Novel Human Neutral Sphingomyelinase 2 Inhibitors as Potential Therapeutics for Alzheimer’s Disease

Abstract

Neutral sphingomyelinase 2 (nSMase2) catalyzes the cleavage of sphingomyelin to phosphorylcholine and ceramide, an essential step in the formation and release of exosomes from cells that is critical for intracellular communication. Chronic increase of brain nSMase2 activity and related exosome release have been implicated in various pathological processes, including the progression of Alzheimer’s disease (AD), making nSMase2 a viable therapeutic target. Recently, we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)-pyrrolidin-3-yl)carbamate 1 (PDDC), the first nSMase2 inhibitor that possesses both favorable pharmacodynamics and pharmacokinetic (PK) parameters, including substantial oral bioavailability, brain penetration, and significant inhibition of exosome release from the brain in vivo. Herein we demonstrate the efficacy of 1 (PDDC) in a mouse model of AD and detail extensive structure–activity relationship (SAR) studies with 70 analogues, unveiling several that exert similar or higher activity against nSMase2 with favorable pharmacokinetic properties.

Graphical Abstract

INTRODUCTION

Exosomes are extracellular vehicles (EVs) that are involved in intracellular communication through the trafficking of protein, lipid, and genetic material in both physiological and pathological processes.^1–3 Neutral sphingomyelinase 2 (nSMase2) is a phosphodiesterase that catalyzes the hydrolysis of sphingomyelin (SM) to phosphorylcholine and ceramide; nSMase2 is an important regulator of exosome biogenesis by controlling the availability of ceramide, a pivotal component of exosomal membrane architecture.⁴ Although transient increases in nSMase2 activity are part of normal physiological processes, chronic increases in nSMase2 activity result in exosome dysfunction.⁵ There are a number of diseases that have been associated with increases in nSMase2 activity and/or exosome dysfunction, including Alzheimer’s disease (AD),^1,6–9 HIV associated neurocognitive disorders (HAND),^10–14 amyotrophic lateral sclerosis (ALS),¹⁵ and metastatic cancer.^16,17 Moreover, by use of a variety of animal models by independent investigators, genetic and pharmacological inhibition of nSMase2 has been shown to reduce exosome secretion.^6–8,18 As a result, there has been an interest in identifying nSMase2 inhibitors that could be used for the treatment of these diseases.^19,20 To date, nSMase2 inhibitors have shown poor drug-like properties that have prevented their advancement into clinical development; they exhibit high molecular weight, potencies at the μM level, poor aqueous solubility, metabolic stability, bioavailability, and/or brain penetration.^19,20 Recently, after a high throughput screen for nSMase2 inhibitors and subsequent hit optimization we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate 1 (PDDC), a new, potent (IC50 = 300 nM) nSMase2 inhibitor that inhibits exosome secretion in vitro and shows brain penetration, inhibition of exosome secretion from brain, and target engagement in an acute in vivo model of inflammation.²¹ In the present work, we extend our previous findings through a systematic and extensive SAR effort around 1 (PDDC) in order to improve on potency of inhibition while maintaining the compound’s metabolic stability. Further, we follow up on the ability of 1 (PDDC), as representative of this chemical series, to show efficacy in reversing cognitive impairment in a chronic model of AD.

RESULTS

Chemistry.

The most critical part of the present study is the detailed SAR reported on a newly identified nSMase inhibitor termed 1 (PDDC), which has been identified by our team originally via high-throughput screening of human nSMase2.²¹ The original screening hit compounds 3 and 4 differed only in the configuration of their chiral center (Figure 1). Despite the fact that these compounds are related to our phosphatidylinositol 4-kinase IIIβ (PI4KB) inhibitors reported recently (e.g., compound 2),^22–24 they are devoid of the kinase activity due to the bulky pyrrolidine ring and lack of essential hydrogen bond donor that prohibit interaction with the hinge region of the kinase (Figure 1). In addition, the compounds do not exert inhibition of other PI4Ks or cytotoxicity (Figure 1, Supporting Information Table S3). Since 3 was the more active against nSMase2 in comparison with its enantiomer 4, our further explorations were focused on this enantiomer and its derivatives.

Figure 1.

Identification of the HTS hit compounds 3 and 4 and their optimization to our tool compound 1 (PDDC).

Here, we report extensive and systematic modifications prepared within our SAR exploration. This series of 70 compounds can be divided into several groups. Initially, we modified the central core of the molecule (Figure 1, in blue). Second, we studied the influence of southern moiety modifications (Figure 1, in red). Next, we focused on modification of the northern part of the molecule (Figure 1, in green). Finally, we explored the effect of the removal of methyl substituents of the central core. For the AD mice studies, we selected compound 1 (PDDC) as a tool inhibitor. Our SAR study has, however, unveiled several other derivatives that share similar or even higher potency against human nSMase 2 with similar metabolic stability. These compounds can be regarded as potential candidates for selection of a lead structure for clinical development.

Synthesis of the Compounds with Modified Central Core.

The central imidazo[1,2-b]pyridazine core was substituted by several bicyclic heterocycles including purine (6), furo[3,2-b]pyridine (7), thieno[3,2-d]pyrimidine (8), thieno[3,4-d]pyrimidine(9) following the procedures depicted in Schemes 1–4.

Scheme 1. Preparation of the Compounds with Purine Central Core^a.

^aReagents and conditions: (a) (1) (3R)-3-(tert-butoxycarbonylamino)pyrrolidine, DIPEA, CH₃CN, 75 °C, (2) TFA, CH₂Cl₂, (3) Ac₂O, Et₃N, CH₃CN, 88% over three steps.

Scheme 4. Preparation of the Target Thieno[3,4-d]pyrimidine Derivative 9^a.

^aReagents and conditions: (a) NBS, DMF, 2 h, 87%; (b) 11(R), DIPEA, CH₃CN, 0.5 h at 0 °C, 1 h at rt, 87%; (c) (3,4-dimethoxyphenyl)boronic acid, 1,4-dioxane, H₂O, K₂CO₃, Pd(PPh₃)₄, 93 °C, overnight, 60%; (d) DABAL-Me₃, Pd₂(dba)₃, X-Phos, THF, rt to 65 °C, overnight, 68%.

The purine derivative 6 was prepared from compound 10, following a reported procedure.²⁵ Initially, the pyrrolidine side chain protected by Boc group was installed onto the purine moiety by nucleophilic displacement of chlorine atom at position 6. Subsequently, the Boc group was substituted by the acetyl group (Scheme 1).

The synthesis of furane containing bicyclic heterocycle 7 started by methylation of 2,4-dibromo-6-methylpyridin-3-ol 13 followed by Sonogashira coupling. The crucial cyclization–iodination step, which was promoted by iodine monochloride, yielded iodinated derivative 16 used in the following cross-coupling reaction with 3,4-dimethoxyphenylboronic acid to give compound 17. Finally, we introduced the northern part of the molecule by nucleophilic substitution of bromine atom to obtain the final derivative 7 (Scheme 2). Regioselectivity of Sonogashira reaction (formation of compound 15) was confirmed by NMR spectroscopy. In the ROESY spectrum of compound 7, cross-peaks between H-6 on the central core and 2′-CH₂ and 5′-CH₂ groups on pyrrolidine ring corresponding to their through-space interaction have been found.

Scheme 2. Preparation of the Compounds with Furo[3,2-b]pyridine Central Core^a.

^aReagents and conditions: (a) CH₃I, K₂CO₃, acetone, 84%; (b) trimethyl(prop-1-yn-1-yl)silane, TBAF–THF, CuI, Pd(PPh₃)₂Cl₂, Et₃N, toluene, 70%; (c) ICl, CH₂Cl₂, 75%; (d) 3,4-dimethoxyphenylboronic acid, Na₂CO₃, Pd(dppf)Cl₂, dioxane–H₂O, 95 °C, 43%; (e) 11(R), DIPEA, n-butanol, 130 °C, 67%.

Bromination of compound 18 and subsequent Suzuki cross coupling were also the initial steps in the synthesis of thieno[3,2-d]pyrimidine derivative 8 (Scheme 3). Removing of the protecting methyl groups from compound 20 and successful chlorination were essential for installing the pyrrolidine part of the molecule, leading to derivative 23. The target compound 8 was then synthesized by palladium-catalyzed introduction of a methyl group utilizing DABAL-Me₃ as the methyl group source.

Scheme 3. Preparation of the Target Thieno[3,2-d]pyrimidine Derivative 8^a.

^aReagents and conditions: (a) NBS, AcOH, CHCl₃, 60 °C, overnight, 74%; (b) (3,4-dimethoxyphenyl)boronic acid, 1,4-dioxane, H₂O, K₂CO₃, Pd(PPh₃)₄, 93 °C, overnight, 85%; (c) NaI, AcOH, 80 °C, 2 h; (d) POCl₃, N,N-dimethylaniline, 110 °C, 3 h, 72%; (e) 11(R), DIPEA, CH₃CN, 80 °C, sealed vessel, overnight, 94%; (f) DABAL-Me₃, Pd₂(dba)₃, X-Phos, THF, rt to reflux, overnight, 82%.

Finally, the thieno[3,4-d]pyrimidine derivative 9 was gained by consecutive bromination and amination of compound 24 followed by two cross-coupling reaction steps leading to introduction of 3,4-dimethoxyphenyl and methyl groups, respectively.

Synthesis of the Compounds with Southern Part Modified.

We have used a modular approach starting from compound 31(R) for preparation of the most derivatives modified in the southern part of the molecule with imidazo[1,2-b]pyridazine core, which was synthesized as depicted in Scheme 5. Dichloroderivative 28 was used as a starting material.²² Halogen atom in position 8 was replaced by (R)-N-(pyrrolidin-3-yl)acetamide (11(R)) to give derivative 29(R). Second chlorine atom in position 6 was then converted to a methyl by treatment with trimethylaluminum–DABCO complex to afford compound 30(R), which was in the last step iodinated by NIS in dichloromethane. Compound 31(R) served as a starting material for synthesis of majority of the final compounds. The opposite enantiomer 31(S) was obtained in the same way using (R)-N-(pyrrolidin-3-yl)acetamide (12(S)).

Scheme 5. Preparation of Iodinated Precursors 31(R), 31(S), and 33^a.

^aReagents and conditions: (a) 11(R) or 12(S), DIPEA, CH₃CN, 80 °C, sealed vessel, 16 h, 92%; (b) DABAL-Me₃, Pd₂(dba)₃, X-Phos, THF, rt to 80 °C, overnight, 42%; (c) NIS,CH₂Cl₂, AcOH, 0 °C to rt 85%; (d) 11(R), DIPEA, CH₃CN, 80 °C, sealed vessel, 16 h, quant.

Alternative starting material 33 bearing chlorine in position 6 and iodine in position 3 was prepared from derivative 32²² using same conditions as in the preparation of 29(R).

The southern position was modified by either Suzuki or Stille coupling. Method A utilizing appropriate boronic acids was used for preparation of compounds 3, 34–42, and 46–48. In contrast, the compounds 43–45 were obtained by cross-coupling reaction with stannane derivatives (Scheme 6). The Suzuki reaction was also applied to preparation of the chloro derivative 49 (Scheme 7).

Sheme 6. Modifications of Southern Part of the Molecule by Means of Cross-Coupling Reactions^a.

^aReagents and conditions. Method A: Ar-B(OH)₂, Na₂CO₃, Pd(dppf)Cl₂, dioxane/H₂O (4:1), 95 °C, 18 h. Method B: Ar-SnBu₃, Pd(PPh₃)₄, DMF, 100 °C, 16 h.

Scheme 7. Preparation of 49 from 33 by Suzuki Cross Coupling^a.

^aReagents and conditions: (a) 3,4-dimethoxyphenylboronic acid, Na₂CO₃, Pd(dppf)Cl₂, dioxane/H₂O (4:1), 95 °C, 18 h, 83%.

Synthesis of the Compounds with Modified Northern Part.

Compound 4, which can be regarded as the closest modification of the northern part of the molecule, was prepared in the same way as the main hit 3 using the pyrrolidine 31(S) with the opposite chiral center (see above). The crucial intermediate for synthesis of most of the derivatives with modified northern moiety was obtained by removing the acetyl group from compound 3 under acidic conditions. Four different modifications have been selected, acyl derivatives (51–57), ureas (58 and 59), carbamates (1 and 60–63), and sulfonyl derivatives (65–76), which have been easily accessible by reaction of hydrochloride 50 with various acyl chlorides (or HATU coupling agent and carboxylic acids), isocyanates, carbamates, and sulfonyl chlorides, respectively, under the basic conditions. Compounds 61–63 were prepared by a slightly modified method. First, p-nitrophenylcarbamate was prepared, and this intermediate was immediately treated with appropriate alcohol or phenol (Scheme 8). The opposite enantiomer 1(S) was prepared in the same manner as the lead compound 1 (PDDC). In brief, this reaction sequence started from 31(S) by the Suzuki cross-coupling reaction, which afforded compound 4, followed by removal of acetyl group and subsequent reaction with phenyl chloroformate to give the desired compound 1(S) (Table 1).

Scheme 8. Preparation of Various Derivatives 51–84^a.

^aReagents and conditions: (a) aq HCl, reflux. (b) Method A: R-Cl, Et₃N, CH₂Cl₂. Method B: R-Cl, DIPEA, HATU, DMF, rt. Method C: iPrNCO, Et₃N, CH₃CN, rt. Method D: PhNCO, Et₃N, CH₂Cl₂, rt. Method E: R-Cl, Et₃N, DMAP, CH₂Cl₂, 0 °C to rt. Method F: (1) p-nitrophenyl chloroformate, Et₃N, DMAP, CH₃CN, 0 °C; (2) cyclohexanol (for compound 61), 3-methylphenol (for compound 62), 2-naphthol (for compound 63), Et₃N, 85 °C. Method G: R-Cl, Et₃N, DMAP, CH₂Cl₂, rt. Method H: chlorosulfonyl isocyanate, tert-butanol, Et₃N, CH₂Cl₂, rt. (c) Amine (R-H), DIPEA, CH₃CN, 85 °C; (d) 3,4-dimethoxyphenylboronic acid, Na₂CO₃, Pd(dppf)Cl₂, dioxane/H₂O (4:1), 95 °C, 18 h; (e) AlMe₃, DABCO, X-Phos, Pd₂(dba)₃, THF, 75 °C, 16 h.

Table 1.

Inhibitory Activity of the Compounds against nSMase2 and Stability of Selected Compounds in Mice and Human Microsomes^a

Compnd	Central core	nSMase2 IC50 (μM)	Mouse Liver Microsome (+NADPH)	Human Liver Microsome (+NADPH)	Compnd	R	nSMase2 IC50 (μM)	Mouse Liver Microsome (+NADPH)	Human Liver Microsome (+NADPH)
			% Remaining at 60 min	% Remaining at 60 min				% Remaining at 60 min	% Remaining at 60 min
GW4869		20			30(R)	H	7.5±0.6
3		1.3±0.3			34		1.5±0.1
6		>100	104 ± 2	Not Available	35		1.4±0.3
7		>100			36		0.7±0.02
8		>100			37		2.5±0.04
9		>100			38		2.0±0.2
					39		0.6±0.04
					40		0.2±0.01	32 ± 1	84 ± 1
					41		0.5±0.06
					42		0.5±0.08
					43		0.3±0.04	28 ± 2	50 ± 4
					44		0.9±0.08
					45		1.2±0.05
					46		0.8±0.07
					47		0.2±0.01	72 ± 6	114 ± 4
					48		0.7±0.08
Compnd	R	nSMase2 IC50 (μM)	Mouse Liver Microsome (+NADPH)	Human Liver Microsome (+NADPH)	Compnd	R	nSMase2 IC50 (μM)	Mouse Liver Microsome (±NADPH)	Human Liver Microsome (±NADPH)
			% Remaining at 60 min	% Remaining at 60 min				% Remaining at 60 min	% Remaining at 60 min
1(PDDC)21		0.3±0.02	63 ± 6	103 ± 14	65		0.3±0.04
1(S)		0.3±0.02			66		0.1±0.01	95 ± 1	111 ± 3
50		2.0±0.1			67		0.5±0.07	20 ± 1	43 ± 6
51		0.6±0.06			68		0.09±0.01	2 ± 1	29 ± 1
52		0.9±0.03	117 ± 8	113 ± 8
53		0.1±0.01	52 ± 1	88 ± 1	69		5.0±0.7
54		0.4±0.08			70		0.4±0.02	7 ± 2	32 ± 6
55		0.2±0.03			71		0.2±0.02	15 ± 5	61 ± 3
56		0.1±0.03	6 ± 1	20 ± 2	72		0.2±0.02	5 ± 1	12 ± 3
57		0.4±0.07			73		0.1±0.01	78 ± 2	95 ± 1
58		0.8±0.1			74		0.2±0.02
59		0.2±0.02	87 ± 1	90 ± 1	75		0.3±0.01
60		0.3±0.06			76c		0.07±0.01	1 ± 0	58±4
61		0.3±0.06			77c		8.4±0.3
62		0.2±0.03			78c		5.3±0.6
63		0.3±0.01			79c		1.1±0.2
					80c		0.7±0.06
64		1.2±0.2			81c		1.0±0.07
					82c		0.6±0.02
Compnd	R	nSMase2 IC50 (μM)	Mouse Liver Microsome (+NADPH)	Human Liver Microsome (+NADPH)	Compnd	R1	R2	nSMase2 IC50 (μM)	Mouse Liver Microsome (+NADPH)	Human Liver Microsome (+NADPH)
			% Remaining at 60 min	% Remaining at 60 min					% Remaining at 60 min	% Remaining at 60 min
83c		0.5±0.04			49	Cl	CH3	>100
					93	H	CH3	7.2±0.5
84c		0.9±0.07			98	CH3	H	0.9±0.1	53 ± 6	28 ± 5
					99	H	H	5.7±1.2	68 ± 13	92 ± 14
85		0.3±0.07	8 ± 3	11 ± 0
86		0.7±0.03
87		0.3±0.04
88		0.7±0.06
89		0.4±0.02
90		2.1±0.2
91		2.4±0.3
92		11.3±0.4

^aNegative controls without NADPH > 95% remaining except 87 which was unstable in the absence of NADPH as well.

