carboxylate group of Asp99 (distance 2.92 Å), a key residue for binding of the substrate ATP and the divalent metal ion. The morpholine group is close to Asp99, but not in direct contact with it, in line with basicity not being required for strong binding.

We measured the pharmacokinetic (PK) properties of TDI-11861 in mice to assess its utility as a tool compound for in vivo pharmacologic studies (see Table 6 and further details in the SI). Even though the oral bioavailability of TDI-11861 in CD-1 mice was low (F% = 11 at 10 mg/kg), a higher dose of 50 mg/kg achieved unbound plasma exposures ~10-fold over its cellular IC₅₀ (5.5 nM) out to 4 h and this would support potential in vivo examination of sAC-mediated pharmacology (Figure 4). Indeed, higher systemic levels of TDI-11861 were attained in male mice (7.04 and 0.93 μM at 1- and 4-h post-dosing, respectively, 50 mg/kg, IP), to enable successful male contraceptive studies.¹⁷

Table 6.

Pharmacokinetic Parameters of TDI-11861 (33) in Male CD-1 Mice

parameter	IV administrationa (2 mg/kg)	oral administrationb (10 mg/kg)	oral administrationc (50 mg/kg)
AUC(0–24h) [mM-h]	0.78 ± 0.11	0.41 ± 0.057	5.46 ± 1.5
Cmax [mM]	2.5 ± 0.21d	0.45 ± 0.015	6.1 ± 1.9
Tmax [h]		0.25	0.25
T1/2 [h]	0.93 ± 0.33
Cl [mL/min/kg]	94 ± 12
F [%]		11 ± 1.4

^aDosed as a solution of the free base (0.4 mg/mL) in 20% HPBCD.

^bDosed as a solution of the free base (2 mg/mL) in DMSO/PEG400/PBS (10:40:50).

^cDosed as a solution of the free base (10 mg/mL) in DMSO/PEG400/PBS (10:40:50).

^dInitial concentration. F, bioavailability.

Figure 4.

Exposure of TDI-11861. Unbound plasma concentration of TDI-11861 (33) in CD-1 mice at 50 mg/kg PO.

Further characterization provided additional measures of TDI-11861’s suitability for interrogating sAC pharmacology in vitro and in vivo (Table 6). TDI-11861 exhibits good permeability in a parallel artificial membrane permeability assay (PAMPA), and good solubility (463 nm/s and 79 μg/mL, respectively), consistent with its Log D value (2.89). Importantly, consistent with its predecessor TDI-10229, TDI-11861 retained high selectivity for sAC vs closely related tmAC family members (Figure S1). TDI-11861 did not show appreciable activity against a panel of 322 kinases³⁶ (Table S2) and 46 other relevant drug targets such as GPCRs, ion channels, and nuclear receptors in a Eurofins safety panel (Table S3). Furthermore, TDI-11861 showed no cytotoxicity at 20 μM (200-fold above its IC₅₀ for sAC in cells) and showed no evidence of adduct formation for 2 h in a glutathione trapping study (Table 7).³⁷

Table 7.

Ancillary Profile of TDI-11861 (33)

	TDI-11861 (33)
PAMPA (nm/s)	463
HPLC Log D (pH = 7.4)	2.89
kinetic solubility (μg/mL)	79
cytotoxicity	>20 μM
tmACs selectivity	no activity at 10 μM against ADCY1, ADCY2, ADCY5, ADCY8, ADCY9
mouse plasma protein binding (fu)	0.13
glutathione reactivity	low propensity at 5 μM

Chemistry.

Scheme 1 illustrates the synthetic route employed for 2 which exemplifies the general protocol utilized for the formation of analogues containing the key 6-chloropyrimidine-2-amino moiety. The PMB-protected iodopyrazole 2a was converted into alcohol 2b by transmetalation (i-PrMgCl-LiCL) followed by condensation of the formed organomagnesium with benzaldehyde. The alcohol in 2b was subsequently deoxygenated (TMSCl/NaI) to furnish 2c. The ester 2c was homologated into the keto-ester 2d (ethyl acetate, NaH, THF), and subsequent condensation with guanidine carbonate provided 2e. Treatment of 2e with POCl₃ provided the protected PMB pyrazole 2f. Final PMB deprotection of 2f provided 2.

Scheme 1. Synthesis of 2^a.

^aReagents and conditions: (a) i-PrMgCl-LiCl, benzaldehyde; (b) TMSCl, NaI; (c) ethyl acetate, NaH, THF; (d) guanidine carbonate, EtOH; (e) POCl₃, dioxane; (f) CAN, acetonitrile/water.

Scheme 2 illustrates the preparation of the difluoromethylpyrazole analogue 4. The PMB group in 2c is removed via treatment of TFA to afford 4a. The NH group in 4a is converted into the difluoromethyl-substituted intermediate 4b using CF₂BrC(O)OEt/NaH. The ester 4b can be transformed into 4 in a similar manner to that described for 2.

Scheme 2. Synthesis of 4^a.

^aReagents and conditions: (a) TFA; (b) CF₂BrC(O)OEt, NaH, DMF; (c) ethyl acetate, NaH, THF; (d) guanidine carbonate, EtOH; (e) POCl₃, dioxane.

The synthesis of 19 is depicted in Scheme 3. The key alcohol 19b is prepared from the transmetalation of 19a (i-PrMgCl-LiCl) followed by condensation with 2-(2-morpholinoethoxy)benzaldehyde. Alcohol 19b can be converted into 19 in a similar manner to that described previously.

Scheme 3. Synthesis of 19^a.

^aReagents and conditions: (a) i-PrMgCl-LiCl, 2-(2-morpholinoethoxy)benzaldehyde; (b) TMSCl, NaI; (c) ethyl acetate, LHMDS, THF; (d) guanidine carbonate, EtOH; (e) POCl₃, dioxane.

The preparation of 20 is depicted in Scheme 4. Iodopyrazole 19a was converted into the benzyl intermediate 20b using conditions previously outlined ((a) metalation; (b) aldehyde condensation; (c) TMSCl/NaI deoxygenation). Ester 20b was transformed into the 6-chloropyrimidine-2-amino intermediate 20e using conditions outlined ((a) keto-ester formation; (b) condensation with guanidine carbonate; (c) POCl₃). The alkene in 20e was converted into the aldehyde 20g using a two-step protocol ((a) dihydroxylation; (b) periodate cleavage). Reductive amination of aldehyde 20g with the appropriate amine provided 20.

Scheme 4. Synthesis of 20^a.

^aReagents and conditions: (a) i-PrMgCl-LiCl, 2-(allyloxy)benzaldehyde; (b) TMSCl, NaI; (c) ethyl acetate, LHMDS, THF; (d) guanidine carbonate, EtOH; (e) POCl₃, dioxane; (f) NMO, K₂OsO₄; (g) NaIO₄; (h) NaBH₃CN, MeOH, THF, 6-oxa-3-azabicyclo[3.1.1]heptane hydrochloride.

The preparation of 33 is outlined in Scheme 5. The key NH-allyloxy-substituted pyrazole 33d was prepared from 33a following those previously depicted utilizing THP to transiently protect the pyrazole NH for the metalation step. The NH pyrazole 33d was converted into the difluoromethylpyrazole intermediate 33e using diethyl-(bromodifluoromethyl)phosphonate/KF. The ester 33e was converted into the 6-chloropyrimidine-2-amino intermediate 33h using steps like those already depicted. Aldehyde 33j was prepared from alkene 33h in two steps ((a) dihydroxylation; (b) periodate cleavage). Reductive amination of aldehyde 33j with (R)-morpholine-3-ylmethanol yielded 33 (TDI-11861).

Scheme 5. Synthesis of TDI-11861 (33)^a.

^aReagents and conditions: (a) DHP, PTSA, THF; (b) i-PrMgCl-LiCl, 2-(allyloxy)benzaldehyde; (c) TMSCl, NaI; (d) diethyl-(bromodifluoromethyl)phosphonate, KF, MeCN; (e) ethyl acetate, LHMDS, THF; (f) guanidine carbonate, EtOH; (g) POCl₃, dioxane; (h) NMO, OsO₄; (i) NaIO₄; (j) NaBH(OAc)₃, DCE, TEA, (R)-morpholine-3-ylmethanol hydrochloride.