We have also tried to substitute the 3-aminopyrrolidine motive by various other cyclic and acyclic amines as shown in Scheme 8. We focused mostly on pyrrolidine analogues due to the extensive activity of compound 76c in our preliminary experiments. The synthesis started by substitution of chlorine atom in position 8 under S_NAr conditions (DIPEA, CH₃CN, or ethanol). Dimethoxyphenyl substituent was then introduced to position 3 by Suzuki coupling, which was followed by methylation using trimethylaluminum–DABCO complex as a methylation reagent.

Further modifications on pyrrolidine ring were achieved under Mitsunobu conditions applied on the hydroxy derivative 83 (Scheme 9). We prepared S-acetyl derivative 85 and azido compound 86 using this methodology. Huisgen cycloaddition of the azido compound 86 with phenylacetylene afforded triazolo derivative 87 under the standard conditions (copper sulfate/sodium ascorbate). Morpholino derivative 88 was prepared by closing the morpholino ring on the amino group of 50 using bis 2,2′-dichloroethyl ether. Acetylation (Ac₂O) of the hydroxy group in compound 82 yielded O-acetyl derivative 89.

Scheme 9. Preparation of Further Derivatives 85–92 with Modifications in Northern Part of the Molecule^a.

^aReagents and conditions: (a) thioacetic acid, DIAD, PPh₃, THF, 0 °C to rt 27%; (b) (PhO)₂PON₃, DIAD, PPh₃, THF, 0 °C to rt, 79%; (c) phenylacetylene, CuSO₄·5H₂O, sodium ascorbate, THF–H₂O, 69%; (d) bischlorodiethyl ether, NaI, DIPEA, DMF, 110 °C, 31%; (e) Ac₂O, Et₃N, DMAP, CH₂Cl₂, rt, 89%; (f) (1) TFA, CH₂Cl₂, rt; (2) cyclohexanecarbonyl chloride (for compound 91) or Ac₂O (for compound 92), Et₃N, CH₂Cl₂, rt.

We prepared two more piperidine-based derivatives starting from Boc-protected analogue 90, which was converted to cyclohexanoyl and acetyl derivatives 91 and 92, respectively.

Finally, we have tried to remove, successively, the methyl substituents on the central core. Compound 93 was obtained by catalytic hydrogenation of the compound 49. Compound 98 with no substituent in position 2 was prepared by following the synthetic route described in Scheme 10. First, chlorine in position 8 in compound 95 (prepared by condensation of 94 with aq chloroacetaldehyde) was replaced by (R)-N-(pyrrolidin-3-yl)acetamide and the obtained substrate 96 was then converted to the chloro intermediate 97 by Suzuki coupling. Position 6 was then methylated with trimethylaluminum–DABCO complex in a similar fashion as previously. Finally, the derivative 99 was obtained by hydrogenation of compound 97.

Scheme 10. Modification of the Central Core Substitutions^a.

^aReagents and conditions: (a) H₂, Pd/C, MeOH/DMF rt; (b) (1) aq chloroacetaldehyde, iPrOH, 80 °C, (2) NBS, CHCl₃, 75 °C; (c) 11(R), DIPEA, CH₃CN, 85 °C; (d) 3,4-dimethoxyphenylboronic acid, Na₂CO₃, Pd(dppf)Cl₂, dioxane/H₂O (4:1), 95 °C; (e) DABAL-Me₃, Pd₂(dba)₃, X-Phos, THF, rt to 80 °C.

Novel Inhibitors of Human nSMase2.

We used the fluorescence-based assay described previously to characterize the potency of this series of inhibitors in vitro.²⁶ The inhibitory potencies of the compounds ranged from completely inactive derivatives (e.g., derivative 6) to highly active inhibitors with nanomolar potencies (e.g., compound 66) (Table 1).

Further characterization of the selected derivatives was performed using in vitro metabolic stability assays in both mice and human microsomes using the assays previously described.²⁷ These data are summarized in Table 1.

1 (PDDC) Pharmacokinetics in 5XFAD Mice.

The pharmacokinetic profile of 1 (PDDC) was previously reported by our group in normal mice.²¹ These studies were repeated in 5XFAD mice harboring presenilin and amyloid mutations related to familial Alzheimer’s disease (FAD), and it was confirmed that there are no differences in the pharmacokinetics in 5XFAD mice versus normal mice. Moreover, the studies were conducted using the intraperitoneal (ip) route of administration to ensure sufficient brain exposures were achieved for efficacy studies. Aged 11-month hemizygous 5XFAD mice received an ip dose of 10 mg/kg 1 (PDDC) and were sacrificed at 0.25, 0.5, 1, 3, and 6 h postdose (Figure 2A). 1 (PDDC) exhibited excellent pharmacokinetics, with the plasma C_max and AUC_0–t of 7.70 ± 0.75 nmol/mL and 22.1 ± 1.33 h·nmol/mL, respectively, whereas the brain C_max and AUC_0–t were 3.89 ± 0.75 and 13.0 ± 0.94 h·nmol/mL, respectively. The AUC_brain:AUC_plasma ratio of 0.6 showed modest brain penetration (Figure 2B) and is similar to that described in wild type mice.

Figure 2.

(A) 11-month old 5XFAD mice were administered a terminal intraperitoneal dose of 10 mg/kg 1 (PDDC) and sacrificed at 0.25, 0.5, 1, 3, and 6 h postdose. (B) Pharmacokinetic parameters following ip administration at 10 mg/kg.

1 (PDDC) Effect on Weight Changes and Fear Conditioning in 5XFAD Mice.

We evaluated body weight and fear conditioning in hemizygous 5XFAD mice and littermate wild type (WT) mice that received daily vehicle or 1 (PDDC) (10 mg/kg ip daily for 5 months). WT mice gained approximately 20% body weight throughout the study. As compared to WT mice, 5XFAD mice treated with vehicle (two-way ANOVA P < 0.001) or 1 (PDDC) (two-way ANOVA P < 0.05) gained significantly less body weight, but 5XFAD mice treated with 1 (PDDC) gained significantly more weight than 5XFAD + vehicle mice (two-way ANOVA P < 0.05, Figure 3).

Figure 3.

Effect of 1 (PDDC) on weight changes in AD mice. WT and 5XFAD mice were administered daily ip vehicle or 10 mg/kg 1 (PDDC) from age 3 months (study day 0) to 8 months (study day 150). WT mice gained significantly more weight than both 5XFAD cohorts of mice. 5XFAD mice treated with 1 (PDDC) gained more weight than 5XFAD treated with vehicle. Significantly different from WT + vehicle at P < 0.05 (*), P < 0.01 (**), P < 0.001 (***); significantly different from 5XFAD + vehicle at P < 0.05 (+), P < 0.01 (++), P < 0.001 (+++), n = 13−16.

Fear conditioning tests conducted at 8 months of age demonstrated equivalent freezing behaviors between groups on the first day of testing, when mice are trained to associate the conditioned stimulus (CS) tone with the unconditioned stimulus (US) footshock (Figure 4A). On the second day of testing, significant impairments in contextual fear memory were measured in 5XFAD mice versus WT mice (P < 0.01, Figure 4B). 5XFAD mice treated daily for 5 months with 1 (PDDC), however, performed like WT mice and froze at significantly higher levels than 5XFAD + vehicle mice (P < 0.05), indicating improvements in memory. Cued fear memory measured by freezing behavior in response to the CS was measured on the third day of testing (Figure 4C). Trend reductions in freezing behavior were observed in 5XFAD mice treated with vehicle versus WT mice during both the acclimation period and the CS presentation. 5XFAD mice treated with 1 (PDDC) froze significantly more than 5XFAD + vehicle mice during CS presentation (P < 0.05), demonstrating improved fear memory. Male and female mice were utilized, and two-way ANOVA to measure the effects of sex on test performance revealed no within-group differences between males and females, with the exception of the 5XFAD + vehicle mice on the 100–200 s bin on the second day of testing. Although both male and female 5XFAD + vehicle mice displayed significantly worse contextual memory versus WT + vehicle controls (P < 0.01), male 5XFAD + vehicle mice experienced a more significant decrease in memory performance versus female counterparts (P < 0.01).

Figure 4.

Fear conditioning. At 8 months of age and following 5 months of daily vehicle or 1 (PDDC) treatment, WT and 5XFAD mice underwent fear conditioning tests. On day 1 (A), mice were trained to associate the conditioned stimulus (CS) tone to the unconditioned stimulus (US) 0.5 mA footshock. On day 2 (B), 5XFAD + vehicle mice had significantly lower contextual freezing rates as compared to WT + vehicle mice, while 5XFAD + 1 (PDDC) mice performed like WT controls. On day 3 (C), 5XFAD + 1 (PDDC) mice exhibited significantly higher freezing rates during CS presentation versus 5XFAD + vehicle mice. Significantly different from WT + vehicle at P < 0.01 (**), P < 0.001 (***); significantly different from 5XFAD + vehicle at P < 0.05 (+), P < 0.01 (++); n = 13–16.

DISCUSSION

The hydrolase nSMase2 is a potential therapeutic target for the treatment of neurodegenerative diseases associated with release of extracellular vesicles from cells. One major obstacle in the validation of nSMase2 as a pharmacological target has been the lack of drug-like inhibitors. Prototype inhibitors exhibiting IC₅₀ > 1 μM (GW4869 and cambinol), are insoluble (GW4869), have molecular weight of >500 (GW4869), or exhibit poor metabolic stability (cambinol).^26,28 Moreover, neither GW4869 nor cambinol has been amenable to improvement through structural modifications.²⁶

Through high throughput screening and subsequent hit optimization we have recently identified PDDC, a selective, noncompetitive nSMase2 inhibitor with IC₅₀ of 0.3 μM. PDDC is metabolically stable, orally available, penetrates the brain, and was shown to be an inhibitor of extracellular vesicle release from astrocytes and from brain in an acute model of inflammation.²¹ However, the compound does not exert a significant inhibition of the catalytic domain of nSMase2 expressed in E. coli (data not shown), which might be attributed to the transmembrane character of the protein. In the present study we advance our work with the PDDC chemical series in two significant ways. First, we carried out a systematic, extensive SAR to improve PDDC’s inhibitory potency while maintaining metabolic stability, and second, using PDDC as representative of the chemical series, we demonstrate it is efficacious in a chronic model of AD.

SAR Studies Identified nSMAse2 Inhibitors That Are 3- to 4-Fold More Potent than PDDC.

We found that the central core of PDDC must be conserved to maintain activity. Apart from compounds based on imidazo[1,2-b]pyridazine scaffold, none of the alternative central cores examined exerted significant activity against nSMase2. Therefore, subsequent structural modifications of the northern and southern moieties retained the imidazo[1,2-b]pyridazine moiety.

Our data show that the southern moiety plays a critical role in inhibitory activity. Complete removal of the southern moiety led to a significant drop of activity by almost 1 order of magnitude (compound 30(R) IC₅₀ = 7.5 μM). The activity of compounds bearing an aromatic moiety at this position ranged from 0.2 μM (e.g., compound 47) to 2.5 μM (compound 37). The most active derivatives contained the phenyl group substituted at the para position such as compounds 40 and 47 with methylthio or trifluoromethoxy group, respectively.

We also investigated the impact of substituents in the northern part of the structure. Among the derivatives bearing amide or urea moiety attached to the pyrrolidine, compounds 53, 55, 56, and 59 exhibited IC₅₀ < 0.3 μM. In contrast, all the carbamate derivatives (1 and 60–63) stayed at the 0.3 μM inhibitory potency level. Moreover, a number of sulfonamides derived from the amine derivative 50 showed improvement of inhibitory activity against nSMase2. Surprisingly, complete removal of pyrrolidine substituent led to the most active compound in the series: compound 76c. Further expansion of the ring using morpholine instead of pyrrolidine ring led to a significant drop of activity by 2 orders of magnitude (compound 77c). Any further substitution on the pyrrolidine ring at position 3 did not lead to higher inhibitory activity except compounds 85 and 87 displaying the 0.3 μM level of activity. In addition to inhibitory activity, the substituent on the pyrrolidine ring seems to regulate selectivity. We routinely carry out a counterscreening assay that utilizes the coupling enzymes including alkaline phosphatase, choline oxidase, and horseradish peroxidase without nSMase2. Alkaline phosphatase, being a phosphodiesterase like nSMase2, provides initial selectivity data within the phosphodiesterase family of enzymes (Table S2). Thus, we can tell, for example, compound 1 is over 300-fold more selective for nSMase2 vs alkaline phosphatase than compound 60 which exhibits similar potency but is 60-fold more selective. Finally, removal of methyl substituent from the central core led either to the comparable inhibitory potency as in the hit compounds, as in case of derivative 98, or to a significant drop of activity, compounds 93 and 99. SAR results are summarized in Figure 5.

Figure 5.

SAR derived from this series of nSMase2 inhibitors.

In summary, the aromatic central core is crucial to observe nSMase2 inhibition while a wide range of substitutions on the northern and southern moieties are tolerated. PDDC is a noncompetitive inhibitor with respect to sphingomyelin²¹ while its potency of inhibition is reduced with increasing concentrations of phosphatidyl serine, suggesting it binds to one of two hydrophobic segments on the N-terminus of the protein (data not shown). Even though the crystal structure of nSMase2 containing these segments is not available, the SAR evidence suggests the aromatic central core defines the major discriminating interaction with the enzyme; this interaction can be further enhanced or diminished through electron donating/withdrawing effects or steric substituents on the northern and southern moieties.

New Potent nSMase2 Inhibitors Are Metabolically Stable.

Further characterization of the potent inhibitors from each SAR category was performed using stability studies in mice and human microsomes. Among the analogues with modified southern moiety only 47 bearing a 4-trifluoromethoxyphenyl exerted reasonable metabolic stability. Compounds modified in the northern moiety, e.g., 52, 53, 59, PDDC, 66, and 73, were also generally stable. Metabolite identification (MET-ID) of PDDC in both human and mouse liver microsomes (Figure S1) confirmed the formation of O-demethylated metabolite (m/z = 474.2135), albeit with a low abundance, suggesting O-dealkylation as a minor metabolic pathway. One of the most active compounds 76c, containing the unsubstituted pyrrolidine, was metabolically unstable. However, sulfonamide derivatives at the northern moiety (66 and 73) were almost 3-fold more potent than 1 (PDDC) and also completely stable in both mice and human microsomes. These compounds are being evaluated in in vivo pharmacokinetic and efficacy studies.

PDDC Reversed Cognitive Impairment in 5XFAD Mice.

Following confirmation of PDDC brain penetration in 5XFAD mice (AUC_brain/AUC_plasma = 0.6), in vivo efficacy studies were initiated. Because nSMase2 has been implicated in the spreading of tau and amyloid β pathology in AD,²⁹ we wanted to determine if long-term PDDC therapy could successfully treat cognitive impairment in AD mice. Cognitive deficits are well documented in the 5XFAD mouse model of AD, and the fear conditioning test demonstrates reproducible impairments in this strain beginning at 5–6 months of age.^30,31 Knockdown of nSMase2 in the 5XFAD model improves both pathology and cognition as measured by performance on the fear conditioning test.⁸ Previous studies have also reported that this model and another model of AD are sensitive to nSMase2 inhibition via GW4869 using pathological end points.^6,7 Ours is the first study, however, to measure the effects of nSMase2 inhibition on cognitive outcomes. A limitation of the present study is a lack of direct comparison of the in vivo efficacy of the previously utilized GW4869 and PDDC in the present study, and future studies that include both behavioral and pathological outcomes will include this control.