CONCLUSIONS

sAC inhibition represents an interesting drug target for a number of indications.³⁸ We have previously reported a potent inhibitor of sAC (TDI-10229) with a cellular IC₅₀ of 92 nM and reasonable druglike properties. In this study, we have detailed the design process and experimental protocols used to engineer and evaluate improved sAC affinity through structure-based drug design, leading to the identification of TDI-11861. This includes use of both SPR and jump dilution activity recovery assays to measure inhibitors with extended residence times. Ample precedent exists showing the pharmacologic benefits of inhibitors with long residence times.^39–43 The use of systemically dosed sAC inhibitors as male contraceptives may represent a limiting example where, for complete fertility inhibition, long residence times are an essential compound property. This is because, upon mating, an sAC inhibitor present in sperm and semen from injected males will be diluted when the ejaculate is deposited into the female’s inhibitor-free female genital tract. These developments are of interest because novel nonhormonal contraceptive strategies for males represent a key unmet medical need.^16,17

In this study, we improved residence time in a consistent manner by optimizing the intrinsic binding affinity through increased protein-ligand contacts. This was achieved through structure-based design tools leveraging new and existing crystal structures. The application of ensemble molecular docking with Glide⁴⁴ and relative binding free-energy calculations with FEP+²⁹ will be detailed in a future study. Relative to our starting point TDI-10229, TDI-11861 shows improved sAC binding affinity (3.3 nM compared to 160.0 nM), improved sAC cellular affinity (5.5 nM relative to 92 nM), long residence time (3181 s versus to 25 s), and a higher solubility (79 μg/mL compared to 1.3 μg/mL). Overall, TDI-11861 represents a next-generation sAC inhibitor with excellent potency, extended residence time, and improved druglike properties. While enhanced bioavailability or plasma half-life may need to be improved for some applications, it is a promising lead compound for further investigation. TDI-11861 enables enhanced interrogation of sAC biology and the utility of sAC inhibition in medicine.

EXPERIMENTAL SECTION

In Vitro Cyclase Activity Assay.

Assays for sAC activity using 5 nM purified protein⁴⁵ were performed at 30 °C in 100 μL reactions containing 4 mM MgCl₂, 2 mM CaCl₂, 1 mM ATP, 40 mM NaHCO₃, 50 mM Tris pH 7.5, and 3 mM DTT. Purified sAC protein was preincubated with the indicated inhibitors for 15 min at room temperature (23 °C). Each reaction was initiated with ~1,000,000 counts of α−³²P labeled ATP. Generated cAMP was purified using sequential Dowex and Alumina chromatography as previously described.⁴⁶

Jump Dilution.

Jump dilutions assays were performed using a modified version of the in vitro cyclase activity assay. All assays were done in the presence of 0.03% BSA and each had a final DMSO concentration of 1%. At the start of each assay, 25 nM recombinant purified GST-tagged sAC_t protein was preincubated with the indicated inhibitors for 15 min at room temperature (23 °C). Each inhibitor was used at a concentration 10-fold above their IC₅₀ values. Following the preincubation period, 1 μL of the enzyme-inhibitor solution was added to 99 μL of a reaction solution containing the following: 2 mM ATP, 10 mM Mn²⁺, 50 mM Tris pH 7.5, 3 mM DTT, and ~4,000,000 counts of α−³²P labeled ATP. After the 100-fold dilution step, each inhibitor was present at a concentration 10-fold below their IC₅₀ values, this was done to minimize inhibitor rebinding during the reaction. The reactions were stopped every 6 min over the course of 1 h and generated cAMP was measured using the sequential chromatography method referenced above. Using Prism (GraphPad, version 9.3.1), the data was fit to the equation: $\%\text{total cAMP formed}=v_{s}t+\left(\left(v_0-v_s\right)\left(1-e^{\left(-k_{\text{obs}}t\right)}\right)\right)/k_{\text{obs}}$, with k_obs being an estimate of the dissociation rate constant (k_off). To calculate v_s (uninhibited enzyme velocity) and v₀ (inhibited enzyme velocity), the jump dilution assay was performed in the presence of only DMSO and an excess concentration of inhibitor, respectively.

Cell-Based Cyclase Activity Assay.

Rat 4–4 cells were generated and functionally authenticated in our laboratory as previously described⁹ and grown in DMEM + 10% FBS. 1.25 × 10⁶ 4–4 cells were seeded per well of a 24-well plate and incubated for 24 h at 37 °C, 5% CO₂. One hour before the experiment the media was aspirated and replaced with 300 μL fresh media. For 10 min, in duplicate wells, cells were preincubated with sAC inhibitor at the indicated concentrations or 0.7% DMSO as control. For cAMP accumulation, cells were incubated with 500 μM IBMX for 5 min. To stop the reaction and to lyse the cells, the media was aspirated and replaced with 250 μL 0.1 M HCl. After shaking the plate for 5 min the cell lysate was transferred to a fresh tube and centrifuged at 1000g for 5 min. The supernatant was used for cAMP quantification using the Direct cAMP Elisa kit (Enzo) following the manufacturer’s instructions.

Measuring Binding Kinetics via Surface Plasmon Resonance.

Association (k_on) and dissociation rate constants (k_off) for the inhibitors were obtained at 25 °C with a Biacore 8K instrument (Cytiva) using a single-cycle kinetics protocol. In PBS-P+ buffer (1 mM KH₂PO₄, 150 mM NaCl, 6 mM Na₂HPO₄, 0.05% (w/v) P20 Surfactant), 50 μg/mL of recombinant purified His-tagged sAC_t protein was covalently immobilized and captured on a Series S Sensor NTA chip (Cytiva) using Ni²⁺-His-tag chelation followed by amine coupling with a 1:1 mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. After coupling, 1 M ethanolamine followed by 350 mM EDTA were, respectively, used to block any remaining reactive groups on the surface of the chip and to strip the Ni²⁺. Following chip preparation, TBS-P+ running buffer (50 mM Tris, 150 mM NaCl, and 0.05% P20 Surfactant) supplemented with 1% DMSO was flowed over the surface of the chip. After a stable baseline was obtained, five different concentrations of the indicated inhibitors were sequentially injected into a single channel for 120 s at a flow rate of 50 μL/min and then, at the same flow rate, allowed to dissociate for 600 s in the presence of running buffer. For TDI-10229, the following concentrations were used: 5000, 1250, 312.5, 78.125, and 19.53125 nM. For all other inhibitors, the following concentrations were used: 1000, 250, 62.5, 15.625, and 4 nM. All experiments were performed in parallel in reference channels lacking immobilized protein. To process the collected data, responses from the reference channels were subtracted from the responses from the active channels. From the reference-subtracted data, fitted curves and k_d and k values were determined using the Biacore 8K Insight Evaluation Software Version 2.0 (Cytiva) using a 1:1 binding kinetics model.

Log D Determination.

Log D 7.4, which is a partition coefficient between 1-octanol and aqueous buffer pH 7.4, of the compounds was measured using the chromatographic procedure whose condition was developed based on a published method.^47,48

Parallel Artificial Membrane Permeability Assay (PAMPA).

Donor wells were filled with 200 μL of PRISMA HT buffer (pH 7.4, pION, Inc.) containing 10 μM test compound. The filter on the bottom of each acceptor well was coated with 4 μL of a GIT-0 Lipid Solution (pION, Inc.) and filled with 200 μL of Acceptor Sink Buffer (pION, Inc.). The acceptor filter plate was put on the donor plate and incubated for 3 h at room temperature. After the incubation, the amount of test compound in both the donor and acceptor wells was measured by LC/MS/MS.

Kinetic Solubility.

Small volumes of compound solution dissolved in DMSO were added to the aqueous buffer solution. After incubation, precipitates were separated by filtration. The solubility was determined by UV absorbance of each filtrate.