Both male and female mice were utilized in the present 5XFAD cognition study. While other laboratories have reported that male 5XFAD mice experience more significant behavioral deficits versus female counterparts,⁸ we only observed significant within-group differences in performance due to sex at one time point of fear conditioning testing, the 100–200 s bin of the contextual test, in the 5XFAD + vehicle mice. Although no within-group sex differences were observed at any other time point of testing for 5XFAD + vehicle mice or at any time point of fear conditioning testing for either WT + vehicle or 5XFAD + PDDC mice, future directions will include increasing the sample size of groups to determine if the male sex drives more impairments in 5XFAD + vehicle mice as previously reported.⁸

Herein we report that daily dosing of PDDC in 5XFAD mice significantly reversed their impairment in contextual fear memory when tested at 8 months of age, resulting in cognitive responses like those observed in WT mice. In tandem, PDDC treatment also increased body weight in 5XFAD mice. There is clear evidence of weight loss in both human AD³² and mouse models of the disease, including 5XFAD.³³ There is no single cause for this phenomenon, but decreased food intake and higher resting energy expenditure are possible contributors.³² A recent study showed increased glucose consumption in the brains of 5XFAD mice with concurrent microglial and astrocyte activation and amyloid β accumulation.³⁴ It is reasonable to hypothesize, therefore, that metabolic demands increase in 5XFAD mice with CNS inflammation and amyloid pathology, which could contribute to the stalled weight gain of 5XFAD mice compared to WT controls observed here and in other studies. Therefore, if PDDC impacts microglial or astrocyte activation and/or amyloid deposition, body weight would be positively impacted as observed here following PDDC treatment in 5XFAD mice. Future pathology studies are planned to explore this hypothesis, with a focus on the amygdala based on the present behavioral findings in the fear conditioning test. Future studies to measure the effects of PDDC treatment on glial and amyloid pathology will also take into account the effects of male or female sex, as previous studies have observed more severe phenotypes in male 5XFAD mice⁸ and a more pronounced amelioration of amyloid deposition due to the nSMase2 inhibitor GW4869 in males versus females.⁷

Even though head-to-head comparisons of compounds in vivo are hard to make, positive efficacy results using PDDC in the 5XFAD model are consonant with a previous study, in another laboratory, also using the 5XFAD model and another nSMase2 inhibitor GW4869 where reduction of amyloid plaque formation was observed.⁷ Though GW4869 has been used as tool compound in vivo, it has extremely poor solubility in both aqueous and organic solvents, has low potency (IC₅₀ = 1 μM), and has never been characterized in the brain to show actual target engagement of nSMase2 following systemic dosing. We have also recently identified DPTIP, a more potent nSMase2 inhibitor (IC₅₀ = 30 nM) from a different chemical series.³⁵ We did not conduct direct comparison in vivo with DPTIP because this compound is not orally bioavailable and shows poor brain penetration vs PDDC. In short, the present study reports on a novel class of structurally distinct, orally bioavailable nSMase2 inhibitors that could be clinically developed for AD.

CONCLUSION

We report on a systematic and extensive SAR study of a novel series of nSMase2 inhibitors initially identified by high-throughput screening. Our SAR study provided 70 novel analogues and led to numerous derivatives with submicromolar nSMase2 inhibitory activity. On the basis of potency and microsomal stability, we identified several promising candidates for further development. Using PDDC as a representative tool compound of this series, we explored its efficacy in a chronic mouse model of AD. We report significant improvement of cognition in 5XFAD mice following 5 months of daily PDDC treatment. These results pave the way toward the development of a novel treatment for this debilitating neurodegenerative disorder.

EXPERIMENTAL SECTION

General Instrumental Methods and Chemical Procedures.

NMR spectra (δ, ppm; J, Hz) were recorded on a Bruker Avance III-400 instrument (400.0 MHz for ¹H and 101 MHz for ¹³C) using inverse broadband probe with ATM module (5 mm BBO-¹H Z-GRD) or Bruker Avance III-400 instrument with broadband PRODIGY cryoprobe with ATM module (5 mm CPBBO BB-¹H/¹⁹F/D Z-GRD). The NMR experiments were performed in DMSO-d₆ or CDCl₃ and referenced to the solvent signal (δ 2.50 and 39.70, respectively, 7.26 and 77.16). Mass spectra were measured on a LTQ Orbitrap XL (Thermo Fisher Scientific) using electrospray ionization (ESI). Column chromatography was performed on silica gel 60 (Fluka) and thin-layer chromatography (TLC) on silica gel 60 F254 foils (Merck). Solvents were evaporated at 2 kPa and bath temperature of 30–60 °C; the compounds were dried at 13 Pa and 50 °C. For all the tested compounds satisfactory elemental analysis was obtained supporting >95% purity as also visible in the NMR spectra. Optical rotation was measured on polarimeter Autopol IV (Rudolph Research Analytical) at 589 nm wavelength in chloroform or DMSO. UPLC samples were measured on Waters UPLC H-Class Core System (column Waters Acquity UPLC BEH C18 1.7 μm, 2.1 mm × 100 mm), Waters Acquity UPLC PDA detector, mass spectrometer Waters SQD2, and MassLynx mass spectrometry software. For reverse-phase flash column chromatography, C-18 RediSep Rf columns (Teledyne ISCO) were used.

(R)-N-(1-(9-(3,4-Dimethoxyphenyl)-2,8-dimethyl-9H-purin-6-yl)pyrrolidin-3-yl)acetamide (6).

To a mixture of 6-chloro-9-(3,4-dimethoxyphenyl)-2,8-dimethyl-9H-purine³⁶ 10 (100 mg, 0.31 mmol) and DIPEA (89 μL, 0.51 mmol) in ethanol (3 mL) was added tert-butyl (3R)-pyrrolidin-3-ylcarbamate (80 mg, 0.42 mmol), and the reaction mixture was heated at 80 °C for 16 h, cooled down, and the solvent was evaporated. The residue was chromatographed on silica gel column (100 g, ethyl acetate–ethanol 10:1). Fractions containing the product were evaporated and dissolved in a mixture of trifluoroacetic acid and dichloromethane (6 mL, 1:5, v/v). Reaction mixture was stirred at rt for 2 h, evaporated, twice coevaporated with acetonitrile (2 × 10 mL), and redissolved in acetonitrile (7 mL). Et₃N (0.2 mL, 1.43 mmol), catalytic amount of DMAP, and acetic anhydride (50 μL, 0.53 mmol) were sequentially added to this solution. Reaction mixture was stirred at rt for 2 h, and then methanol (1 mL) was added and the mixture was evaporated. Residue was purified by column chromatography (50 g, ethyl acetate–acetone–ethanol–water 18:3:2.5:1.5) to obtain 112 mg (88%) of the product. The obtained solid was recrystallized from ethyl acetate. [α]_D²⁰ –15.0 (c 0.306, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.15 (d, J = 6.5 Hz, 1H, NHCO), 7.12 (d, J = 8.5 Hz, 1H, H-5″), 7.04 (d, J = 2.4 Hz, 1H, H-2″), 6.94 (dd, J = 8.5, 2.4 Hz, 1H, H-6″), 4.32 (br s, 1H, H-2′), 4.26 – 3.57 (br s, 4H, H-1′, 4′), 3.84 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 2.34 (s, 3H, CH₃), 2.33 (s, 3H, CH₃), 2.15 (br s, 1H, H-3′a), 1.88 (br s, 1 H, H-3′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 160.5 (C-2), 153.0 (C-4), 151.8 (C-6), 149.2 and 149.1 (C-4″, C-3″), 147.5 (C-8), 127.4 (C-1″), 120.0 (C-6″), 116.5 (C-5), 112.0 (C-5″), 111.7 (C-2″), 56.0 and 55.9 (2 × OCH₃), 48.8* (C-2′), 30.0* (C-3′), 25.9 (CH₃), 22.8 (COCH₃), 14.4 (CH₃) (two CH₂ groups from pyrrolidine were not detected due to the signal broadening caused by the slow exchange). HRMS calcd for C₂₁H₂₇N₆O₃ m/z: 411.2139 (M + H)⁺, found 411.2138.

2,4-Dibromo-3-methoxy-6-methylpyridine (14).

To a solution of compound 13³⁷ (5.34 g, 20 mmol) in acetone (195 mL) were sequentially added methyl iodide (1.87 mL, 30 mmol) and potassium carbonate (4.13 g, 30 mmol) at rt. The reaction mixture was stirred for 16 h and then diluted with ethyl acetate (150 mL), and the suspension was filtrated. The obtained filtrate was evaporated, and the residue was chromatographed on silica gel column (200 g, cyclohexane–ethyl acetate 20:1). An amount of 4.049 g (72%) of the product was obtained as a colorless oil which solidified upon standing. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.66 (d, J = 0.5 Hz, 1H), 3.81 (s, 3H), 2.41 (d, J = 0.5 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 155.7, 148.8, 136.0, 127.8, 60.9, 22.6. UPLC–MS: t = 4.55 (M + H, 280/282/284).

4-Bromo-3-methoxy-6-methyl-2-(prop-1-yn-1-yl)pyridine (15).

Suspension of starting material 14 (1.94 g, 6.92 mmol), CuI (66 mg, 0.34 mmol), Pd(PPh₃)₂Cl₂ (124 mg, 0.17 mmol) in toluene (21 mL), triethylamine (4.92 mL, 34.6 mmol), and TBAF (1 M in THF, 10.3 mL) was degassed at 0 °C for 15 min, and then the flask was filled with argon. To this mixture trimethylsilylpropyne (1.43 mL, 7.61 mL) was added and the reaction mixture was stirred for 48 h, then diluted with brine (75 mL) and extracted with ethyl acetate (200 mL). The organic phase was dried over Na₂SO₄ and evaporated. The residue was purified by silica gel column chromatography (250 g, petroleum ether–ethyl acetate 10:1) which furnished compound 15 (1.17 g, 70%) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.57 (s, 1H), 3.84 (s, 3H), 2.37 (s, 3H), 2.13 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 154.77, 152.8, 137.3, 127.1, 126.8, 92.0, 76.1, 61.0, 40.1, 23.0, 4.1. UPLC–MS: t = 3.58 (M + H, 239.7/242.4).

7-Bromo-3-iodo-2,5-dimethylfuro[3,2-b]pyridine (16).

To a solution of derivative 15 (1.16 g, 4.8 mmol) in dry dichloromethane (20 mL) was added a solution of ICl in dichloromethane (1 M, 10 mL) dropwise during 5 min under argon atmosphere, and the reaction mixture was stirred for 24 h at rt. After that, the second portion of ICl (1 M, 3 mL) was added and stirring continued for another 17 h. Reaction was poured onto a saturated solution of Na₂S₂O₃ (200 mL), and then the water phase was extracted with ethyl acetate (300 mL). The organic phase was dried over Na₂SO₄ and evaporated. The residue was purified by silica gel column chromatography (200 g, toluene–ethyl acetate 30:1). An amount of 1.5 g (88%) of the product 16 was obtained as a brownish solid. After several attempts to further purify this compound it was not possible to obtain pure ¹H NMR. The compound was then immediately used in the next synthetic step. UPLC–MS: t = 5.04 (M + H, 352.1/354.1).

7-Bromo-3-(3,4-dimethoxyphenyl)-2,5-dimethylfuro[3,2-b]pyridine (17).

A suspension of starting material 16 (700 mg, 1.99 mmol), 3,4-dimethoxyphenylboronic acid (434 mg, 2.39 mmol), sodium carbonate (317 mg, 2.99 mmol) in dioxane–water (35 mL, 4:1) was three times purged with argon. Then Pd(dppf)Cl₂ (162 mg, 0.2 mmol) was added, and again the flask was purged with argon. Reaction mixture was then heated to 95 °C (bath) overnight, cooled down, and diluted with brine (100 mL). Water phase was extracted with ethyl acetate (3 × 150 mL). Combined organic phases were dried over Na₂SO₄ and evaporated. Residue was chromatographed on silica gel column (200 g, petroleum ether–ethyl acetate 10:1) to obtain 309 mg (43%) of the compound 17. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.49 (s, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.27 (dd, J = 8.3, 2.0 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 3.81 (2s, 6H), 2.65 (s, 3H), 2.55 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 156.4, 155.2, 148.8, 148.4, 147.2, 143.0, 123.0, 121.6, 121.2, 116.7, 112.9, 112.5, 112.2, 55.8, 23.9, 14.0. UPLC–MS: t = 5.21 (M + H, 362.2/364.2).

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,5-dimethylfuro[3,2-b]pyridin-7-yl)pyrrolidin-3-yl)acetamide (7).

A solution of bromo derivative 17 (100 mg, 0.28 mmol), (R)-N-(pyrrolidin-3-yl)acetamide 11(R) (53 mg, 0.42 mmol), DIPEA (0.1 mL, 0.56 mmol) in n-butanol (2 mL) was heated at 130 °C for 30 h, then cooled down and evaporated. Residue was purified by column chromatography on silica gel (50 g, ethyl acetate → ethyl acetate–acetone–ethanol–water 17:3:3:2) to yield 92 mg (80%) of the product 7. The sample for analysis was obtained by crystallization from acetone. [α]_D²⁰ +6.5 (c 0.245, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.18 (d, J = 6.8 Hz, 1H, NH), 7.42 (d, J = 2.0 Hz, 1H, H-2″), 7.27 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.06 (d, J = 8.4 Hz, 1H, H-5″), 6.21 (s, 1H, H-6), 4.48–4.27 (m, 1H, H-3′), 3.85 (dd, J = 10.5, 6.2 Hz, 1H, H-2′a), 3.80 (s, 6H, OCH₃), 3.75–3.69 (m, 1H, H-5′a), 3.63 (td, J = 9.6, 8.8, 5.0 Hz, 1H, H-5′b), 3.49 (dd, J = 10.5, 4.1 Hz, 1H, H-2′b), 2.56 (s, 3H, 2-CH₃), 2.38 (s, 3H, 6-CH₃), 2.27–2.09 (m, 1H, H-4′a), 2.00–1.86 (m, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (CO), 154.3 (C-8), 152.4 (C-2), 148.6 and 147.9 (C-3″, C-4″), 146.5 (C-5), 138.4 (C-4), 134.3 (C-7), 124.5 (C-1″), 121.4 (C-6″), 115.8 (C-3), 113.1 (C-2″), 112.1 (C-5″), 102.0 (C-6), 55.8 and 55.7 (3″-OCH₃, 4″-OCH₃), 54.2 (C-2′), 48.7 (C-3′), 46.9 (C-5′), 30.7 (C-4′), 24.6 (2-CH₃), 22.8 (COCH₃), 14.00 (6-CH₃). HRMS calcd for C₂₃H₂₈O₄N₃ (M + H)⁺ 410.2074; found, 410.2069.

7-Bromo-2,4-dimethoxy-6-methylthieno[3,2-d]pyrimidine (19).

N-Bromosuccinimide (1.13 g, 6.25 mmol) was added to a solution of 2,4-dimethoxy-6-methylthieno[3,2-d]pyrimidine 18 (1.05 g, 5 mmol) in chloroform (5 mL) and acetic acid (5 mL), and the mixture was stirred at 60 °C overnight. The reaction mixture was diluted with chloroform (50 mL) and washed with saturated solution of potassium hydrogen carbonate (2 × 40 mL), solution of sodium thiosulfate (40 mL), dried over sodium sulfate, and evaporated. The residue was purified by flash chromatography on silica gel (40 g) in 20–40% ethyl acetate in pentane. An amount of 1.07 g (74%) of bromo derivative 19 was obtained. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 4.13 (s, 3H), 4.10 (s, 3H), 2.59 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 165.1, 164.4, 160.5, 144.3, 109.4, 107.7, 55.3, 54.6, 16.4. HRMS calcd for C₈H₈O₂N₂BrS (M + H)⁺ 290.9620; found, 290.9620.

7-(3,4-Dimethoxyphenyl)-2,4-dimethoxy-6-methylthieno[3,2-d]pyrimidine (20).