GSH (Glutathione) Reactivity Assay.

Two microliters of 1 mM compound are spiked into 398 μL of 0.1 M potassium phosphate buffer containing 5 mM GSH to achieve a final compound concentration of 5 μM. A similar buffer solution was prepared minus GSH that was used as the compound control sample. The samples were incubated at 37 °C, and an aliquot (50 μL) was taken at different time points (0, 10, 30, 60, and 120 min) and quenched with cold acetonitrile (400 μL) containing internal standards and ethyl maleimide solution (50 μL of a 20 mM solution in acetonitrile). The samples were vortexed, and the supernatants were subjected to LCMS analysis. The remaining percentage at each time point was determined using the peak area ratios from the extracted ion chromatograms. The remaining percentage at t min (%) = peak area ratio t min/peak area ratio 0 min × 100.

Metabolic Stability.

Liver microsomes were purchased from Sekisui XenoTech, LLC. (Kansas City, KS). The microsomes (0.2 mg protein/mL) and the compounds (1 μM) were mixed in phosphate buffer (pH 7.4). The reactions were initiated by adding an NADPH-generating system (a mixture of MgCl₂, β-NADP⁺, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase) to the mixtures before incubation. Incubations were conducted at 37 °C and terminated by adding acetonitrile. The zero-time incubations, which served as the controls, were terminated by adding acetonitrile before adding an NADPH-generating system. After the samples were mixed and centrifuged, the compound concentration in the supernatant fractions was measured by LC/MS/MS.

In Vivo Pharmacokinetic Analysis in Mice.

The free base of TDI-11861 (33) was dosed in adult, male CD-1 mice (n = 3 each arm). The IV dose in mice (2 mg/kg) was administered as a solution at 0.4 mg/mL in 20% HPBCD. The PO doses in mice (10 and 50 mpk) were administered as a solution at 2 and 10 mg/mL concentrations, respectively, in DMSO/PEG400/PBS (10:40:50). The dosing volume for all studies in mice was 5 mL/kg. Serial blood samples were collected after dosing at selected time points up to 24 h. After centrifugation to harvest plasma, the concentrations of TDI-11861 (33) were determined by liquid chromatography coupled with tandem mass spectrometry. The standard PK parameters in mice (Cl, T_1/2, AUC, and F) were calculated using the 2 mpk IV and 10 mpk PO arms. The reported mean values were calculated from individual animals in each arm. All animal studies included as part of this manuscript were performed at Pharmaron in accordance with guidelines defined by “Institutional Animal Care and Use Committee (IACUC)”.

Crystal Structure Determination.

The protein construct comprising the catalytic domains of human sAC (hsAC-cat) was expressed, purified, and crystallized as described,⁴⁹ with a micro-seeding step added to improve the reproducibility of high-quality crystal growth. In short, hsAC-cat with a C-terminal His-tag was expressed in insect cells; purified through affinity, ion exchange, and size exclusion chromatography; and crystallized in hanging drops with 0.1 M sodium acetate pH 5.0, 0.2 M trisodium citrate, 14% (w/v) PEG 4000, and 10% (v/v) glycerol as reservoir solution. Crystals were transferred to a drop containing 0.1 M sodium acetate pH 5.0, 0.2 M trisodium citrate, 15% (w/v) PEG 4000, 20% (v/v) glycerol, 10% DMSO (v/v), soaked with 5 mM ligand for 24 h, and flash-frozen in liquid nitrogen.

Diffraction data were collected at 100 K at BESSY beamline 14.1 operated by Helmholtz-Zentrum Berlin⁵⁰ and processed with XDSapp.⁵¹ Phases were determined through Patterson searches with Phaser⁵² using hsAC apo (PDB ID 4CLL) as a search model. The crystallographic model was then rebuilt in Coot⁵³ and refined with Phenix.⁵⁴

EXPERIMENTAL SECTION

Synthetic Materials and Methods.

Reagents and solvents were obtained from commercial sources and used without further purification. ¹H NMR spectra (400 MHz) were collected on a Varian spectrometer. ¹³C NMR spectra (126 MHz) were collected on a Bruker spectrometer. Chemical shifts are reported in ppm relative to residual solvent peak in the indicated solvent, and for ¹H NMR spectra, multiplicities, coupling constants in hertz, and numbers of protons are indicated. Purities of all reported compounds were greater than 95% based on HPLC chromatograms obtained on an Agilent 1200 LCMS system. The Supporting Information contains details surrounding the preparation of 5–14, 16–18, and 21–32.

Preparation of 2. Ethyl 4-[hydroxyl(phenyl)methyl]-1-[(4-methoxyphenyl)methyl]pyrazole-3-carboxylate.

To a mixture of ethyl 4-iodo-1-[(4-methoxyphenyl)methyl]pyrazole-3-carboxylate (3.0 g, 7.8 mmol, 1 equiv) in THF (30 mL) was added i-PrMgCl-LiCl (1.3 M, 6.3 mL, 1.05 equiv) dropwise at −15 °C under N₂. After stirring at −15 °C for 30 min, benzaldehyde (907 mg, 8.55 mmol, 864 μL, 1.1 equiv) was added to the mixture dropwise at −15 °C. The resulting reaction mixture was stirred at 15 °C for 12 h under N₂. The reaction mixture was quenched with saturated aqueous NH₄Cl solution (100 mL). The mixture was extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (ISCO; 80 g SepaFlash Silica Flash Column, gradient elution of 0–25% ethyl acetate/petroleum ether @ 100 mL/min) which furnished the title compound (1.9 g, 5.19 mmol, 67%). ¹H NMR: (400 MHz, chloroform-d) δ 7.39–7.17 (m, 5H), 7.09 (br d, J = 8.2 Hz, 2H), 6.91–6.69 (m, 3H), 5.92 (br d, J = 4.0 Hz, 1H), 5.17 (s, 2H), 4.64 (br d, J = 4.6 Hz, 1H), 4.48–4.28 (m, 2H), 3.73 (s, 3H), 1.35 (br t, J = 7.0 Hz, 3H).

Ethyl 4-benzyl-1-[(4-methoxyphenyl)methyl]pyrazole-3-carboxylate.

To a solution of NaI (4.66 g, 31.1 mmol, 6 equiv) in MeCN (20 mL) was added TMSCl (3.38 g, 31.1 mmol, 3.95 mL, 6 equiv) under N₂. After stirring at 15 °C for 10 min, a solution of ethyl 4-[hydroxyl(phenyl)methyl]-1-[(4-methoxyphenyl)methyl]pyrazole-3-carboxylate (1.9 g, 5.2 mmol, 1 equiv) in MeCN (10 mL) was added. The mixture was stirred at 15 °C for 2 h under N₂. The reaction mixture was quenched with saturated, aqueous Na₂SO₃ solution (150 mL). The mixture was extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (80 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by gradient flash chromatography (ISCO; 20 g SepaFlash Silica Flash Column, gradient elution of 0–20% ethyl acetate/petroleum ether @ 75 mL/min) which furnished the title compound (1.6 g, 4.6 mmol, 88%).

Ethyl 3-[4-benzyl-1-[(4-methoxyphenyl)methyl]pyrazol-3-yl]-3-oxo-propanoate.

A mixture of ethyl 4-benzyl-1-[(4-methoxyphenyl)-methyl]pyrazole-3-carboxylate (390 mg, 1.11 mmol, 1 equiv) in THF (10 mL) was cooled to 0 °C and then NaH (134 mg, 3.34 mmol, 60 wt % dispersion in oil, 3 equiv) was added. After stirring for 20 min, ethyl acetate (686 mg, 7.79 mmol, 0.76 mL, 7 equiv) was added dropwise at 0 °C. The mixture was stirred at 70 °C for 2 h under N₂. The reaction mixture was quenched with saturated NH₄Cl solution (80 mL). The aqueous phase was extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0–15% ethyl acetate/petroleum ether gradient @ 45 mL/min) to furnish the title compound (250 mg, 57%) as a yellow oil. ¹H NMR: (400 MHz, DMSO-d₆) δ 7.64 (s, 1H), 7.31–7.13 (m, 7H), 6.96–6.85 (m, 2H), 5.27 (s, 2H), 4.06 (q, J = 7.1 Hz, 2H), 4.01 (s, 2H), 3.96 (s, 2H), 3.74–3.69 (m, 3H), 1.13 (t, J = 7.1 Hz, 3H).