A mixture of bromo derivative 19 (289 mg, 1.00 mmol), 3,4-dimethoxyboronic acid (209 mg, 1.15 mmol), 1,4-dioxane (12 mL), and aqueous 1 M potassium carbonate solution (3 mL) was degassed, tetrakis(triphenylphosphine)palladium (58 mg, 0.05 mmol) was added, and the mixture was degassed again and then heated under argon to 93 °C overnight. The mixture was diluted with ethyl acetate (60 mL), washed with water (30 mL), dried over sodium sulfate, and evaporated. Flash chromatography of the residue on silica gel (40 g) in 20–40% ethyl acetate in pentane afforded 260 mg (75%) of 20. ¹H NMR (400 MHz, CDCl₃, 25 °C) δ 7.18 (d, J = 2.0, 1H), 7.06 (dd, J = 8.2, 2.0, 1H), 6.98 (d, J = 8.2, 1H), 4.14 (s, 3H), 3.98 (s, 3H), 3.94 (s, 3H), 3.91 (s, 3H), 2.64 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 165.4, 164.0, 162.2, 148.6, 148.5, 144.8, 132.9, 126.2, 122.7, 113.7, 111.0, 110.0, 56.0, 54.9, 54.3, 16.1. HRMS calcd for C₁₇H₁₈O₄N₂NaS (M + Na)⁺ 369.0878; found, 369.0880.

7-(3,4-Dimethoxyphenyl)-6-methylthieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (21).

A mixture of 20 (260 mg, 0.75 mmol), acetic acid (3.2 mL), and sodium iodide (322 mg, 2.15 mmol) was heated at 80 °C for 2 h and, after cooling, poured into ice–water (50 g). The precipitate was filtered off, washed with water, and dried in vacuum. An amount of 271 mg (85%) of yellowish powder was obtained. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 11.16 (brs, 1H), 10.80 (brs, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.2, 2.0 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 2.35 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 158.9, 152.0, 148.9, 148.9, 145.4, 145.0, 129.1, 123.9, 122.5, 113.7, 112.1, 110.8, 55.7, 15.1. HRMS calcd for C₁₅H₁₅O₄N₂S (M + H)⁺ 319.0747; found, 319.0748.

2,4-Dichloro-7-(3,4-dimethoxyphenyl)-6-methylthieno[3,2-d]pyrimidine (22).

A mixture of 21 (159 mg, 0.5 mmol), POCl₃ (1.40 mL, 15 mmol), and N,N-dimethylaniline (82 μL, 0.65 mmol) was stirred at 110 °C for 3 h, cooled to room temperature, and poured into a mixture of potassium hydrogen carbonate solution and ice. The precipitate was filtered off, washed with water, and dried in vacuum. Flash chromatography of this crude product on silica gel (12 g) in 15–40% ethyl acetate in pentane afforded 127 mg (72%) of dichloro derivative 22. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.02–7.00 (m, 3H), 3.95 (s, 3H), 3.92 (s, 3H), 2.70 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 163.0, 16.4, 154.5, 150.2, 149.4, 149.1, 134.1, 128.0, 124.3, 122.9, 113.4, 111.4, 56.2, 56.1, 16.4. HRMS (ESI): calcd for C₁₅H₁₂O₂N₂Cl₂NaS (M + H)⁺ 376.9889; found, 376.9889.

(R)-N-{1-[2-Chloro-7-(3,4-dimethoxyphenyl)-6-methylthieno[3,2-d]pyrimidin-4-yl]pyrrolidin-3-yl}acetamide (23).

A mixture of 21 (533 mg, 1.5 mmol), (R)-N-(pyrrolidin-3-yl)acetamide (264 mg, 2.06 mmol), ethyldiisopropylamine (0.40 mL, 2.30 mmol), and acetonitrile (11 mL) was heated to 80 °C in a closed vial overnight, cooled to room temperature, and the precipitated product was filtered off and washed with acetonitrile and ether. An amount of 639 mg (95%) of 23 was obtained. ¹H NMR (400 MHz, CDCl₃, 25 °C) δ 6.96–6.93 (m, 2H), 6.92–6.91 (m, 1H), 6.75 (d, J = 7.0, 1H), 4.52–4.44 (m, 1H), 3.95–3.69 (m, 4H), 3.92 (s, 3H), 3.88 (s, 3H), 2.54 (s, 3H), 2.16–2.07 (m, 1H), 2.04–1.89 (m, 1H). ¹³C NMR (101 MHz, CDCl₃, 25 °C) δ 170.4, 160.2, 157.0, 156.4, 149.0, 149.0, 144.6, 133.2, 125.7, 123.0, 113.7, 112.4, 111.3, 56.2, 53.2, 49.1, 46.1, 23.3, 15.3. HRMS (ESI): calcd for C₂₁H₂₄O₃N₄ClS (M + H)⁺ 447.12522; found, 447.12522.

(R)-{1-[7-(3,4-Dimethoxyphenyl)-2,6-dimethylthieno[3,2-d]pyrimidin-4-yl]pyrrolidin-3-yl}acetamide (8).

Over 10 min a 2 M solution of trimethylaluminum in hexane (0.5 mL) was added to a solution of 1,4-diazabicyclo[2.2.2]octane (56 mg, 0.5 mmol) in tetrahydrofuran (2 mL), and the mixture was stirred for 30 min. Then a solution of 23 (223 mg, 0.5 mmol), Pd₂(dba)₃ (28 mg), and X-Phos (29 mg) in tetrahydrofuran (6 mL) was added. The mixture was heated to reflux overnight, cooled to 0 °C, and a saturated solution of NH₄Cl was added dropwise. The mixture was filtered through a Celite pad, the filter was washed with tetrahydrofuran, and the collected filtrates were evaporated. Chromatography of the residue on silica gel in ethyl acetate–ethanol–acetone–water (40:6:3:1) afforded 175 mg (82%) of a white crystalline compound 8. ¹H NMR (400 MHz, CDCl₃, 25 °C) δ 7.02 (d, J = 1.9 Hz, 1H, H-2″), 7.00 (dd, J = 8.2, J = 1.9 Hz, 1H, H-6″), 6.95 (d, J = 8.2 Hz, 1H, H-5″), 6.56 (brd, J = 6.6 Hz, 1H, NH), 4.57–4.51 (m, 1H, H-3′), 3.96 (dd, J = 5.8, J = 11.2, 1H, H-2′a), 3.92 (s, 3 H, 3″-OCH₃), 3.90–3.79 (m, 2H, H-5′), 3.88 (s, 3H, 4″-OCH₃), 3.72 (dd, J = 2.6, J = 11.2, 1H, H-2′b), 2.53 (s, 3H, 8-CH₃), 2.47 (s, 3H, 2-CH₃), 2.23–2.12 (m, 1H, H-4′a), 2.04–1.98 (m, 1H, H-4′b), 1.96 (s, 3H, COCH₃). ¹³C NMR (101 MHz, CDCl₃, 25 °C) δ 170.2 (CO), 163.6 (C-2), 159.6 (C-6), 155.9 (C-4), 148.8 (C-3″), 1487 (C-4″), 142.3 (C-8), 133.3 (C-9), 126.5 (C-1″), 123.0 (C-6″), 114.0 (C-2″), 111.2 (C-5″), 56.1 (3″-OCH₃), 56.0 (4″-OCH₃), 53.0 (C-2′), 49.2 (C-3′), 45.7 (C-5′), 31.4 (C-4′), 26.00 (2-CH₃), 23.3 (COCH₃), 15.3 (8-CH₃). HRMS (ESI): calcd for C₂₂H₂₇O₃N₄S (M + H)⁺ 427.1798; found, 427.1799.

7-Bromo-2,4-dichlorothieno[3,4-d]pyrimidine (25).

To a solution of compound 24 (1.025 g, 5 mmol) in dry DMF (20 mL), N-bromosuccinimide (890 mg, 5 mmol) was added in one portion at 0 °C, and the reaction mixture was stirred at room temperature for 2 h. The solution was diluted with EtOAc and H₂O (40 mL each), and the layers were separated. The aqueous layer was extracted with ethyl acetate (1 × 40 mL), and the combined organic phase was dried over Na₂SO₄ and evaporated. The residue was purified by silica gel column chromatography (hexane–ethyl acetate 6:1), yielding compound 25 (1.237g, 87%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃, 25 °C) δ (ppm): 8.31 (s, 1H). ¹³C NMR (125 MHz, CDCl₃, 25 °C) δ 159.4, 154.9, 148.5, 126.3, 124.5, 107.6.

(R)-N-[1-(7-Bromo-2-chlorothieno[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl]acetamide (26).

A mixture of 25 (284 mg, 1 mmol), (R)-N-(pyrrolidin-3-yl)acetamide (167 mg, 1.3 mmol), ethyldiisopropylamine (0.280 μL, 2.0 mmol), and acetonitrile (4 mL) was stirred at 0 °C for 30 min and at room temperature for an hour. The deposited solid was filtered off and washed with water and ethyl acetate. An amount of 326 mg (87%) of an almost white crystalline compound was obtained. ¹H NMR (400 MHz, CDCl₃, 25 °C) δ 8.76 (s, 1H), 8.22 (d, J = 6.6, 1H), 4.51–4.29 (m, 1H), 4.26–3.64 (m, 4H), 3.35 (s, 3H), 2.34–2.24 (m, 1H), 2.16–2.00 (m, 1H). ¹³C NMR (101 MHz, CDCl₃, 25 °C) δ 169.5, 157.5, 155.8, 149.4, 127.4, 119.0, 100.4, 54.8, 49.6, 47.2, 22.8. HRMS (ESI): calcd for C₁₂H₁₂ON₄BrClNaS (M + Na)⁺ 396.9496; found, 396.9497; C₁₂H₁₃ON₄BrClS (M + H)⁺ 374.9677; found, 374.9677.

(R)-N-{1-[2-Chloro-7-(3,4-dimethoxyphenyl)thieno[3,4-d]pyrimidin-4-yl]pyrrolidin-3-yl}acetamide (27).

A mixture of bromo derivative 26 (113 mg, 0.3 mmol), 3,4-dimethoxyboronic acid (57 mg, 0.31 mmol), 1,4-dioxane (3.7 mL), and aqueous 1 M potassium carbonate solution (0.92 mL) was degassed, tetrakis(triphenylphosphine)palladium (18 mg, 0.02 mmol) was added, and the mixture was degassed again and then heated under argon to 93 °C overnight. The mixture was diluted with chloroform (20 mL), washed with water (2 × 10 mL), dried over sodium sulfate, and evaporated. Chromatography of the residue on silica gel 12 g in ethyl acetate–acetone–ethanol–water (40:6:3:1) afforded 78 mg (60%) of a yellowish crystalline product. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.38 (s, 1H), 8.23 (d, J = 6.5, 1H), 7.71–7.67 (m, 2H), 7.04 (d, J = 8.4, 1H), 4.42–4.35 (m, 1H), 4.05–3.88 (m, 3H), 3.85 (s, 3H), 3.80 (s, 3H), 3.77–3.72 (m, 1H), 2.25–2.16 (m, 1H), 1.99–1.92 (m, 1H), 1.83 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.5, 156.6, 154.3, 148.7, 148.7, 135.0, 128.9, 126.8, 120.8, 115.8, 112.2, 111.8, 55.8, 55.7, 46.0, 22.8. HRMS (ESI): calcd for C₂₀H₂₁O₃N₄ClNaS (M + Na)⁺ 455.0915; found, 455.0915; C₂₀H₂₂O₃N₄ClS (M + H)⁺ 433.1096; found, 433.1095.

(R)-N-{1-[7-(3,4-Dimethoxyphenyl)-2-methylthieno[3,4-d]pyrimidin-4-yl]pyrrolidin-3-yl}acetamide (9).

Over 10 min a 2 M solution of trimethylaluminum in hexane (0.5 mL) was added to a solution of 1,4-diazabicyclo[2.2.2]octane (17 mg, 0.15 mmol) in tetrahydrofuran (2 mL), and the mixture was stirred for 30 min. Then a solution of 27 (65 mg, 0.15 mmol), Pd₂(dba)₃ (8.5 mg), and X-Phos (8.7 mg) in tetrahydrofuran (6 mL) was added, and the mixture was heated at 65 °C overnight. After cooling to 0 °C a saturated solution of NH₄Cl was added dropwise. The mixture was filtered through a Celite pad, the filter was washed with tetrahydrofuran, and collected filtrates were evaporated. Chromatography of the residue on silica gel in ethyl acetate–ethanol–acetone–water (38:6:3:1) afforded 42 mg (68%) of a yellowish crystalline compound 9. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.40 (brs, 1H, NH), 8.21 (brs, 1H, H-7), 8.03 (d, J = 2.2, 1H, H-2″), 7.59 (dd, J = 8.4, J = 2.2, 1H, H-6″), 7.02 (d, J = 8.4, 1H, H-5″), 4.46–3.67 (m, 5H, H-2′, H-3′, H-5′), 3.84, 3.82 (s, 3H each, 3″-OCH₃, 4″-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.26–1.90 (m, 2H, H-4′), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 170.0 (CO), 161.3 (C-2), 156.1 (C-6), 148.8 (C-3″), 148.2 (C-4″), 127.5 (C-9), 126.7 (C-1″), 121.1 (C-7), 120.5 (C-5), 119.8 (C-6″), 112.2 (C-2″), 111.5 (C-5″), 55.8, 55.6 (3″-OCH₃, 4″-OCH₃), 54.7 (C-2′), 26.5 (2-CH₃), 22.8 (COCH₃). HRMS (ESI): calcd for C₂₁H₂₅O₃N₄S (M + Na)⁺ 413.16419; found, 413.16436.

(R)-N-(1-(6-Chloro-2-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (29(R)).

A mixture of starting material 28 (600 mg, 2.97 mmol), (R)-N-(pyrrolidin-3-yl)acetamide 11(R) (457 mg, 3.56 mmol), and DIPEA (0.79 mL, 4.54 mmol) in acetonitrile (10 mL) was heated at 85 °C for 16 h, then cooled down, and evaporated. Residue was purified by column chromatography on silica gel (100 g, ethyl acetate → ethyl acetate–ethanol 7:1) to yield 864 mg (quantitative). The sample for analysis was obtained by crystallization from methanol. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.5 Hz, 1H), 7.75 (d, J = 0.9 Hz, 1H), 5.91 (s, 1H), 4.48–3.85 (br s + m, 3H), 3.80–3.30 (br s, 1H), 2.30 (d, J = 0.9 Hz, 3H), 2.23–2.07 (m, 1H), 1.96–1.83 (m, 1H), 1.81 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4, 146.5, 139.7, 142.0, 132.0, 114.8, 92.7, 22.7, 14.5 (the signals of the pyrrolidine ring were not detected due to signal broadening). HRMS calcd for C₁₃H₁₇ClN₅O m/z: 294.1116 (M + H)⁺, found 294.1117.

(R)-N-(1-(2,6-Dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (30(R)).

To a solution of DABCO (448 mg, 4 mmol) in 15 mL of freshly distilled tetrahydrofuran, AlMe₃ (2 M in hexanes, 4 mL, 8 mmol) was added dropwise, and the mixture was stirred at room temperature for 30 min under argon atmosphere. A solution of 29(R) (1.4 g, 4.77 mmol), Pd₂(dba)₃ (224 mg, 0.27 mmol), and X-Phos (234 mg, 0.49 mmol) in 80 mL of freshly distilled tetrahydrofuran was subsequently added to the solution, and the reaction mixture was stirred at 75 °C overnight under argon atmosphere. The mixture was cooled to 0 °C, quenched with saturated NH₄Cl (16 mL), diluted with acetone and ethyl acetate, and filtered through Celite. The Celite pad was thoroughly washed with acetone and ethyl acetate. The filtrate was evaporated and the residue was purified by silica gel column chromatography (200 g, chloroform–ethanol 10:1) yielding compound 30(R) (1.42 g, 92%) as an off-white solid. Analytical sample was obtained by crystallization from ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.16 (d, J = 6.6 Hz, 1H), 7.62 (d, J = 0.8 Hz, 1H), 5.71 (s, 1H), 4.37–4.27 (m, 1H), 4.03 (br s, 1H), 3.77 (br s, 3H), 2.28 (d, J = 0.8 Hz, 3H), 2.27 (s, 3H), 2.19–2.08 (m, 1H), 1.93–1.84 (m, 1H), 1.81 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4, 151.6, 138.7, 141.0, 132.6, 113.8, 93.6, 55.2, 48.8, 47.6, 30.5, 22.7, 21.6, 14.6. HRMS calcd for C₁₄H₂₀N₅O m/z: 274.1662 (M + H)⁺, found 274.1663.

(R)-N-(1-(3-Iodo-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (31(R)).