2-Amino-4-[4-benzyl-1-[(4-methoxyphenyl)methyl]pyrazol-3-yl]-1H-pyrimidin-6-one.

To a solution of ethyl 3-[4-benzyl-1-[(4-methoxyphenyl)methyl]pyrazol-3-yl]-3-oxo-propanoate (250 mg, 0.637 mmol, 1 equiv) in EtOH (5 mL) was added guanidine carbonate (172 mg, 0.956 mmol, 1.5 equiv). The mixture was stirred for 24 h at 85 °C under N₂. The reaction mixture was diluted with water (20 mL) and then concentrated under reduced pressure to remove the EtOH. The solution was adjusted to pH = 5 via addition of aqueous HCl (2 N). The mixture was filtered, and the residue was dried under reduced pressure to furnish the title compound (150 mg, 61%) as a white solid.

4-[4-Benzyl-1-[(4-methoxyphenyl)methyl]pyrazol-3-yl]-6-chloropyrimidin-2-amine.

To a solution of 2-amino-4-[4-benzyl-1-[(4-methoxyphenyl)methyl]pyrazol-3-yl]-1H-pyrimidin-6-one (150 mg, 0.387 mmol, 1 equiv) in dioxane (3 mL) was added POCl₃ (890 mg, 5.81 mmol, 0.54 mL, 15 equiv) dropwise. The reaction mixture was stirred at 75 °C for 12 h under N₂. The reaction mixture was quenched with saturated NaHCO₃ solution (80 mL). The organic layer was separated, and aqueous phase was extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (60 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0–25% ethyl acetate/petroleum ether gradient @ 45 mL/min) to furnish the title compound (40 mg, 0.099 mmol, 25% yield) as a yellow oil.

4-(4-Benzyl-1H-pyrazol-3-yl)-6-chloro-pyrimidin-2-amine (2).

To a solution of 4-[4-benzyl-1-[(4-methoxyphenyl)methyl]pyrazol-3-yl]-6-chloro-pyrimidin-2-amine (30 mg, 0.074 mmol, 1 equiv) in MeCN (1 mL) and H₂O (1 mL) was added CAN (122 mg, 0.222 mmol, 3 equiv) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h and then warmed to 20 °C. The reaction mixture was stirred at 20 °C for 12 h. The reaction mixture was quenched with saturated aqueous NaHCO₃ solution (5 mL), diluted with H₂O (20 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (60 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by preparative-HPLC (column: Waters Xbridge 150 × 25 mm, 5 μm; mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B%: 35–55%, 10 min) which furnished the title compound 2 as a white solid (4.4 mg, 21%). ¹H NMR: (400 MHz, DMSO-d₆) δ 7.55 (s, 1H), 7.31–7.18 (m, 4H), 7.16–7.06 (m, 3H), 7.04 (s, 1H), 4.28 (s, 2H); LCMS: (MH⁺) 286.1.

Preparation of 4. Ethyl 4-benzyl-1H-pyrazole-3-carboxylate.

Ethyl 4-benzyl-1-[(4-methoxyphenyl)methyl]pyrazole-3-carboxylate (1.53 g, 4.37 mmol, 1 equiv) was dissolved in TFA (20 mL). The mixture was stirred at 85 °C for 12 h. The reaction mixture was concentrated under reduced pressure to remove TFA. The reaction mixture was diluted with H₂O (80 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (60 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (ISCO; 20 g SepaFlash Silica Flash Column, gradient elution of 0–20% ethyl acetate/petroleum ether @ 100 mL/min), which furnished the title compound (950 mg, 62%) as a white solid. ¹H NMR: (400 MHz, DMSO-d₆) δ 13.82–13.17 (m, 1H), 7.69–7.41 (m, 1H), 7.33–7.13 (m, 5H), 4.33–4.18 (m, 2H), 4.04 (br s, 2H), 1.33–1.22 (m, 3H).

4-Benzyl-1-(difluoromethyl)pyrazole-3-carboxylate.

A mixture of ethyl 4-benzyl-1H-pyrazole-3-carboxylate (830 mg, 3.60 mmol, 1 equiv) in DMF (10 mL) was cooled to 0 °C. Sodium hydride (433 mg, 10.8 mmol, 60 wt % dispersion in oil, 3 equiv) was added. After stirring for 20 min, ethyl 2-bromo-2,2-difluoro-acetate (878 mg, 4.33 mmol, 0.556 mL, 1.2 equiv) was added dropwise at 0 °C. The mixture was stirred at 15 °C for 2 h under N₂. The reaction mixture was quenched with saturated, aqueous NH₄Cl (80 mL). The mixture was extracted with ethyl acetate (60 mL × 3). The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (ISCO; 12 g SepaFlash Silica Flash Column, gradient elution of 0–3% ethyl acetate/petroleum ether @ 36 mL/min) which furnished the title compound (220 mg, 0.78 mmol, 22%) as a yellow oil. ¹H NMR: (400 MHz, DMSO-d₆) δ 8.13 (s, 1H), 8.02–7.70 (m, 1H), 7.31–7.26 (m, 2H), 7.24–7.16 (m, 3H), 4.28 (q, J = 6.7 Hz, 2H), 4.08–4.04 (m, 2H), 1.28–1.23 (m, 3H).

Ethyl 3-[4-benzyl-1-(difluoromethyl)pyrazol-3-yl]-3-oxo-propanoate.

A mixture of ethyl acetate (396 mg, 4.50 mmol, 0.44 mL, 7 equiv) in THF (2 mL) was cooled to 0 °C. Sodium hydride (31 mg, 0.77 mmol, 60 wt % dispersion in oil, 1.2 equiv) was added. After stirring for 2 min, ethyl 4-benzyl-1-(difluoromethyl)pyrazole-3-carboxylate (180 mg, 0.64 mmol, 1 equiv) in THF (1 mL) was added dropwise at 0 °C. The mixture was stirred at 50 °C for 1 h under N₂. The reaction mixture was quenched with saturated aqueous NH₄Cl (20 mL). The mixture was extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0–3% ethyl acetate/petroleum ether gradient at 35 mL/min) to furnish the title compound (100 mg, 49%) as a yellow oil.

2-Amino-4-[4-benzyl-1-(difluoromethyl)pyrazol-3-yl]-1H-pyrimidin-6-one.

To a solution of ethyl 3-[4-benzyl-1-(difluoromethyl)-pyrazol-3-yl]-3-oxo-propanoate (80 mg, 0.25 mmol, 1 equiv) in EtOH (2 mL) was added guanidine carbonate (134 mg, 0.745 mmol, 3 equiv). The mixture was stirred at 85 °C for 12 h under N₂. The reaction mixture was diluted with H₂O (10 mL) and concentrated under reduced pressure to remove EtOH. The mixture was extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (30 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to furnish the title compound (80 mg, crude, ~100%) as a white solid.

4-[4-Benzyl-1-(difluoromethyl)pyrazol-3-yl]-6-chloro-pyrimidin-2-amine (4).

POCl₃ (507 mg, 3.31 mmol, 0.31 mL, 15 equiv) was added into a solution of 2-amino-4-[4-benzyl-1-(difluoromethyl)-pyrazol-3-yl]-1H-pyrimidin-6-one (70 mg, 0.22 mmol, 1 equiv) in dioxane (2 mL) at 15 °C. Then the mixture was heated to 75 °C and stirred for 2 h under N₂. The reaction mixture was adjusted to pH 7 by the addition of a NaOH solution (1 N). The reaction mixture was extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (60 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex Gemini-NX 150 × 30 mm × 5 μm; mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B%: 38–68%, 6 min) to furnish the title compound 4 (14 mg, 0.041 mmol, 19% yield) as a white solid. ¹H NMR: (400 MHz, DMSO-d₆) δ 8.05 (s, 1H), 7.97–7.65 (m, 1H), 7.32–7.22 (m, 6H), 7.20–7.13 (m, 1H), 7.01 (s, 1H), 4.29 (s, 2H); LCMS: (MH⁺) 336.1.

Preparation of 19. 4-[Hydroxy-[2-(2-morpholinoethoxy)phenyl]-methyl]-1-methyl-pyrazole-3-carboxylate.

To a solution of methyl 4-iodo-1-methyl-pyrazole-3-carboxylate (1.35 g, 5.07 mmol, 1 equiv) in THF (15 mL) was added isopropropylmagnesium chloride–lithium chloride complex (1.3 M, 4.3 mL, 1.1 equiv) at −10 °C under N₂. After stirring at −10 °C for 0.5 h, a solution of 2-(2-morpholinoethoxy)benzaldehyde (1.40 g, 5.95 mmol, 1.17 equiv) in THF (5 mL) was added to the mixture dropwise. After the addition, the mixture was allowed to warm slowly to 25 °C and stirred at 25 °C for 12 h. The reaction mixture was diluted with sat. NH₄Cl solution (100 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic layer was washed with brine (70 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, petroleum ether/ethyl acetate = 1:1 to 1:0 followed by 5% MeOH as an additive) to furnish the title compound (800 mg, 2.13 mmol, 42% yield) as a yellow oil. ¹H NMR: (chloroform-d, 400 MHz) δ 7.26 (s, 1H), 7.17 (dt, J = 1.7, 7.8 Hz, 1H), 7.08 (dd, J = 1.4, 7.8 Hz, 1H), 6.87–6.82 (m, 2H), 6.44 (s, 1H), 4.21–4.09 (m, 2H), 3.88 (s, 3H), 3.80 (s, 3H), 3.62 (s, 4H), 2.69–2.62 (m, 2H), 2.46–2.36 (m, 4H).

Methyl 1-methyl-4-[[2-(2-morpholinoethoxy)phenyl]methyl]-pyrazole-3-carboxylate.

To a solution of NaI (2.87 g, 19.2 mmol, 6 equiv) in MeCN (10 mL) was added TMSCl (2.08 g, 19.2 mmol, 2.43 mL, 6 equiv) under N₂. After 10 min of stirring at 25 °C, a solution of methyl 4-[hydroxy-[2-(2-morpholinoethoxy)phenyl]-methyl]-1-methyl-pyrazole-3-carboxylate (1.20 g, 3.20 mmol, 1 equiv) in MeCN (6 mL) was added. The mixture was stirred at 25 °C under N₂ for 1 h. The reaction mixture was diluted with sat. aq. Na₂SO₃ (70 mL). The solution was extracted with ethyl acetate (50 mL × 3). The combined organic layer was washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO₂, petroleum ether/ethyl acetate = 1:1 to 1:0 and then 5% MeOH additive) to furnish the title compound (1.1 g, 3.1 mmol, 96% yield) as a yellow oil. ¹H NMR: (chloroform-d, 400 MHz) δ 7.24–7.15 (m, 2H), 6.99 (s, 1H), 6.95–6.84 (m, 2H), 4.24 (br t, J = 4.8 Hz, 2H), 4.10 (s, 2H), 3.93 (s, 3H), 3.88 (s, 3H), 3.76 (br d, J = 4.0 Hz, 4H), 2.92 (br s, 2H), 2.69 (br s, 4H).

Ethyl 3-[1-methyl-4-[[2-(2-morpholinoethoxy)phenyl]methyl]-pyrazol-3-yl]-3-oxo-propanoate.

To a solution of methyl 1-methyl-4-[[2-(2-morpholinoethoxy)phenyl]methyl]pyrazole-3-carboxylate (990 mg, 2.75 mmol, 1 equiv) and ethyl acetate (1.70 g, 19.3 mmol, 1.89 mL, 7 equiv) in THF (10 mL) was added LiHMDS (1 M, 8.26 mL, 3 equiv) at −40 °C quickly in one portion. The mixture was stirred at −40 °C for 2 h under N₂. The reaction mixture was diluted with sat. NH₄Cl solution (100 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic layer was washed with brine (90 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, petroleum ether/ethyl acetate = 1:0) to furnish the title compound (900 mg, 79%) as a yellow oil. ¹H NMR: (chloroform-d, 400 MHz). δ 7.20 (d, J = 7.5 Hz, 2H), 6.99 (s, 1H), 6.92–6.83 (m, 2H), 4.22 (d, J = 7.1 Hz, 2H), 4.15–4.10 (m, 4H), 4.04 (s, 2H), 3.84 (s, 3H), 3.71–3.65 (m, 4H), 2.78 (t, J = 5.6 Hz, 2H), 2.57–2.48 (m, 4H), 1.31–1.26 (m, 3H).

2-Amino-4-[1-methyl-4-[[2-(2-morpholinoethoxy)phenyl]-methyl]pyrazol-3-yl]-1H-pyrimidin-6-one.

Ethyl 3-[1-methyl-4-[[2-(2-morpholinoethoxy)phenyl]methyl]pyrazol-3-yl]-3-oxo-propanoate (800 mg, 1.93 mmol, 1 equiv) and guanidine carbonate (1.04 g, 5.78 mmol, 3 equiv) were taken up in anhydrous EtOH (16 mL). The mixture was stirred for 36 h at 85 °C under N₂ during which time a white precipitate formed. The reaction mixture was diluted with water (10 mL) and then concentrated under the reduced pressure to remove the EtOH. The pH of the solution was adjusted to 5 by the addition of aqueous HCl (4 N). Then, the pH of the solution was adjusted to 9 by the addition of aqueous NH₃. The solution was filtered, and the residue was dried under reduced pressure to afford the title compound (400 mg, 51%) as a light, yellow solid.

4-Chloro-6-[1-methyl-4-[[2-(2-morpholinoethoxy)phenyl]-methyl]pyrazol-3-yl]pyrimidin-2-amine (19).

POCl₃ (2.24 g, 14.6 mmol, 1.36 mL, 15 equiv) was added into a solution of 2-amino-4-[1-methyl-4-[[2-(2-morpholinoethoxy)phenyl]methyl]pyrazol-3-yl]-1H-pyrimidin-6-one (400 mg, 0.975 mmol, 1 equiv) in dioxane (8 mL) at 25 °C. The mixture was heated to 75 °C for 16 h. The reaction mixture was added slowly to an aq. NaHCO₃ solution (saturated, 110 mL) to quench the excess POCl₃. The solution was extracted with ethyl acetate (60 mL × 5). The organic layer was washed with brine (70 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex Synergi C18 150 × 25 × 10 μm; mobile phase: [water (0.2% formic acid)-ACN]; B%: 20–50%, 10 min) to furnish the title compound (157 mg, formic acid salt, 34%) 19 as an off-white solid. ¹H NMR: (DMSO-d₆, 400 MHz). δ 8.13 (s, 1H), 7.26 (s, 1H), 7.21 (dd, J = 1.6, 7.4 Hz, 1H), 7.19–7.12 (m, 1H), 7.06 (br s, 2H), 6.98 (s, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.84 (t, J = 7.3 Hz, 1H), 4.20 (s, 2H), 4.05 (t, J = 5.6 Hz, 2H), 3.80 (s, 3H), 3.53–3.48 (m, 4H), 2.63 (br t, J = 5.3 Hz, 2H), 2.40 (br s, 4H); ¹³C NMR (126 MHz, DMSO) δ 163.83, 163.68, 160.42, 158.42, 158.18, 144.21, 132.56, 130.70, 129.91, 127.91, 122.17, 121.01, 116.64, 112.18, 104.57, 66.43, 53.96, 40.59, 40.50, 40.43, 40.34, 40.26, 40.17, 40.09, 40.00, 39.92, 39.83, 39.67, 39.50, 39.44, 25.67; LCMS: (M + H⁺): 429.2. HRMS calcd for C₂₁H₂₆N₆ClO₂ (M + H⁺) 429.1801; found: 429.1800.