A solution of compound 30(R) (1.22 g, 4.5 mmol) in dichloromethane (50 mL) with acetic acid (0.19 mL) was cooled down to 0 °C, then N-iodosuccinimide (1.1 g, 4.89 mmol) was added in one portion and the reaction mixture was stirred overnight (0 °C → rt). Reaction mixture was diluted with ethyl acetate (700 mL) and washed with saturated aqueous NaHCO₃ (200 mL) and saturated aqueous Na₂S₂O₃ (200 mL). Organic phase was dried over Na₂SO₄ and evaporated. The residue was purified by silica gel column chromatography (200 g, chloroform–ethyl acetate 20:1 → 15:1) which furnished compound 31(R) (1.53 g, 85%) as an off-white solid. Recrystallization from hot acetone yielded an analytically pure sample. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.16 (d, J = 6.6 Hz, 1H), 5.84 (s, 1H), 4.38–4.27 (m, 1H), 4.01 (br s, 1H), 3.77 (br s, 3H), 2.34 (s, 3H), 2.31 (s, 3H), 2.19–2.09 (m, 1H), 1.93–1.83 (m, 1H), 1.81 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4, 152.2, 140.8, 142.6, 135.2, 94.7, 70.9, 55.3, 48.8, 30.5, 22.8, 21.5, 15.1 (one CH₂ peak was not detected). HRMS calcd for C₁₄ H₁₉IN₅O m/z: 400.0629 (M + H)⁺, found 400.0630.

(S)-N-1-(6-Chloro-3-iodo-2-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (31(S)).

This compound has been prepared according to the method described for the synthesis of compound 31(R) from compound 28. As a reagent in nucleophilic substitution (R)-N-(pyrrolidin-3-yl)acetamide 12(S) was used. The physical and spectral properties are identical to those for compound 31(R).

(R)-N-(1-(6-Chloro-3-iodo-2-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (33).

Compound 32 (2 g, 6.1 mmol), (R)-N-(pyrrolidin-3-yl)acetamide (860 mg, 6.71 mmol), DIPEA (1.3 mL, 7.3 mmol) was dissolved in a mixture of acetonitrile (40 mL) and ethanol (10 mL). Reaction mixture was heated at 85 °C for 16 h and cooled down. The precipitated product was filtered-off and washed with acetonitrile (2.436 g, 96%). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.91 (br s, 1H), 5.99 (s, 1H), 4.44–4.35 (m, 1H), 4.09 (br s, 1H), 3.94–3.79 (m, 3H), 2.35 (s, 3H), 2.19–2.14 (m, 1H), 1.98–1.90 (m, 1H), 1.83 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.0, 146.8, 141.8, 143.4, 134.6, 93.5, 71.1, 47.9, 55.1, 48.4, 30.1, 22.3, 14.6. HRMS calcd for C₁₃H₁₆ClIN₅O m/z: 420.0083 (M + H)⁺, found 420.0082.

General Procedure for Suzuki Coupling with Compound 31(R) (Method A).

A suspension of starting material 31(R) (1 mmol), appropriate boronic acid (1.5 mmol), sodium carbonate (2 mmol) in dioxane–water (10 mL, 4:1) was three times purged with argon. Then Pd(dppf)₂Cl₂ (0.1 mmol) was added, and again the flask was purged with argon. Reaction mixture was then heated to 95 °C overnight, cooled down and diluted with ethyl acetate or chloroform (300 mL). The suspension was dried over Na₂SO₄ and evaporated. The final compound was isolated by column chromatography and then crystallized.

General Procedure for Stille Coupling with Compound 31(R) (Method B).

A mixture of compound 31(R) (0.5 mmol) and tributyltin reagent (0.65 mmol) in DMF (10 mL) was degassed and purged with argon. To this mixture Pd(PPh₃)₄ (58 mg, 10%) was added, and reaction mixture was purged with argon and then heated to 100 °C for 16 h. Reaction mixture was cooled down, diluted with ethyl acetate (300 mL), dried over Na₂SO₄, and evaporated. Product was isolated by column chromatography and then crystallized.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (3).

Prepared by method A from 3,4-dimethoxyphenylboronic acid in 64%. Chromatography: CHCl₃–ethanol 15:1, crystallization from methanol). [α]_D²⁰ +6.5 (c 0.245, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.94 (d, J = 6.1 Hz, 1H, NH), 7.32 (d, J = 2.0 Hz, 1H, H-2″), 7.19 (dd, J = 2.0, J = 8.3 Hz, 1H, H-6″), 7.07 (d, J = 8.3 Hz, 1H, H-5″), 5.78 (s, 1H, H-5), 4.41–4.35 (m, 1H, H-3), 4.12 (m, 1H, H-2a), 3.93 (m, 1H, H-5a), 3.86–3.76 (m, 8H, H-2b, H-5b, 3″-OCH₃, 4″-OCH₃), 2.41 (s, 3H, 2-CH₃), 2.30 (s, 3H, 6-CH₃), 2.24–2.16 (m, 1H,H-4a), 1.98–1.90 (m, 1H, H-4b), 1.84 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.0 (COCH₃), 151.0 (C-6), 148.5 (C-3), 148.4 (C-4), 141.0 (C-8), 136.3 (C-2), 131.7 (C-9), 123.6 (C-3), 122.4 (C-1), 121.9 (C-6″), 113.9 (C-2″), 112.3 (C-5″), 93.6 (C-7), 55.8, 55.7 (3″-OCH₃, 4″-OCH₃), 54.8 (C-2′), 48.5 (C-3′), 47.4 (C-5′), 30.3 (C-4′), 22.3 (COCH₃), 21.2 (6-CH₃), 14.6 (2-CH₃). HRMS calcd for C₂₂H₂₇N₅O₃ m/z: 410.2187 (M + H)⁺, found 410.2186.

(S)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (4).

(S)-Enantiomer was prepared following the same reaction sequence as for compound 3. As an amine was used commercially available (S)-N-(pyrrolidin-3-yl)acetamide. All the spectra were identical to those for (R)-enantiomers. [α]_D²⁰ –2.0 (c 0.245, CHCl₃).

(R)-N-(1-(2,6-Dimethyl-3-phenylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (34).

Prepared by method A from benzeneboronic acid in 93% yield. Chromatography: (1) CH₂Cl₂–ethanol 15:1, (2) reverse-phase flash chromatography (C18, 30 g, water/acetonitrile 30% to 100%). Product was lyophilized. [α]_D²⁰ +3.8 (c 0.312, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.6 Hz, 1H, NH), 7.72–7.59 (m, 2H, H-2″), 7.56–7.46 (m, 2H, H-3″), 7.36 (ddt, J = 8.0, 6.8, 1.3 Hz, 1H, H-4″), 5.82 (s, 1H, H-5), 4.36 (h, J = 5.7 Hz, 1H, H-3′), 4.08 (br s, 1 H) and 3.82 (br s, 3H, H-2′, H-5′), 2.39 (s, 3H, 8-CH₃), 2.28 (s, 3H, 2-CH₃), 2.22–2.11 (m, 1H, H-4′a), 1.91 (ddt, J = 12.7, 7.5, 5.2 Hz, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.5 (C-6), 141.2 (C-8), 137.0 (C-2), 132.2 (C-9), 129.7 (C-1″), 129.3, 128.3 (C-2″, C-3″), 127.3 (C-4″), 123.9 (C-3), 94.2 (H-7), 55.2 (C-2′), 48.8 (C-3′), 47.7 (C-5′), 30.5 (C-4′), 22.7 (COCH₃), 21.6 (6-CH₃), 14.9 (2-CH₃). HRMS calcd for C₂₀H₂₄N₅O m/z: 350.1975 (M + H)⁺, found 350.1972.

(R)-N-(1-(2,6-Dimethyl-3-(3,4,5-trimethoxyphenyl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (35).

Prepared by method A from 3,4,5-trimethoxybenezeneboronic acid in 65% yield. Chromatography: CHCl₃–ethanol 15:1, crystallization from ethyl acetate. [α]_D²⁰ +12.5 (c 0.240, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.6 Hz, 1H, NH), 6.99 (s, 2H, H-2″), 5.81 (s, 1H, H-7), 4.39–4.30 (m, 1H, H-3′), 4.08 (br s, 1H) and 3.81 (s + br s, 9H, H-2′, H-5′, 3″-OCH₃), 3.73 (s, 3H, 4″-OCH₃), 2.43 (s, 3H, 2-CH₃), 2.30 (s, 3H, 6-CH₃), 2.12–2.23 (m, 1H, H-4′a), 1.95–1.86 (m, 1H, H-4′b), 1.82 (s, 1H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 152.8 (C-3″), 151.5 (C-6), 141.2 (C-8), 137.0 (C-4″), 136.9 (C-2), 132.1 (C-9), 125.1 (C-1″), 123.8 (C-3), 106.9 (C-2″), 94.0 (C-7), 60.3 (4″-OCH₃), 56.1 (3″-OCH₃), 55.3 (C-2′), 48.8 (C-3′), 47.7* (C-5′), 30.4 (C-4′), 22.8 (COCH₃), 21.7 (6-CH₃), 15.2 (2-CH₃). HRMS calcd for C₂₃H₃₀N₅O₄ m/z: 440.2292 (M + H)⁺, found 440.2291.

(R)-N-(1-(2,6-Dimethyl-3-(4-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (36).

Prepared by method A from 4-trifluoromethylbenzeneboronic acid in 85% yield. Chromatography: (1) CH₂Cl₂–ethanol 15:1, (2) reverse-phase flash chromatography (C18, 30 g, water/acetonitrile 30% to 100%). Product was lyophilized. [α]_D²⁰ +10.2 (c 0.313, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.6 Hz, 1H, NH), 7.97–7.90 (m, 2H) and 7.87–7.82 (m, 2H, H-2″, H-3″), 5.87 (s, 1H, H-7), 4.36 (h, J = 5.7 Hz, 1H, H-3′), 4.08 (br s, 1 H) and 3.82 (br s, 3H, H-2′, H-5′), 2.43 (s, 3H, 2-CH₃), 2.30 (s, 3H, 6-CH₃), 2.23–2.11 (m, 1H, H-4′a), 1.97–1.87 (m, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.9 (C-6), 141.2 (C-8), 138.2 (C-2), 133.9 (C-9), 132.8 (C-3), 129.5 (C-2″), 127.3 (q, J = 32 Hz, C-4″), 125.5 (q, J = 3.7 Hz, C-3″), 124.5 (q, J = 273 Hz, CF₃), 122.4 (C-1″), 94.7 (C-7), 55.3 (C-2′), 48.8 (C-3′), 47.8 (C-5′), 30.5 (C-4′), 22.7 (COCH₃), 21.6 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₁H₂₂N₅OF₃Na m/z: 440.1669 (M + Na)⁺, found 440.1666.

(R)-N-(1-(3-(4-Cyanophenyl)-2,6-dimethylimidazo[1,2-b]-pyridazin-8-yl)pyrrolidin-3-yl)acetamide (37).

Prepared by method A from 4-cyanobenzeneboronic acid in 72% yield. Chromatography: (1) CH₂Cl₂–ethanol 15:1, (2) reverse-phase flash chromatography (C18, 30 g, water/acetonitrile 30% to 100%). Product was lyophilized. [α]_D²⁰ +16.8 (c 0.310, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.6 Hz, 1H, NH), 7.93 (s, 4H, C-2″, C-3″), 5.89 (s, 1H, H-7), 4.36 (h, J = 5.7 Hz, 1H, H-3′), 4.08 (br s, 1 H) and 3.82 (br s, 3H, H-2′, H-5′), 2.44 (s, 3H, 2-CH₃), 2.31 (s, 3H, 6-CH₃), 2.16 (dq, J = 13.8, 7.5, 6.4 Hz, 1H, H-4′a), 1.91 (dq, J = 12.5, 5.9 Hz, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 152.0 C-6), 141.2 (C-8), 138.7 (C-2), 134.5 (C-1″or C-4″), 133.0 (C-9), 132.20 and 129.3 (C-2″, C-3″), 122.2 (C-3), 119.1 (C-1″ or C-4″), 109.2 (CN), 95.0 (C-7), 55.3 (C-2′), 48.7 (C-3′), 48.2* (C-5′), 30.4 (C-4′), 22.7 (COCH₃), 21.6 (6-CH₃), 15.3 (2-CH₃). HRMS calcd for C₂₁H₂₂N₆ONa m/z: 397.1747 (M + Na)⁺, found 397.1746.

(R)-N-(1-(3-(4-Fluoro-3-methoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)-pyrrolidin-3-yl)-acetamide (38).

Prepared by method A from 4-fluoro-3-methoxyphenylboronic acid in 72% yield. Chromatography: (1) CH₂Cl₂–ethanol 15:1, (2) reverse-phase flash chromatography (C18, 50 g, water/acetonitrile 30% to 100%). Product was lyophilized. [α]_D²⁰ +10.6 (c 0.311, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.6 Hz, 1H, NH), 7.47 (dd, J = 8.4, 2.0 Hz, 1H, H-2″), 7.32 (dd, J = 11.5, 8.4 Hz, 1H, H-5″), 7.20 (ddd, J = 8.4, 4.5, 2.1 Hz, 1H, H-6″), 5.83 (s, 1H, H-7), 4.35 (h, J = 5.8 Hz, 1H, H-3), 4.08 (br s, 1 H) and 3.87 (s, 3H) and 3.83 (br s, 3H, H-2′, H-5′, OCH₃), 2.41 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.17 (dq, J = 13.7, 7.3 Hz, 1H, H-4′a), 1.91 (dq, J = 12.5, 6.1 Hz, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 152.0 (C-6), 151.0 (d, J = 245.9 Hz, C-4″), 147.2 (d, J = 10.7 Hz, C-3″), 141.2 (C-8), 137.1 (C-2), 132.2 (C-1″), 126.8 (d, J = 3.8 Hz, C-1″), 123.08 (C-3), 122.2 (d, J = 6.7 Hz, C-6″), 115.8 (d, J = 18.3 Hz, C-5″), 114.9 (d, J = 1.9 Hz, C-2″), 94.2 (C-7), 56.2 (OCH₃), 55.3 (C-2′), 48.8 C-3′), 47.7 (C-5′), 30.4 (C-4′), 22.8 (COCH₃), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₁H₂₅N₅O₂F m/z: 398.1987 (M + H)⁺, found 398.1983.

(R)-N-(1-(3-(3-Fluoro-4-methoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (39).

Prepared by method A from 3-fluoro-4-methoxybenzeneboronic acid in 49% yield. Chromatography: CHCl₃–ethanol 15:1, crystallization from ethanol. [α]_D²⁰ +8.1 (c 0.247, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.6 Hz, 1H, NH), 7.54 (dd, J = 12.9, 2.1 Hz, 1H, H-2″), 7.42 (ddd, J = 8.5, 2.1, 1.1 Hz, 1H, H-6″), 7.28 (t, J ~ 9.1 Hz, 1H, H-5″), 5.81 (s, 1H, H-7), 4.38–4.29 (m, 1H, H-3′), 4.06 (br s, 1H) and 3.81 (br s, 3H H-2′, H-5′), 3.90 (s, 3H, OCH₃), 2.38 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.21–2.10 (m, 1H, H-4′a), 1.96–1.85 (m, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.6 (C-6), 151.2 (d, J = 243 Hz, C-3″), 146.4 (d, J = 10.6 Hz, C-4″), 141.1 (C-8), 137.0 (C-2), 132.1 (C-9), 125.7 (d, J = 3.3 Hz, H-6″), 122.6 (d, J = 1.0 Hz, C-3), 122.5 (d, J = 7.5 Hz, C-1″), 116.5 (d, J = 19.0 Hz, C-2″), 113.8 (d, J = 2.3 Hz, C-5″), 94.2 (C-7), 56.2 (OCH₃), 55.3 (C-2′), 48.8 (C-3′), 47.5* (C-5′), 30.5 (C-4′), 22.8 (COCH₃), 21.7 (6-CH₃), 14.9 (2-CH₃). HRMS calcd for C₂₁H₂₅N₅O₂F m/z: 398.1987 (M + H)⁺, found 398.1988.

(R)-N-(1-(2,6-Dimethyl-3-(4-(methylthio)phenyl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (40).

Prepared by method A from 4-(methylthio)benzeneboronic acid in 61% yield. Chromatography: CHCl₃–acetone 4:1, crystallization from ethyl acetate. [α]_D²⁰ +6.1 (c 0.231, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.16 (d, J = 6.6 Hz, 1H, NH), 7.60 (d, J = 8.5 Hz, 1H) and 7.37 (d, J = 8.5 Hz, 1H, H-2″, H-3″), 5.81 (s, 1H, H-7), 4.39–4.30 (m, 1H, H-3′), 4.05 (br s, 1H) and 3.81 (br s, 3H, H-2′, H-5′), 2.53 (s, 3H, SCH₃), 2.38 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6-CH₃), 2.21–2.10 (m, 1H, H-4′a), 1.97–1.86 (m, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.5 (C-6), 141.1 (C-8), 137.3 (C-2), 136.9 (C-1″ or C-4″), 132.2 (C-9), 129.7 and 125.8 (C-2″, C-3″), 126.2 (C-1″ or C-4″), 123.5 (C-3), 94.1 (C-7), 55.2 (C-2′), 48.8 (C-3′), 47.7 (C-5′), 30.5 (C-4′), 22.7 (COCH₃), 21.6 (6-CH₃), 14.9 (2-CH₃), 14.8 (SCH₃). HRMS calcd for C₂₁H₂₅N₅OSNa m/z: 418.1672 (M + Na)⁺, found 418.1673.

(R)-N-(1-(3-(3-(N,N-Dimethylsulfamoyl)phenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (41).

Prepared by method A (3-(N,N-dimethylsulfamoyl)phenyl)boronic acid in 50% yield. Chromatography: CHCl₃–ethanol 15:1, crystallization from ethyl acetate (freezer). [α]_D²⁰ +18.0 (c 0.239, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.6 Hz, 1H, NH), 8.14 (dt, J = 1.8, 1.0 Hz, 1H, H-2″), 7.99 (dt, J = 7.7, 1.5 Hz, 1H, H-6″), 7.80–7.74 (m, 1H, H-5″), 7.72 (dt, J = 7.9, 1.5 Hz, 1H, H-4″), 5.88 (s, 1H, H-7), 4.40–4.31 (m, 1H, H-3′), 4.07 (br s, 1H) and 3.83 (br s, 3H, H-2′, H-5′), 2.70 (s, 6H, N(CH₃)₂), 2.46 (s, 3H, 2-CH₃), 2.27 (s, 3H, 6-CH₃), 2.22–2.11 (m, 1H, H-4′a), 1.96–1.87 (m, 1H, H-4′b), 1.82 (s, 3H (COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.9 (C-6), 141.2 (C-8), 138.0 (C-2), 134.6 (C-1″ or C-3″), 133.1 (C-6″), 132.7 (C-9), 130.7 (C-1″ or C-3″), 129.5 C-5″), 127.7 (C-2″), 126.0 (C-4″), 122.1 (C-3), 94.7 (C-7), 55.1* (C-2′), 48.7 (C-3′), 47.8* (C-5′), 37.8 (N(CH₃)₃), 30.5* (C-4′), 22.7 (COCH₃), 21.5 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₂H₂₈N₆O₃SNa m/z: 479.1836 (M + Na)⁺, found 479.1835.

(R)-N-(1-(3-(6-Methoxypyridin-3-yl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (42).

Prepared by method A from 2-methoxy-5-pyridineboronic acid in 45% yield. Chromatography: (1) CHCl₃–ethanol 20:1 → 15:1, (2) CHCl₃–acetone 1:1, crystallization from ethyl acetate (freezer). [α]_D²⁰ +15.8 (c 0.291, DMSO). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.48–8.34 (m, 1H, H-2″), 8.18 (d, J = 6.6 Hz, 1H, NH), 7.98 (dd, J = 8.6, 2.4 Hz, 1H, H-5″), 6.96 (dd, J = 8.6, 0.8 Hz, 1H, H-4″), 5.81 (s, 1H, H-7), 4.40–4.29 (m, 1H, H-3′)), 4.07 (br s, 1 H) and 3.81 (br s, 3H, H2′, H-5′), 3.91 (s, 3H, OCH₃), 2.37 (s, 3H, 2-CH₃), 2.27 (s, 3H, 6-CH₃), 2.22–2.10 (m, 1H, H-4′a), 1.97–1.86 (m, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.5 (COCH₃), 162.7 (C-3″), 151.7 (C-6), 147.1 (C-2″), 141.2 (C-8), 140.0 (C-5″), 137.1 (C-2), 132.4 (C-9), 121.0 (C-3), 119.4 (C-1″), 110.3 (C-4″), 94.3 (C-7), 55.3 (C-2′), 53.5 (OCH₃), 48.8 (C-3′), 48.0* (C-5′), 30.5 (C-4′), 22.8 (COCH₃), 21.6 (6-CH₃), 14.7 (2-CH₃). HRMS calcd for C₂₀H₂₅N₆O₂ m/z: 381.2034 (M + H)⁺, found 381.2036.

(R)-N-(1-(2,6-Dimethyl-3-(thiophen-2-yl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (43).

Prepared by method B from 2-(tributylstannyl)thiophene in 68% yield. Chromatography: CHCl₃–acetone 3:2, crystallization from ethyl acetate. [α]_D²⁰ +6.7 (c 0.210, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.7 Hz, 1H, NH), 7.63 (dd, J = 3.7, 1.2 Hz, 1H, H-4″), 7.60 (dd, J = 5.2, 1.2 Hz, 1H, H-2″), 7.21 (dd, J = 5.2, 3.7 Hz, 1H, H-3″), 5.87 (s, 1H, H-7), 4.36 (h, J = 5.6 Hz, 1H, H-3′), 4.06 (br s, 1H) and 3.82 (br s, 3H, H-2′, H-5′), 2.54 (s, 3H, 2-CH₃), 2.39 (s, 3H, 6-CH₃), 2.22–2.11 (m, 1H, H-4′a), 1.96–1.86 (m, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.7 (C-6), 141.0 (C-8), 136.9 (C-2), 131.9 (C-5″), 130.6 (C-9), 126.9, 124.9, 125.3 (C-2″, C-3″, C-4″), 119.4 (C-3), 94.2 (C-7), 55.3 (C-2′), 48.8 (C-3′), 47.8 (C-5′), 30.4 (C-4′), 22.7 (COCH₃), 21.6 (2-CH₃), 16.3 (6-CH₃). HRMS calcd for C₁₈H₂₂N₅OS m/z: 356.1540 (M + H)⁺, found 356.1536.

(R)-N-(1-(3-(Furan-2-yl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (44).

Prepared by method B from 2-(tributylstannyl)furane in 52% yield. Chromatography: CHCl₃–acetone 3:2, prior to crystallization, product was decolorized with active carbon, crystallization from ethyl acetate. [α]_D²⁰ +3.0 (c 0.234, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.7 Hz, 1H, NH), 7.80 (dd, J = 1.8, 0.9 Hz, 1H, H-2″), 7.21 (dd, J = 3.3, 0.9 Hz, 1H, H-4″), 6.66 (dd, J = 3.3, 1.8 Hz, 1H, H-3″), 5.86 (s, 1H, H-7), 4.35 (h, J = 5.7 Hz, 1H, H-3′), 3.45–4.25 (2 × br s, 4 H, H-2′, H-5′), 2.58 (s, 2H, 2-CH₃), 2.38 (s, 2H, 6-CH₃), 2.16 (dtd, J = 13.5, 7.7, 6.0 Hz, 1H, H-4′a), 1.91 (ddt, J = 12.6, 7.4, 5.2 Hz, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 152.0 (C-6), 145.0 (C-2″), 141.9 (C-5″), 140.9 (C-8), 136.8 (C-2), 132.5 (C-9), 116.7 (C-3), 111.4 and 107.7 (C-3″, C-4″), 94.3 (C-7), 55.3 (C-2′), 48.8 (C-3′), 30.5 (C-4′), 22.7 (COCH₃), 21.7 (6-CH₃), 15.6 (2-CH₃) (one CH₂ was not detected). HRMS calcd for C₁₈H₂₂N₅O₂ m/z: 340.1768 (M + H)⁺, found 340.1743.

(R)-N-(1-(2,6-Dimethyl-3-(thiazol-2-yl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (45).

Prepared by method B from 2-tributylstannylthiazole in 62% yield. Chromatography: (1) CHCl₃–acetone 3:2, (2) toluene–acetone 1:1, crystallization from acetone–ethanol. [α]_D²⁰ +11.3 (c 0.266, DMSO). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.18 (d, J = 6.6 Hz, 1H, NH), 7.99 (d, J = 3.3 Hz, 1H) and 7.73 (d, J = 3.3 Hz, 1H, H-4″, H-5″), 5.98 (s, 1H, H-7), 4.37 (h, J = 5.6 Hz, 1H, H-3′), 4.28–3.39 (2 × br s, 4 H, H-2′, H-5′), 2.76 (s, 1H, 2-CH₃), 2.46 (s, 1H, 6-CH₃), 2.25–2.11 (m, 1H, H-4′a), 2.00–1.85 (m, 1H, H-4′b), 1.82 (s, 1H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 155.5 (C-2″), 152.0 (C-6), 142.3 (C-4″ or C-5″), 141.0 (C-8), 140.2 (C-2), 132.5 (C-9), 119.7 (C-3), 118.5 (C-4″ or C-5″), 95.1 (C-7), 22.7 (COCH₃), 21.5 (6-CH₃), 16.5 (2-CH₃) (carbon signals of pyrrolidine ring were not detected due to signal broadening). HRMS calcd for C₁₇H₂₁N₆OS m/z: 357.1492 (M + H)⁺, found 357.1469.

(R)-N-(1-(2,6-Dimethyl-3-(3-morpholinophenyl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (46).

Prepared by method A from (3-morpholinophenyl)boronic acid in 82% yield. Chromatography: (1) CHCl₃–acetone 1:1, (2) toluene–acetone 1:2, crystallization from ethyl acetate. [α]_D²⁰ +6.6 (c 0.334, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.18 (d, J = 6.7 Hz, 1H, NH), 7.34 (dd, J = 8.4, 7.6 Hz, 1H, H-5″), 7.21 (dd, J = 2.6, 1.5 Hz, 1H, H-2″), 7.14–7.07 (m, 1H) and 6.96 (ddd, J = 8.4, 2.6, 0.9 Hz, 1H, H-4″, H-6″), 5.80 (s, 1H, H-7), 4.35 (m, 1H, H-3′), 3.82 and 4.08 (2 × br s, 4H, H-2′, H-5′), 3.81–3.73 (m, 4H, OCH₂), 3.20–3.09 (m, 4H, NCH₂), 2.39 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.24–2.11 (m, 1H, H-4′a), 1.95–1.86 (m, 1H, H-4′b), 1.83 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.4 (C-3″), 151.1 (C-6), 141.1 (C-8), 137.0 (C-2), 132.1 (C-9), 130.3 (C-1″), 124.4 (C-3), 128.9, 120.5, 116.2, 114.5 (C-2″, C-4″, C-5″, C-6″), 94.0 (C-7), 66.3 (OCH₂), 55.3 (C-2′), 48.8 (NCH₂), 47.7 (C-5′), 30.5 (C-4′), 22.8 (COCH₃), 21.7 (6-CH₃), 15.0 (2-CH₃) (C-3′ of pyrrolidine ring was not detected due to the signal broadening). HRMS calcd for C₂₄H₃₁N₆O₂ m/z: 435.2503 (M + H)⁺, found 435.2480.

(R)-N-(1-(2,6-Dimethyl-3-(4-(trifluoromethoxy)-phenyl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (47).

Prepared by method A from 4-(trifluoromethoxy)benzeneboronic acid in 84% yield. Chromatography: (1) CHCl₃–acetone 2:1, (2) toluene–acetone 2:3, crystallization from ethyl acetate. [α]_D²⁰ +6.7 (c 0.237, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (d, J = 6.5 Hz, 1H, NH), 7.81 (d, J = 8.3 Hz, 1H) and 7.49 (d, J = 8.3 Hz, 1H, H-2″, H-3″), 5.85 (s, 1H, H-7), 4.40–4.31 (m, 1H, H-3′), 4.08 (br s, 1H) and 3.83 (br s, 3H, H-2′, H-5′), 2.40 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.23–2.10 (m, 1H, H-4′a), 1.98–1.86 (m, 1H, H-4′b) 1.82 (s, 1H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.8 (C-6), 147.3 (d, J = 1.8 Hz, C-4″), 141.2, (C-8), 137.5 (C-2), 132.5 (C-9), 131.0 (C-3″), 129.1 (C-1″), 122.5 (C-3), 121.1 (C-2″), 120.3 (d, J = 255.4 Hz, OCF₃), 94.5 (C-7), 55.2 (C-2′), 48.8 (C-3′), 47.7 (C-5′), 30.4 (C-4′), 22.8 (COCH₃), 21.6 (6-CH₃), 14.9 2-CH₃). HRMS calcd for C₂₁H₂₃F₃N₅O₂ m/z: 434.1798 (M + H)⁺, found 434.1799.

(R)-N-(1-(3-(1H-Indol-6-yl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (48).

Prepared by method A from indole-6-boronic acid in 40% yield. Chromatography: (1) CHCl₃–acetone 1:1, (2) toluene–acetone 1:2, (3) flash chromatography on RP column (C18, H₂O–acetonitrile + 0.5% HCOOH, 30% to 50%). [α]_D²⁰ +16.8 (c 0.262, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 11.17 (br s, 1H, NH-indole), 8.18 (d, J = 6.7 Hz, 1H, NH), 7.68 (dt, J = 1.6, 0.8 Hz, 1H, H-7″), 7.63 (d, J = 8.2 Hz, 1H, H-4″), 7.41 (dd, J = 3.0, 2.4 Hz, 1H, H-2″), 7.22 (dd, J = 8.2, 1.5 Hz, 1H, H-5″), 6.47 (ddd, J = 3.0, 2.0, 1.0 Hz, 1H, H-3″), 5.80 (s, 1H, H-7), 4.41–4.31 (m, 1H, H-3), 4.10 (br s, 1H) and 3.84 (br s, 3H, H-2′, H-5′), 2.40 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6-CH₃), 2.23–2.13 (m, 1H, H-4′a), 1.98–1.87 (m, 1H, H-4′b), 1.83 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (COCH₃), 151.3 (C-6), 141.2 (C-8), 136.4 (C-2), 135.9 (C-7″a), 131.8 (C-9), 127.1 (C-3″a), 126.3 (C-2″), 125.4 (C-3), 122.1 (C-6″), 120.9 (C-5″), 119.7 (C-4″), 112.7 (C-7″), 101.2 C-3″), 93.8 (C-7), 55.3 (C-2′), 48.8 (C-3′), 47.7 (C-5′), 30.5 (C-4′), 22.8 (COCH₃), 21.7 (6-CH₃), 14.9 (2-CH₃). HRMS calcd for C₂₂H₂₅N₆O m/z: 389.2084 (M + H)⁺, found. 389.2124.

(R)-N-(1-(6-Chloro-3-(3,4-dimethoxyphenyl)-2-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (49).

A suspension of starting material 33 (420 mg, 1 mmol), 3,4-dimethoxyphenylboronic acid (218 mg, 1.2 mmol), sodium carbonate (159 mg, 1.5 mmol) in dioxane–water (10 mL, 4:1) was three times purged with argon. Then Pd(dppf)₂Cl₂ (41 mg, 0.05 mmol) was added, and again the flask was purged with argon. The reaction mixture was then heated to 95 °C for 2 h, cooled down, and diluted with ethyl acetate (300 mL). The mixture was dried over Na₂SO₄ and evaporated. Product was isolated by column chromatography and then crystallized. Yield: 83%. Chromatography: CHCl₃–ethanol 20:1, crystallization from methanol. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.96 (d, J = 6.2 Hz, 1H), 7.23 (d, J = 2.0 Hz, 1H), 7.17 (dd, J = 2.0, J = 8.3 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 5.94 (s, 1H), 4.40 (m, 1H), 4.15 (br s, 1H), 3.98–3.86 (m, 3H), 3.85 (s, 3H), 3.81 (s, 3H), 2.41 (s, 3H), 2.25–2.16 (m, 1H), 1.98–1.92 (m, 1H), 1.84 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.1, 148.9, 148.7, 146.2, 142.0, 137.1, 131.1, 124.5, 122.1, 121.2, 113.9, 112.4, 92.6, 55.9, 55.7, 55.1, 47.8, 48.5, 30.2, 22.3, 14.4. HRMS calcd for C₂₁H₂₅ClN₅O₃ m/z: 430.1640 (M + H)⁺, found 430.1640.

(R)-1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-amine Hydrochloride (50).

Acetamide 3 (2.87 g, 7 mmol) was heated to reflux overnight in a mixture of conc HCl (40 mL) and water (40 mL). The reaction mixture was evaporated to dryness, coevaporated with ethanol (2 × 100 mL), and then triturated with isopropanol (with heating and sonication). The precipitated solid was filtered off, washed with isopropanol and diethyl ether, and directly used without further purification. An amount of 2.19 g of the hydrochloride salt was obtained. UPLC–MS: t = 3.08 (M + H, 368.3).

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)benzamide (51).