Preparation of 20. 4-[(2-Allyloxyphenyl)-hydroxy-methyl]-1-methyl-pyrazole-3-carboxylate.

To a solution of methyl 4-iodo-1-methyl-pyrazole-3-carboxylate (5.00 g, 18.8 mmol, 1 equiv) in THF (50 mL) was added i-PrMgCl-LiCl (1.3 M, 15.2 mL, 1.05 equiv) at −15 °C. The mixture was stirred at −15 °C for 0.5 h under N₂. 2-Allyloxybenzaldehyde (3.05 g, 18.8 mmol, 1 equiv) in THF (5 mL) was added to the mixture at −15 °C. The whole mixture was stirred at 25 °C for another 11.5 h under N₂. The reaction mixture was poured into a saturated ammonium chloride solution (150 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (80 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 80 g SepaFlash Silica Flash Column, eluent of 0–50% ethyl acetate/petroleum ether gradient @ 100 mL/min) to furnish the title compound (3.80 g, 12.6 mmol, 67% yield) as a light-yellow oil. ¹H NMR: (400 MHz, chloroform-d) δ 7.53 (dd, J = 1.2, 7.5 Hz, 1H), 7.26–7.22 (m, 1H), 7.00 (t, J = 7.5 Hz, 1H), 6.95 (s, 1H), 6.86 (d, J = 8.2 Hz, 1H), 6.39 (d, J = 3.9 Hz, 1H), 6.05–5.91 (m, 1H), 5.34–5.18 (m, 2H), 4.77 (br d, J = 4.6 Hz, 1H), 4.53 (d, J = 5.1 Hz, 2H), 3.96 (s, 3H), 3.85 (s, 3H).

Methyl 4-[(2-allyloxyphenyl)methyl]-1-methyl-pyrazole-3-carboxylate.

To a solution of NaI (10.1 g, 67.5 mmol, 6 equiv) in MeCN (15 mL) was added TMSCl (7.33 g, 67.5 mmol, 8.56 mL, 6 equiv) under N₂. The mixture was stirred at 25 °C for 10 min. Methyl 4-[(2-allyloxyphenyl)-hydroxy-methyl]-1-methyl-pyrazole-3-carboxylate (3.4 g, 11.3 mmol, 1 equiv) was dissolved in MeCN (20 mL) and added to the mixture. The whole mixture was stirred at 25 °C for 1 h under N₂. The reaction mixture was poured into saturated sodium sulfite solution (150 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (80 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, eluent of 0–20% ethyl acetate/petroleum ether gradient @ 75 mL/min) to furnish the title compound (3.1 g, 96%) as a yellow oil. ¹H NMR: (400 MHz, chloroform-d) δ 7.23–7.14 (m, 2H), 7.04 (s, 1H), 6.92–6.82 (m, 2H), 6.03 (tdd, J = 5.1, 10.4, 17.2 Hz, 1H), 5.37 (qd, J = 1.5, 17.3 Hz, 1H), 5.25 (dd, J = 1.4, 10.6 Hz, 1H), 4.55 (td, J = 1.5, 5.0 Hz, 2H), 4.14 (s, 2H), 3.92 (s, 3H), 3.86 (s, 3H).

Ethyl 3-[4-[(2-allyloxyphenyl)methyl]-1-methyl-pyrazol-3-yl]-3-oxo-propanoate.

To a solution of methyl 4-[(2-allyloxyphenyl)-methyl]-1-methyl-pyrazole-3-carboxylate (3.10 g, 10.8 mmol, 1 equiv) and EtOAc (6.68 g, 75.8 mmol, 7.42 mL, 7 equiv) in THF (30 mL) was added LiHMDS (1 M, 32.5 mL, 3 equiv) at −40 °C quickly in one portion. The mixture was stirred at −40 °C for 1 h under N₂. The reaction mixture was poured into a saturated, aqueous ammonium chloride solution (200 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (80 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, eluent of 0–15% ethyl acetate/petroleum ether gradient @ 100 mL/min) to furnish the title compound (3.5 g, 95%) as a yellow oil. ¹H NMR: (400 MHz, chloroform-d). δ 7.24 (dd, J = 1.5, 7.4 Hz, 1H), 7.18 (dt, J = 1.8, 7.8 Hz, 1H), 7.01 (s, 1H), 6.91–6.83 (m, 2H), 6.04 (tdd, J = 5.1, 10.4, 17.2 Hz, 1H), 5.36 (qd, J = 1.6, 17.3 Hz, 1H), 5.25 (dd, J = 1.4, 10.6 Hz, 1H), 4.55 (td, J = 1.5, 5.0 Hz, 2H), 4.25–4.19 (m, 2H), 4.15 (s, 2H), 4.04 (s, 2H), 3.83 (s, 3H), 1.30–1.26 (m, 3H).

4-[4-[(2-Allyloxyphenyl)methyl]-1-methyl-pyrazol-3-yl]-2-amino-1H-pyrimidin-6-one.

To a solution of ethyl 3-[4-[(2-allyloxyphenyl)-methyl]-1-methyl-pyrazol-3-yl]-3-oxo-propanoate (3.50 g, 10.2 mmol, 1 equiv) in EtOH (30 mL) was added guanidine carbonate (5.53 g, 30.7 mmol, 3 equiv). The mixture was stirred at 85 °C for 12 h under N₂. The reaction mixture was diluted with water (10 mL), and the mixture concentrated under reduced pressure to remove the EtOH. The yellow precipitate formed was collected while the pH of the remaining aqueous phase was adjusted to 5 by the addition of aq. HCl solution (1 N). The combined yellow precipitate was triturated with EtOAc/MeOH (5:1, 10 mL). The solid was collected by filtration and dried under reduced pressure to furnish the title compound (1.0 g, 29%) as a yellow solid. The material was used directly in the next step without further purification. LCMS: (M + H⁺): 338.1.

4-[4-[(2-Allyloxyphenyl)methyl]-1-methyl-pyrazol-3-yl]-6-chloropyrimidin-2-amine.

To a solution of 4-[4-[(2-allyloxyphenyl)-methyl]-1-methyl-pyrazol-3-yl]-2-amino-1H-pyrimidin-6-one (900 mg, 2.67 mmol, 1 equiv) in dioxane (10 mL) was added POCl₃ (6.14 g, 40.0 mmol, 3.72 mL, 15 equiv). The mixture was stirred at 75 °C for 12 h under N₂. The reaction mixture was concentrated under reduced pressure to remove POCl₃. The mixture was dissolved in EtOAc (30 mL), and the resulting solution was added slowly to a saturated, aqueous sodium bicarbonate solution (150 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (80 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0–30% ethyl acetate/petroleum ether gradient @ 75 mL/min) to furnish the title compound as (0.7 g, 74%) as a yellow solid. ¹H NMR: (400 MHz, chloroform-d) δ 7.24 (s, 1H), 7.21–7.11 (m, 2H), 7.07 (s, 1H), 6.90–6.85 (m, 2H), 6.04 (tdd, J = 5.1, 10.4, 17.2 Hz, 1H), 5.38 (qd, J = 1.6, 17.3 Hz, 1H), 5.25 (qd, J = 1.4, 10.6 Hz, 1H), 5.12 (br s, 2H), 4.57 (td, J = 1.5, 5.0 Hz, 2H), 4.26 (s, 2H), 3.88 (s, 3H).

3-[2-[[3-(2-Amino-6-chloro-pyrimidin-4-yl)-1-methyl-pyrazol-4-yl]methyl]phenoxy]propane-1,2-diol.