Hydrochloride 50 (200 mg, 0.5 mmol) was suspended in dichloromethane (15 mL), and triethylamine (349 μL, 0.25 mmol) and benzoyl chloride (87 μL, 0.75 mmol) were added, and the reaction mixture was stirred for 16 h and evaporated. The residue was chromatographed on silica gel column (100 g, CHCl₃–acetone 10:1), and 203 mg (86%) of the product was obtained as a foam. Analytical sample was obtained after crystallization from ethyl acetate. [α]_D²⁰ –57.8 (c 0.301, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.65 (d, J = 6.6 Hz, 1H, NH), 8.04–7.83 (m, 2H, o-Bz), 7.56–7.50 (m, 1H, p-Bz), 7.50–7.43 (m, 1H, m-Bz), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.5 Hz, 1H, H-5″), 5.82 (s, 1H, H-7), 4.62 (p, J = 6.1 Hz, 1H), 4.23 (br s, 1H) and 3.93 (br s, 3H, H-2′, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4′-OCH₃), 2.40 (s, 3H, 2-CH₃), 2.33–2.21 (m, 4H, 6-CH₃, H-4′a), 2.11 (dq, J = 12.8, 6.5 Hz, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 166.7 (COCH3), 151.4 (C-2), 148.4 (C-3″), 148.3 (C-4″), 141.2 (C-8), 136.6 (C-2), 134.4 (i-Ph), 132.0 (C-9), 131.4 (p-Ph), 128.4 (m-Ph), 127.6 (o-Ph), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.3 (C-2″), 111.8 (C-5″), 93.9 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 54.9 (C-2′), 49.6 (C-3′), 47.9 (C-5′), 30.5 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₇H₃₀N₅O₃ m/z: 472.2343 (M + H)⁺, found 472.2344.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-2,2,2-trifluoroacetamide (52).

Hydrochloride 50 (160 mg, 0.4 mmol) was suspended in dichloromethane (10 mL). Triethylamine (300 μL, 0.2 mmol) and trifluoroacetic anhydride (83 μL, 0.6 mmol) were added, the reaction mixture was stirred for 16 h, and the solvent was evaporated. The residue was chromatographed on a silica gel column (100 g, CHCl₃–acetone 8:1), and 120 mg (65%) of the product was obtained as a foam. [α]_D²⁰ –5.5 (c 0.234, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 9.72 (d, J = 6.1 Hz, 1H, NH), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.83 (s, 1H, H-7), 4.55–4.44 (m, 1H, H-3′)), 4.18 (br s, 1H) and 3.87 (br s, 3H, H-2′, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″OCH₃), 2.40 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.29–2.22 (m, 1H, H-4′a), 2.11–2.01 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 156.5 (q, J = 36.4 Hz, COCF₃), 151.4 (C-8), 148.4 (C-3″), 148.3 (C-4″), 141.1 (C-8), 136.6 (C-2), 131.9 (C-9), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 116.0 (q, J = 288 Hz, CF₃), 113.3 (C-2″), 111.8 (C-5″), 94.1 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 54.1 (C-2′), 49.8 (C-3′), 47.6 (C-5′), 30.0 (C-4′), 21.7 (6-CH₃), 15.0 (C-2). HRMS calcd for C₂₂H₂₅F₃N₅O₃ m/z: 464.1904 (M + H)⁺, found 464.1906.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)hexanamide Hydrochloride (53).

Hydrochloride 50 (200 mg, 0.5 mmol) was suspended in dichloromethane (10 mL). Triethylamine (350 μL, 0.25 mmol) and hexanoyl chloride (105 μL, 0.75 mmol) were added, the reaction mixture was stirred for 16 h, and the solvent was evaporated. The residue was chromatographed on a silica gel column (100 g, CHCl₃–acetone 5:1), and the obtained semisolid was treated with HCl in ether (4 M) to form a white solid (149 mg, 59%). [α]_D²⁰ +48.5 (c 0.344, DMSO). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.23 (d, J = 6.7 Hz, 1H, NH), 7.25 (d, J = 1.9 Hz, 1H, H-2″), 7.19 (dd, J = 8.3, 1.9 Hz, 1H, H-6″), 7.12 (d, J = 8.4 Hz, 1H, H-5″), 6.22 (s, 1H, H-7), 4.46–4.38 (m, 1H, H-3′), 4.06 (br s, 2H) and 3.88 (br s, 2H) and 3.71 (br s, 1H, H-2′, H-5′), 3.84 (s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.50* (s, 3H, 2-CH₃), 2.36 (s, 3H, 6-CH₃), 2.27–2.15 (m, 1H, H-4′a), 2.15–2.05 (m, 2H, COCH₂), 2.04–1.93 (m, 1H, H-4′b), 1.55–1.46 (m, 2H, COCH₂CH₂), 1.32–1.16 (m, 4H, CH₂CH₂CH₃), 0.84 (t, J = 7.0 Hz, 3H, CH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 172.5 (CO), 153.5* (C-2), 149.2 and 148.6 (C-2″, C-3″), 139.8 (C-2), 132.9 (C-2), 124.9 (C-3), 124.7* (C-1″), 122.8 (C-6″), 113.6 (C-2″), 111.9 (C-5″), 55.8 (3″-OCH₃, 4″-OCH₃), 55.3 (C-2′), 48.6 (C-3′), 48.1 (C-5′), 35.4 (COCH), 31.1 (hexyl-CH₂), 30.4 (C-4′), 25.1 (hexyl-CH₂), 22.0 (hexyl-CH₂), 21.4 (2-CH₃), 14.0 (hexyl-CH₃), 12.7* (8-CH₃). HRMS calcd for C₂₆H₃₅N₅O₃Na m/z: 488.2632 (M + Na)⁺, found 488.2629.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-2-phenylacetamide (54).

Hydrochloride 50 (200 mg, 0.5 mmol) was suspended in dichloromethane (10 mL). Triethylamine (350 μL, 0.25 mmol) and 2phenylacetyl chloride (100 μL, 0.75 mmol) were added, the reaction mixture was stirred for 16 h, and the solvent was evaporated. The residue was chromatographed on a silica gel column (100 g, CHCl₃–acetone 5:1) to afford 179 mg (74%) of the product. Analytical sample was obtained after crystallization from ethyl acetate. [α]_D²⁰ –39.2 (c 0.278, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.43 (d, J = 6.6 Hz, 1H, NH), 7.32–7.19 (m, 6H, H-2″, o-Ph, m-Ph, p-Ph), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.81 (s, 1H, H-7), 4.41–4.33 (m, 1H, H-3′), 4.07 (br s, 1H) and 3.83 (s, 3H) and 3.81 (s, 3H) and 3.78 (br s, 3H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 3.42 (s, 2H, CH₂Ph), 2.39 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.24–2.12 (m, 1H, H-4′a), 1.97–1.87 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 170.3 (CO), 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.2 (C-8), 136.6 and 136.5 (C-1″, i-Ph), 131.9 (C-5), 129.1 and 128.4 (o-Ph, m-Ph), 126.5 (p-Ph), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 94.0 (C-7), 55.7 (2 x, 3″-OCH₃, 4″-OCH₃), 55.2 (C-2′), 48.9 (C-3′), 47.8 (C-5′), 42.3 (CH₂Ph), 30.6 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₈H₃₂N₅O₃ m/z: 486.2500 (M + H)⁺, found 486.2449.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-2-phenoxyacetamide (55).

Hydrochloride 50 (200 mg, 0.5 mmol) was suspended in dichloromethane (10 mL), and triethylamine (350 μL, 0.25 mmol) and 2-phenoxyacetyl chloride (104 μL, 0.75 mmol) were added. The reaction mixture was stirred for 16 h, and then the solvent was evaporated. The residue was chromatographed on a silica gel column (100 g, toluene–acetone 4:1). Product (172 mg, 69%) was isolated as a foam. [α]_D²⁰ –38.3 (c 0.243, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.40 (d, J = 6.9 Hz, 1H, NH), 7.33–7.23 (m, 3H, H-2″, H-arom), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 7.00–6.89 (m, 3H, H-arom), 5.80 (s, 1H, H-7), 4.55–4.43 (m, 3H, H-3′, CH₂O), 4.13 (br s, 1H) and 3.96–3.36 (m, 9H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.24–2.16 (m, 1H, H-4′a), 2.06–1.96 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 168.0 (CO), 158.0 (i-Ph), 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.1 (C-8), 136.6 (C-2), 131.9 (C-9), 129.6 (C-arom), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 121.3 (p-Ph), 114.8 (C-arom), 113.2 (C-2″), 111.8 (C-5″), 93.9 (C-7), 67.0 (CH₂O), 55.7 (3″-OCH₃, 4″-OCH₃), 54.8 (C-2′), 48.7 (C-3′), 47.6 (C-5′), 30.3 (C−4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₈H₃₂N₅O₄ m/z: 502.2449 (M + H)⁺, found 502.2406.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-2,2-difluoro-2-phenylacetamide (56).

To a solution of 50 (320 mg, 0.8 mmol), DIPEA (0.6 mL, 4 mmol), and 2,2-difluoro-2-phenylacetic acid (206 mg, 1.2 mmol) in DMF (10 mL) was added HATU (365 mg, 0.96 mmol). The reaction mixture was stirred for 24 h, and then solvent was evaporated. The residue was chromatographed on a silica gel column (150 g, toluene–ethyl acetate 2:1). Fractions containing product were evaporated and rechromatographed (120 g, toluene–acetone 8:1) to afford product (202 mg, 48%). Solids were recrystallized from ethyl acetate in freezer. [α]_D²⁰ ~0 (c 0.224, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 9.29 (d, J = 6.8 Hz, 1H, NH), 7.66–7.45 (m, 5H, Ph), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.5 Hz, 1H, H-5″), 5.81 (s, 1H, H-7), 4.51–4.41 (m, 1H, H-3′), 4.14 (br s, 1H) and 3.89 (br s, 3H, H-2′, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.27–2.16 (m, 1H, H-4′a), 2.08–1.95 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSOd₆, 25 °C) δ 163.6 (t, J = 31.3 Hz, CO), 163.3 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.1 (C-8), 136.6 (C-2), 133.4 (t, J = 25.6 Hz, i-Ph), 131.2 (C-9), 129.0 (p-Ph), 125.4 (t, J = 5.9 Hz, o-Ph), 123.9 (C-3), 122.0 (C-1″), 114.9 (t, J = 251 Hz, CF₂), 113.2 (C-2″), 111.8 (C-5″), 94.1 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 54.1 (C-2′), 49.4 (C-3′), 47.8 (C-5′), 30.2 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₈H₃₀F₂N₅O₃ m/z: 522.2311 (M + H)⁺, found 522.2203.

(S)-N-((R)-1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-2-methoxy-2-phenylacetamide (57).

To a solution of 50 (160 mg, 0.4 mmol), DIPEA (0.3 mL, 2 mmol), and (S)-2-methoxy-2-phenylacetic acid (100 mg, 0.6 mmol) in DMF (5 mL) was added HATU (182.5 mg, 0.48 mmol). The reaction mixture was stirred for 14 h, and then the solvent was evaporated. The residue was chromatographed on a silica gel column (75 g, toluene–acetone 3:1) to afford product (154 mg, 75%) as a foam. [α]_D²⁰ +97.5 (c 0.279, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.44 (d, J = 7.1 Hz, 1H, NH), 7.47–7.25 (m, 6H, H-arom, H-2″), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.80 (s, 1H, H-7), 4.66 (s, 1H, CHOCH₃), 4.46–4.36 (m, 1H, H-3′), 4.07 (br s, 1H) and 3.82 (s, 3H) and 3.81 (s, 3H) and 3.78 (br s, 3H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 3.28 (s, 3H, CHOCH₃), 2.39 (s, 3H, 2-CH₃), 2.29 (s, 3H, 8-CH₃), 2.22–2.11 (m, 1H, H-4′a), 2.04–1.93 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 170.1 (CO), 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.2 (C-8), 138.1 (i-Ph), 136.6 (C-2), 131.9 (C-9), 128.4 and 128.2 and 127.2 (o-, m-, p-Ph), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 94.0 (C-7), 83.3 (CHOCH₃), 56.9 (CHOCH₃), 55.7 (3″-OCH₃, 4″-OCH₃), 54.5 C-2′), 48.6 (C-3′), 30.5 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₉H₃₄N₅O₄ m/z: 516.2605 (M + H)⁺, found 516.2562.

(R)-1-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-3-isopropylurea (58).

To a mixture of hydrochloride 50 (150 mg, 0.37 mmol), Et₃N (57 μL, 0.41 mmol) in dichloromethane (7 mL) was added isopropyl isocyanate (55 μL, 0.55 mmol), and the mixture was stirred overnight at room temperature and then quenched with few drops of methanol. The reaction mixture was evaporated and chromatographed on a silica gel column (150 g, CHCl₃–EtOH 20:1). An amount of 79 mg (47%) of the product was obtained. Analytical sample was obtained after crystallization from acetone. [α]_D²⁰ –16.6 (c 0.301, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.27 (d, J = 2.0 Hz, 1H, C-2″), 7.16 (dd, J = 8.3, 2.0 Hz, 1H, C-6″), 7.07 (d, J = 8.4 Hz, 1H, C-5″), 6.09 (d, J = 6.9 Hz, 1H, NHCO), 5.80 (s, 1H, H-7), 5.60 (d, J = 7.7 Hz, 1H, NH-iPr), 4.23 (q, J = 5.8 Hz, 1H, H-3′), 4.03 (bs, 2H) and 3.81 (s, 3H) and 3.78 (s, 3H) and 3.78 (br s, 2H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 3.72–3.62 (m, 1H, CH(CH₃)₂), 2.39 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6-CH₃), 2.14 (dq, J = 13.4, 6.9, 6.3 Hz, 1H, H-4′a), 1.85 (dq, J = 12.7, 6.3 Hz, 1H, H-4′b), 1.03 (d, J = 2.8 Hz, 3H, CH₃), 1.01 (d, J = 2.9 Hz, 3H, CH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 157.2 (CO), 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.2 (C-8), 136.6 (C-2), 131.9 (C-9), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 93.8 (C-7), 56.0 (C-2′), 55.7 (3″-OCH₃, 4″-OCH₃), 49.2 (C-3′), 47.7* (C-5′), 41.0 (CH-(CH₃)₂, 31.1 (C-4′), 23.4 (2 × CH₃), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₄H₃₂N₆O₃Na m/z: 475.2428 (M + Na)⁺, found 475.2429.

(R)-1-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-3-phenylurea (59).

To a mixture of hydrochloride 50 (200 mg, 0.5 mmol), Et₃N (0.14 mL, 1 mmol) in dichloromethane (7 mL) was added phenyl isocyanate (81 μL, 0.74 mmol), and the mixture was stirred overnight at room temperature and then quenched with few drops of methanol. The reaction mixture was evaporated and chromatographed on a silica gel column (150 g, CHCl₃–EtOH 25:1). The fractions containing product were evaporated and rechromatographed (80 g, CHCl₃–acetone 4:1). An amount of 120 mg (49%) of the product was obtained as an off-white solid. [α]_D²⁰ +4.02 (c 0.235, DMSO). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.31 (s, 1H, NHPh), 7.42–7.33 (m, 2H, o-Ph), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.25–7.19 (m, 2H, m-Ph), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 6.90 (tt, J = 7.3, 1.2 Hz, 1H, p-Ph), 6.55 (d, J = 6.8 Hz, 1H, NHCO), 5.84 (s, 1H, H-7), 4.38–4.28 (m, 1H, H-3′), 3.85 (br s, 1H) and 3.81 (br s, 3H) and 3.78 (br s, 6H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 2.40 (s, 3H, 2-CH₃), 2.30 (s, 3H, 6-CH₃), 2.28–2.16 (m, 1H, H-4′a), 2.04–1.91 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 155.1 (CO), 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.2 (C-8), 140.4 (i-Ph), 136.6 (C-2), 131.9 (C-9), 128.9 (C-arom), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 121.4 (p-Ph), 117.8 (C-arom), 113.2 (C-2″), 111.8 (C-5″), 93.9 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 49.3 (C-3′), 30.8 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃) (two CH₂ carbons were not detected). HRMS calcd for C₂₇H₃₁N₆O₃ m/z: 487.2452 (M + H)⁺, found 487.2437.

Procedure for Carbamates 1 and 60.

Hydrochloride 50 (160 mg, 0.4 mmol) was suspended in dry CH₂Cl₂ (10 mL), and the mixture was cooled down to 0 °C. To this suspension were sequentially added Et₃N (0.3 mL, 2.2 mmol) and DMAP (cat.). Appropriate chloroformate (0.6 mmol) was then added dropwise at 0 °C, and the reaction mixture slowly warmed to rt and was stirred overnight. Reaction mixture was evaporated, residue was purified by silica gel chromatography (100 g), and product was then crystallized.

Phenyl(R)-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (1 (PDDC)).