To a solution of 4-[4-[(2-allyloxyphenyl)methyl]-1-methyl-pyrazol-3-yl]-6-chloro-pyrimidin-2-amine (0.75 g, 2.1 mmol, 1 equiv) in THF (48 mL) and H₂O (6 mL) was added NMO (740 mg, 6.32 mmol, 3 equiv) and K₂OsO₄ dihydrate (78 mg, 0.21 mmol, 0.1 equiv) at 25 °C. The mixture was stirred at 25 °C for 12 h under N₂. The reaction mixture was poured into a saturated, aqueous sodium sulfite solution (150 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (80 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0–100% ethyl acetate/petroleum ether (additive 3% MeOH) gradient @ 70 mL/min) to furnish the title compound (700 mg, 86%) as a light-yellow solid. ¹H NMR: (400 MHz, DMSO-d₆) δ 7.53 (s, 1H), 7.18 (d, J = 7.5 Hz, 1H), 7.15–7.09 (m, 1H), 7.06 (s, 2H), 6.98 (s, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.78 (t, J = 7.4 Hz, 1H), 4.99 (d, J = 5.0 Hz, 1H), 4.66 (t, J = 5.7 Hz, 1H), 4.25 (s, 2H), 4.02–3.98 (m, 1H), 3.92–3.86 (m, 1H), 3.85–3.81 (m, 1H), 3.81 (s, 3H), 3.48–3.41 (m, 2H).

2-[2-[[3-(2-Amino-6-chloro-pyrimidin-4-yl)-1-methyl-pyrazol-4-yl]methyl]phenoxy]acetaldehyde.

To a solution of 3-[2-[[3-(2-amino-6-chloro-pyrimidin-4-yl)-1-methyl-pyrazol-4-yl]methyl]-phenoxy]propane-1,2-diol (650 mg, 1.67 mmol, 1 equiv) in dioxane (10 mL) and H₂O (3 mL) was added NaIO₄ (892 mg, 4.17 mmol, 0.231 mL, 2.5 equiv) at 0 °C. The mixture was stirred at 25 °C for 2 h under N₂. The reaction mixture was poured into saturated, aq. Na₂SO₃ (150 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (80 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to furnish the title compound (750 mg, crude) as a light-yellow oil. The material was used directly in next step without further purification.

4-Chloro-6-[1-methyl-4-[[2-[2-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)ethoxy]phenyl]methyl]pyrazol-3-yl]pyrimidin-2-amine (20).

To a solution of 2-[2-[[3-(2-amino-6-chloro-pyrimidin-4-yl)-1-methyl-pyrazol-4-yl]methyl]phenoxy]acetaldehyde (0.11 g, 0.31 mmol, 1 equiv) in MeOH (1 mL) and THF (0.5 mL) was added TEA (37 mg, 0.37 mmol, 0.051 mL, 1.2 equiv) and 6-oxa-3-azabicyclo[3.1.1]-heptane (50 mg, 0.37 mmol, 1.2 equiv, HCl). The mixture was stirred at 25 °C for 2 h under N₂. NaBH₃CN (58 mg, 0.92 mmol, 3 equiv) was added to the mixture. The mixture was stirred at 25 °C for another 2 h under N₂. The reaction mixture was poured into H₂O (100 mL). The mixture was extracted with ethyl acetate (30 mL × 3). The organic phase was washed with brine (50 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC (EtOAc/MeOH = 20:1) to furnish the desired compound. The compound was further purified by prep-HPLC (column: Waters Xbridge BEH C18 100 × 30 mm × 10 μm; mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B%: 25–55%, 8 min) to furnish the title compound as (14 mg, 10%) 20 as a white solid. ¹H NMR: (400 MHz, DMSO-d₆) δ 7.23–7.13 (m, 3H), 7.06 (br s, 2H), 7.00–6.96 (m, 2H), 6.84 (t, J = 7.3 Hz, 1H), 4.33 (d, J = 6.0 Hz, 2H), 4.21 (s, 2H), 4.09 (t, J = 5.6 Hz, 2H), 3.78 (s, 3H), 3.00 (d, J = 11.3 Hz, 2H), 2.87 (t, J = 5.6 Hz, 2H), 2.78 (q, J = 6.4 Hz, 1H), 2.67 (d, J = 11.3 Hz, 2H), 2.09 (d, J = 7.8 Hz, 1H); ¹³C NMR (126 MHz, DMSO) δ 163.8, 163.7, 160.4, 156.7, 144.3, 132.5, 130.6, 130.1, 127.9, 122.3, 120.8, 112.3, 104.6, 79.2, 66.4, 55.8, 55.5, 30.4, 25.6; LCMS: (M + H⁺): 441.2; HRMS calcd for C₂₂H₂₆N₆ClO₂ (M + H⁺) 441.1801; found: 441.1802.

Preparation of TDI-11861 (33). 4-Iodo-1-tetrahydropyran-2-ylpyrazole-3-carboxylate.

To a solution of ethyl 4-iodo-1H-pyrazole-3-carboxylate (20 g, 75 mmol, 1 equiv) in THF (75 mL) were added DHP (19.0 g, 226 mmol, 20.6 mL, 3 equiv) and PTSA (1.29 g, 7.52 mmol, 0.1 equiv). The reaction mixture was stirred at 80 °C for 8 h under N₂. The reaction mixture was concentrated under reduced pressure. Then, the mixture was diluted with H₂O (300 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, gradient eluent of 0–20% ethyl acetate/petroleum ether @ 100 mL/min), which furnished the title compound (24 g, 91%) as a colorless oil.

Ethyl 4-[(2-allyloxyphenyl)-hydroxy-methyl]-1-tetrahydropyran-2-yl-pyrazole-3-carboxylate.

To a solution of ethyl 4-iodo-1-tetrahydropyran-2-yl-pyrazole-3-carboxylate (13.3 g, 38.0 mmol, 1 equiv) in THF (150 mL) was added i-PrMgCl-LiCl (1.30 M, 30.7 mL, 1.05 equiv) at −15 °C under a nitrogen atmosphere. After stirring at −15 °C for 0.5 h, a solution of 2-allyloxybenzaldehyde (6.78 g, 41.8 mmol, 1.1 equiv) in THF (20 mL) was added to the mixture dropwise. After the addition, the mixture was stirred at 60 °C for 8 h and then at 25 °C for 8 h under N₂. The reaction mixture was quenched with saturated, aqueous NH₄Cl (300 mL). The mixture was extracted with ethyl acetate (100 mL × 3). The combined organic layer was washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0–18% ethyl acetate/petroleum ether gradient @ 45 mL/min) to furnish the title compound as a yellow oil (7.7 g, 19.9 mmol, 52%).

Ethyl 4-[(2-allyloxyphenyl)methyl]-1H-pyrazole-3-carboxylate.

To a solution of NaI (16.8 g, 112 mmol, 6 equiv) in MeCN (150 mL) was added TMSCl (12.1 g, 112 mmol, 14.2 mL, 6 equiv) dropwise under N₂. After 10 min of stirring at 25 °C, a solution of ethyl 4-[(2-allyloxyphenyl)-hydroxy-methyl]-1-tetrahydropyran-2-yl-pyrazole-3-carboxylate (7.2 g, 18.6 mmol, 1 equiv) in MeCN (20 mL) was added dropwise. The mixture was stirred at 25 °C under N₂ for 2 h. The reaction mixture was quenched with saturated, aqueous Na₂SO₃ (300 mL). The mixture was extracted with ethyl acetate (100 mL × 3). The combined organic layer was washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 80 g SepaFlash Silica Flash Column, eluent of 0–30% ethyl acetate/petroleum ether gradient @ 100 mL/min) to furnish the title compound as a colorless oil (4.6 g, 91%).

Ethyl 4-[(2-allyloxyphenyl)methyl]-1-(difluoromethyl)pyrazole-3-carboxylate.

To a solution of ethyl 4-[(2-allyloxyphenyl)methyl]-1H-pyrazole-3-carboxylate (4.6 g, 16.1 mmol, 1 equiv) in MeCN (80 mL) were added KF (2.3 g, 40 mmol, 0.94 mL, 2.5 equiv) and 1-[[bromo(difluoro)methyl]-ethoxy-phosphoryl]oxyethane (7.7 g, 29 mmol, 1.8 equiv). The mixture was stirred at 25 °C for 40 h under N₂. The reaction mixture was quenched with saturated, aqueous NH₄Cl (200 mL). The mixture was extracted with ethyl acetate (100 mL × 3). The combined organic layer was washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0–5% ethyl acetate/petroleum ether gradient @ 45 mL/min) to furnish the title compound as a colorless oil (5.2 g, ~100%).

Ethyl 3-[4-[(2-allyloxyphenyl)methyl]-1-(difluoromethyl)pyrazol-3-yl]-3-oxo-propanoate.

To a solution of ethyl 4-[(2-allyloxyphenyl)methyl]-1-(difluoromethyl)pyrazole-3-carboxylate (3.0 g, 8.9 mmol, 1 equiv) and ethyl acetate (5.5 g, 62 mmol, 6.1 mL, 7 equiv) in THF (100 mL) was added LiHMDS (1.0 M, 27 mL, 3 equiv) at −40 °C under N₂. The mixture was stirred at −40 °C for 1 h under N₂. The reaction mixture was quenched with saturated, aqueous NH₄Cl (200 mL). The mixture was extracted with ethyl acetate (100 mL × 3). The combined organic layer was washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, eluent of 0–4% ethyl acetate/petroleum ether gradient @ 75 mL/min) to furnish as a colorless oil (2.5 g, 6.6 mmol, 74%). LCMS: (M + H⁺): 379.1.

4-[4-[(2-Allyloxyphenyl)methyl]-1-(difluoromethyl)pyrazol-3-yl]-2-amino-1H-pyrimidin-6-one.

To a solution of ethyl 3-[4-[(2-allyloxyphenyl)methyl]-1-(difluoromethyl)pyrazol-3-yl]-3-oxo-propanoate (2.5 g, 6.6 mmol, 1 equiv) in EtOH (50 mL) was added guanidine carbonate (3.6 g, 20 mmol, 3 equiv). The mixture was stirred at 85 °C for 12 h under N₂. The reaction mixture was diluted with H₂O (50 mL) and concentrated under reduced pressure to remove EtOH. The mixture was extracted with ethyl acetate (100 mL × 3). The combined organic layer was diluted with THF (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to furnish the title compound as a yellow solid (2.2 g, 89%). LCMS: (M + H⁺): 374.1.

4-[4-[(2-Allyloxyphenyl)methyl]-1-(difluoromethyl)pyrazol-3-yl]-6-chloro-pyrimidin-2-amine.

POCl₃ (13.6 g, 88.4 mmol, 8.21 mL, 15 equiv) was added into a solution of 4-[4-[(2-allyloxyphenyl)methyl]-1-(difluoromethyl)pyrazol-3-yl]-2-amino-1H-pyrimidin-6-one (2.20 g, 5.89 mmol, 1 equiv) in dioxane (5 mL) at 25 °C. The mixture was heated to 75 °C for 12 h under N₂. The reaction mixture was concentrated under reduced pressure to remove the solvent. The reaction mixture was quenched with saturated, aqueous NaHCO₃ (200 mL). The mixture was extracted with ethyl acetate (100 mL × 3). The combined organic layer was washed with brine (100 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0–10% ethyl acetate/petroleum ether gradient @ 75 mL/min) to furnish the title compound as a yellow solid (1.10 g, 2.8 mmol, 48%).

3-[2-[[3-(2-Amino-6-chloro-pyrimidin-4-yl)-1-(difluoromethyl)-pyrazol-4-yl]methyl]phenoxy]propane-1,2-diol.

To a solution of 4-[4-[(2-allyloxyphenyl)methyl]-1-(difluoromethyl)pyrazol-3-yl]-6-chloro-pyrimidin-2-amine (550 mg, 1.40 mmol, 1 equiv) in THF (4 mL) and H₂O (4 mL) were added NMO (460 mg, 3.93 mmol, 0.415 mL, 2.8 equiv) and OsO₄ (71 mg, 0.28 mmol, 0.014 mL, 0.2 equiv) at 0 °C. The reaction mixture was stirred at 25 °C for 2 h under N₂. The reaction mixture was quenched with saturated aqueous Na₂SO₃ (60 mL). The mixture was extracted with ethyl acetate (30 mL × 3). The combined organic layer was washed with brine (20 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0–60% ethyl acetate/petroleum ether gradient @ 50 mL/min) to furnish the title compound as a yellow solid (450 mg, 1.06 mmol, 75%).

2-[2-[[3-(2-Amino-6-chloro-pyrimidin-4-yl)-1-(difluoromethyl)-pyrazol-4-yl]methyl]phenoxy]acetaldehyde.

A mixture of 3-[2-[[3-(2-amino-6-chloro-pyrimidin-4-yl)-1-(difluoromethyl)pyrazol-4-yl]-methyl]phenoxy]propane-1,2-diol (450 mg, 1.06 mmol, 1 equiv) in dioxane (9 mL) and H₂O (3 mL) was added NaIO₄ (565 mg, 2.64 mmol, 0.146 mL, 2.5 equiv) at 0 °C. The reaction mixture was stirred at 20 °C for 2 h under N₂. The reaction mixture was quenched with saturated, aqueous Na₂SO₃ (40 mL). The mixture was extracted with ethyl acetate (30 mL × 3). The combined organic layer was washed with brine (30 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to furnish the title compound as a yellow solid (417 mg, ~100%).

[(3R)-4-[2-[2-[[3-(2-Amino-6-chloro-pyrimidin-4-yl)-1-(difluoromethyl)pyrazol-4-yl]methyl]phenoxy]ethyl]morpholin-3-yl]methanol (33, TDI-11861).

To a solution of 2-[2-[[3-(2-amino-6-chloro-pyrimidin-4-yl)-1-(difluoromethyl)pyrazol-4-yl]methyl]-phenoxy]acetaldehyde (100 mg, 0.254 mmol, 1 equiv) in DCE (3 mL) were added TEA (26 mg, 0.25 mmol, 0.035 mL, 1 equiv) and [(3R)-morpholin-3-yl]methanol (43 mg, 0.28 mmol, HCl salt). After stirring at 20 °C for 3 h, NaBH(OAc)₃ (215 mg, 1.02 mmol, 4 equiv) was added to the mixture. The mixture was stirred at 20 °C for 12 h under N₂. The reaction mixture was quenched with saturated aqueous NaHCO₃ (40 mL). The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic layer was washed with brine (30 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 × 30 mm × 10 μm; mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B%: 25–55%) to furnish the title compound as a white solid (36 mg, 29%). ¹H NMR: (400 MHz, chloroform-d) δ 7.43 (s, 1H), 7.32–7.27 (m, 1H), 7.25–7.19 (m, 1H), 7.16 (s, 0.5H), 7.11–7.06 (m, 1H), 7.01 (s, 0.3H), 6.95–6.88 (m, 2H), 5.53–5.37 (m, 2H), 4.35–4.20 (m, 2H), 4.19–4.11 (m, 1H), 4.10–4.02 (m, 1H), 3.85 (dd, J = 4.1, 11.6 Hz, 1H), 3.80–3.70 (m, 2H), 3.62–3.46 (m, 2H), 3.45–3.36 (m, 1H), 3.29–3.20 (m, 1H), 2.90 (td, J = 2.7, 11.8 Hz, 1H), 2.79–2.66 (m, 2H), 2.59–2.48 (m, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 162.2, 160.7, 159.3, 155.1, 146.6, 128.9, 128.4, 127.2, 126.6, 122.3, 119.2, 110.9, 110.5, 109.0, 107.0, 103.7, 67.4, 65.0, 64.9, 59.9, 57.8, 51.6, 50.4, 23.8; LCMS: (M + H⁺): 495.2. HRMS calcd for C₂₂H₂₆N₆ClF₂O₃ (M + H⁺) 495.1718; found: 495.1715.

ABBREVIATIONS

AC

adenylyl cyclase

IP

intraperitoneal

PO

per os

sAC

soluble adenylyl cyclase

tmAC

transmembrane adenylyl cyclase

PK

pharmacokinetic

Data Availability Statement

Diffraction data and refined model for the sAC/TDI-11861 complex have been deposited with the worldwide PDB (wwPDB) under accession number 8B75.