Yield: 140 mg (72%). Chromatography: toluene–ethyl acetate 3:1. Crystallization: ethyl acetate (freezer). [α]_D²⁰ +11.5 (c 0.234, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.19 (d, J = 6.5 Hz, 1H, NH), 7.43–7.34 (m, 2H, m-Ph), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.25–7.10 (m, 4H, o-Ph, p-Ph, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.82 (s, 1H, H-7), 4.31–4.23 (m, 1H, H-3′), 4.15 (br s, 1H) and 3.92 (br s, 3H, H-2′, H-5′), 3.81 (s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.40 (s, 3H, 2-CH₃), 2.30 (s, 3H, 6-CH₃), 2.28–2.19 (m, 1H, H-4′a), 2.09–2.00 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 154.5 (CO), 151.2 (C-2), 150.9 (i-Ph), 148.2 (C-3″), 148.1 (C-4″), 141.0 (C-8), 136.4 (C-2), 131.7 (C-9), 129.3 (m-Ph), 125.0 (p-Ph), 123.7 (C-3), 122.0 (C-1″), 121.8 (o-Ph, C-6″), 113.1 (C-2″), 111.6 (C-5″), 93.8 (C-7), 55.6 (3″-OCH₃, 4″-OCH₃), 55.2* (C-2′), 55.1* (C-5′), 50.5 (C-3′), 30.2 (C-4′), 21.5 (6-CH₃), 14.8 (2-CH₃). HRMS calcd for C₂₇H₃₀N₅O₄ m/z: 488.2292 (M + H)⁺, found 488.2267.

Phenyl(S)-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-(1(S)).

The physical and spectral properties are identical to those for compound 1 (PDDC).

Benzyl (R)-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (60).

Yield: 82 mg (41%). Chromatography: toluene–acetone 1:1. Crystallization: ethyl acetate. [α]_D²⁰ –18.1 (c 0.238, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.69 (d, J = 6.6 Hz, 1H, NH), 7.42–7.31 (m, 5H, Ph), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.5 Hz, 1H, H-5″), 5.79 (s, 1H, H-7), 5.04 (s, 2H, CH₂O), 4.22 (q, J = 5.8 Hz, 1H), 3.97–3.70 (br s and 2s, 10H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.28 (s, 3H, 8-CH₃), 2.22–2.13 (m, 1H, H-4′a), 1.99–1.89 (m, 1H, H4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 156.0 (CO), 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.1 (C-8), 137.2 (i-Ph), 136.6 (C-2), 131.9 (C-9), 128.5 and 128.0 (o-, m-, p-Ph), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.3 (C-2″), 111.8 (C-5″), 93.9 (C-7), 65.6 (CH₂O), 55.7 (3″-OCH₃, 4″-OCH₃), 55.2 (C-2′), 50.5 (C-3′), 47.7 (C-5′), 30.4 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₈H₃₂N₅O₄ m/z: 502.2449 (M + H)⁺, found 502.2414.

Procedure for Preparation of Carbamates (61–63).

Hydrochloride 50 (320 mg, 0.79 mmol) was suspended in dry acetonitrile (20 mL), and the mixture was cooled down to 0 °C. To this suspension were sequentially added Et₃N (0.68 mL, 3.95 mmol) and DMAP (cat.). p-Nitrophenyl chloroformate (221 mg, 1.1 mmol) was then added in one portion at 0 °C, and the reaction mixture was stirred for 2 h at 0 °C. Then were sequentially added triethylamine (0.4 mL, 2.4 mmol) and an appropriate alcohol (4 mmol), and the reaction mixture was heated to 85 °C (bath) overnight. After evaporation the residue was chromatographed on silica gel column (150 g) followed by purification on reverse-phase flash chromatography (C18, 50 g, water/acetonitrile 40% to 100%). The final compounds were then lyophilized from dioxane.

Cyclohexyl (R)-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (61).

Yield: 139 mg (36%). Chromatography: (1) toluene–ethyl acetate 2:1, (2) reverse-phase flash chromatography (C18, 50 g, water/acetonitrile 40% to 100%). Product was lyophilized. [α]_D²⁰ –2.7 (c 0.255, CHCl₃).¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.47 (d, J = 6.4 Hz, 1H, NH), 7.27 (d, J = 2.0 Hz, 1H, H-2″), 7.16 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.78 (s, 1H, H-7), 4.58–4.43 (br s, 1H, COOCH), 4.22–4.01 (m and br s, 2H, H-3′, H-2′a), 3.82 (2s and br s, 9H, H-2′b, H-5′, 3″-OCH₃, 4″-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6-CH₃), 2.21–2.10 (m, 1H, H-4′a), 1.98–1.89 (m, 1H, H-4′b), 1.87–1.75 (br m, 2H, cyclohexyl), 1.73–1.59 (br m, 2H, cyclohexyl), 1.54–1.44 (br m, 1H, cyclohexyl), 1.38–1.25 (m, 4H, cyclohexyl), 1.24–1.13 (m, 1H, cyclohexyl). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 155.7 (CO), 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.1 (C-8), 136.6 (C-2), 131.9 (C-9), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 111.8 (C-2″), 113.2 (C-5″), 93.8 (C-7), 72.0 (COOCH), 55.8 and 55.7 (3″-OCH₃, 4″-OCH₃), 55.2 (C-2′), 50.3 (C-3′), 47.6 (C-5′), 32.0 (cyclohexyl), 30.4 (C-4′), 23.7 (cyclohexyl), 21.7 (cyclohexyl), 25.1 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₇H₃₆N₅O₄ m/z: 494.2762 (M + H)⁺, found 494.2786.

m-Tolyl (R)-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (62).

Yield: 192 mg (49%). Chromatography: (1) toluene–ethyl acetate 3:1 → 2:1, (2) reverse-phase flash chromatography (C18, 50 g, water–acetonitrile 40% to 100%). Product was lyophilized. [α]_D²⁰ +11.6 (c 0.259, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.16 (d, J = 6.5 Hz, 1H, NH), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.25 (t, J = 7.8 Hz, 1H, H-5‴), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.5 Hz, 1H, H-5″), 7.04–7.00 (m, 1H, H-4‴), 6.95 (d, J = 2.6 Hz, 1H, H-2‴), 6.91 (d, J = 8.4 Hz, 1H, H-6‴), 5.82 (s, 1H, H-7), 4.30–4.22 (m, 1H, H-3′), 4.14 (br s, 1H) and 3.87 (br s, 3H, H-2′, H-5′), 3.82 (s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.40 (s, 3H, 2-CH₃), 2.31 (s, 3H, 3‴-CH₃), 2.30 (s, 3H, 6-CH₃), 2.27–2.18 (m, 1H, H-4′a), 2.09–1.98 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 154.3 (CO), 151.4 (C-6), 151.0 (C-1‴), 148.4 (C-3″), 148.3 (C-4″), 141.1 (C-8), 139.1 (C-3‴), 136.6 (C-2), 131.9 (C-9), 129.1 (C-5‴), 125.9 (C-4‴), 123.9 (C-3), 122.5 (C-2‴), 122.2 (C-1″), 122.0 (C-6″), 119.0 (C-6‴), 113.3 (C-2″), 111.8 (C-5‴), 94.0 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.2 (C-2′), 50.7 (C-3′), 47.7 (C-5′), 30.4 (C-4′), 21.7 (6-CH₃), 21.0 (3‴-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₈H₃₂N₅O₄ m/z: 502.2449 (M + H)⁺, found 502.2498.

Naphthalen-2-yl (R)-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (63).

Yield: 242 mg (45%). Chromatography: (1) toluene–ethyl acetate 3:1 → 2:1, (2) reverse-phase flash chromatography (C18, 50 g, water–acetonitrile 30% to 100%). Product was lyophilized. [α]_D²⁰ +23.4 (c 0.222, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.30 (d, J = 6.5 Hz, 1H, NH), 7.97–7.87 (m, 3H, H-arom), 7.68 (d, J = 2.3 Hz, 1H, H-arom), 7.51 (dddd, J = 14.5, 8.3, 6.9, 1.5 Hz, 2H, H-arom), 7.33 (dd, J = 8.8, 2.4 Hz, 1H, H-arom), 7.29 (d, J = 2.0 Hz, 1H, H-2″), 7.18 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.5 Hz, 1H, H-5″), 5.84 (s, 1H, H-7), 4.35–4.26 (m, 1H, H-3′), 4.17 (br s, 1H) and 3.95 (br s, 3H, H-2′, H-5′), 3.82 (s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.41 (s, 3H, 2-CH₃), 2.31 (s, 3H, 6-CH₃), 2.30–2.22 (m, 1H, H-4′a), 2.12–2.02 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 154.4 (CO), 151.4 (C-6), 148.8 (C-arom), 148.4 (C-3″), 148.3 (C-4″), 141.1 (C-8), 136.6 (C-2), 133.6 (C-arom), 131.9 (C-arom), 130.8 (C-9), 129.2 (C-arom), 127.8 (C-arom), 127.5 (Carom), 126.7 (C-arom), 125.6 (C-arom), 123.9 (C-3), 122.2, (C-arom) 122.1 (C-1″), 122.0 (C-6″), 113.3 (C-2″), 111.8 (C-5″), 94.0 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.1 (C-2′), 50.7 (C-3′), 47.7 (C-5′), 30.4 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₃₁H₃₂N₅O₄ m/z: 538.2449 (M + H)⁺, found 538.2511.

General Procedure for Preparation of the Sulfonates 64–74.

Hydrochloride 50 (160 mg, 0.4 mmol) was suspended in dry CH₂Cl₂ (10 mL), and Et₃N (0.3 mL, 2.2 mmol) and DMAP (cat.) were seqentially added at room temperature. Corresponding sulfonyl chloride (0.6 mmol) was then added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated, the residue was purified by silica gel chromatography (80 g), and the product was then crystallized.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)methanesulfonamide (64).

Yield: 110 mg (67%). Chromatography: CHCl₃–ethanol 20:1, crystallization from acetone. [α]_D²⁰ –11.8 (c 0.263, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.48 (d, J = 5.1 Hz, 1H, NH), 7.29 (d, J = 2.0 Hz, 1H, H-2″), 7.18 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.5 Hz, 1H, H-5″), 5.82 (s, 1H, H-7), 4.11 (br s, 1H) and 4.08 (m, 1 H) and 3.82 (br s, 3H, H-2′, H-3′, H-5′), 3.81 (br s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 3.01 (s, 3H, SO₂CH₃), 2.40 (s, 3H, 2-CH₃), 2.30 (s, 3H, 6-CH₃), 2.29–2.20 (m, 1H, H-4′a), 2.05–1.94 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.4 (C-2), 148.4 (C-3″), 148.3 (C-4″), 136.6 (C-8), 141.0 (C-2), 131.9 (C-9), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 111.8 (C-2″), 113.2 (C-5″), 93.9 (C-7), 55.4 (3″-OCH₃, 4″-OCH₃), 52.3 (C-2′), 47.8 (C-3′), 40.5 (SO₂CH₃), 31.4 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₁H₂₈N₅O₄S m/z: 446.1857 (M + H)⁺, found 446.1857.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)propane-2-sulfonamide (65).

Yield: 66 mg (35%). Chromatography: CHCl₃–acetone 21:3, crystallization from ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.46 (d, J = 7.1 Hz, 1H, NH), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.81 (s, 1H, H-7), 4.15 (br s, 1H) and 4.10–4.00 (m, 1H) and 3.81 (s, 3H, H-2′, H-3′, H-5′), 3.78 (s, 3H) and 3.80 (br s, 3H, 3″-OCH₃, 4″-OCH₃), 3.26 (p, J = 6.8 Hz, 1H, CH(CH₃)₂), 2.40 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6-CH₃), 2.27–2.15 (m, 1H, H-4′a), 2.03–1.92 (m, 1H, H-4′b), 1.27 (d, J = 2.4 Hz, 3H, CH(CH₃)₂), 1.25 (d, J = 2.5 Hz, 3H, CH(CH₃)₂). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 136.6 (C-8), 141.0 (C-2), 131.8 (C-9), 123.9 (C-3), 122.2 (C1″), 122.0 (C-6″), 113.3 (C-2″), 111.8 (C-5″), 93.9 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.5 (C-2′), 52.4 (C-3′), 52.0 (CH(CH₃)₂), 47.7 (C-5′), 31.6 (C-4′), 21.7 (6-CH₃), 16.6 and 16.5 (CH(CH₃)₂), 15.0 (2-CH₃). HRMS calcd for C₂₃H₃₂N₅O₄S m/z: 474.217 (M + H)⁺, found 474.2169.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)cyclopropanesulfonamide (66).

Yield: 131 mg (69%). Chromatography: CHCl₃–acetone 21:4, crystallization from ethyl acetate. [α]_D²⁰ ∼0 (c 0.242, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.52 (d, J = 7.1 Hz, 1H, NH), 7.29 (d, J = 2.0 Hz, 1H, H-2″), 7.18 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.3 Hz, 1H, H-5″), 5.81 (s, 1H, H-7), 4.20 (br s, 1H) and 4.17–4.05 (m, 1H) and 3.85 (br s, 3H, H-2′, H-3′, H-5′), 3.82 (s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.66 (tt, J = 7.8, 5.0 Hz, 1H, cyclopropyl-CH), 2.40 (s, 3H, 2-CH₃), 2.30 (s, 3H, 6-CH₃), 2.29–2.19 (m, 1H, H-4′a), 2.07–1.95 (m, 1H, H-4′b), 1.06–0.95 (m, 4H, cyclopropyl-CH₂). ¹³C NMR (101 MHz, d6-DMSO) δ ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.0 (C-8), 136.6 (C-2), 131.8 (C-9), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 93.9 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.5 (C-2′), 52.3 (C-3′), 47.7 (C-5′), 31.6 (C-4′), 30.2 (cyclopropyl-CH), 21.7 (6-CH₃), 15.00 (2-CH₃), 5.3 and 5.2 (cyclopropyl-CH₂). HRMS calcd for C₂₃H₃₀N₅O₄S m/s: 472.2013 (M + H)⁺, found 472.2014.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)benzenesulfonamide (67).

Yield: 157 mg (77%). Chromatography: CHCl₃–acetone 21:3, crystallization from ethyl acetate–acetone. [α]_D²⁰ +33.1 (c 0.296, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.09 (s, 1H, NH), 7.91–7.81 (m, 2H, o-Ph), 7.70–7.55 (m, 3H, m-, p-Ph), 7.27 (d, J = 2.0 Hz, 1H, C-2″), 7.16 (dd, J = 8.4, 2.0 Hz, 1H, C-6″), 7.07 (d, J = 8.4 Hz, 1H, C-5″), 5.71 (s, 1H, C-7), 4.03–3.62 (2 × br s and m, 5H, H-2′, H3′, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.37 (s, 3H, 2-CH₃), 2.27 (s, 3H, 6-CH₃), 2.09–1.96 (m, 1H, H-4′a), 1.89–1.78 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.3 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.2 (i-Ph), 140.9 (C-8), 136.6 (C-2), 132.7 (p-Ph), 131.7 (C-9), 129.4 and 126.6 (o-, m-Ph), 123.8 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 93.8 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.1 (C-2′), 52.5 (C-3′), 47.3 (C5′), 30.8 (C-4′), 21.7 (6-CH₃), 14.5 (2-CH₃). HRMS calcd for C₂₆H₃₀N₅O₄S m/z: 508.2013 (M + H)⁺, found 508.2019.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-4-methylbenzenesulfonamide (68).

Yield: 175 mg (82%). Chromatography: CHCl₃–acetone 5:1, crystallization from ethyl acetate. [α]_D²⁰ +42.4 (c 0.245, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.99 (d, J = 5.1 Hz, 1H, NH), 7.72–7.68 (m, 2H, o-Ph), 7.42–7.35 (m, 2H, m-Ph), 7.27 (d, J = 2.0 Hz, 1H, H-2″), 7.16 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.3 Hz, 1H, H-5″), 5.71 (s, 1H, H-7), 3.97 and 3.63 (2 × br s and m, 5H, H-2′H-3′, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.38 (s, 3H, p-CH₃), 2.37 (s, 3H, 2-CH₃), 2.27 (s, 3H, 6-CH₃), 2.06–1.96 (m, 1H, H-4′a), 1.90–1.79 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.3 (C-6), 148.4 (C-3″), 148.3 (C-4″), 142.8 (p-Ph), 140.9 (C-8), 138.4 (i-Ph), 136.5 (C-2), 131.7 (C-9), 129.8 (mPh), 126.7 (o-Ph), 123.8 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 93.8 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.1 (C-2′), 52.5 (C-3′), 47.3 (C-5′), 30.7 (C-4′), 21.7 (6-CH₃), 21.2 (p-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₇H₃₂N₅O₄S m/z: 522.2170 (M + H)⁺, found 522.2170.

(R)-N-(4-(N-(1-(3-(3, 4-Dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-sulfamoyl)phenyl)acetamide (69).

Yield: 171 mg (76%). Chromatography: CHCl₃–ethanol 15:1, crystallization from ethyl acetate. [α]_D²⁰ +64.6 (c 0.356, DMSO). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 10.32 (s, 1H, NHCOCH₃), 7.94 (s, 1H, NH-3′), 7.78 (s, 4H, o-, m-Ph), 7.27 (d, J = 2.0 Hz, 1HH-2″), 7.15 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.5 Hz, 1H, H-5″), 5.71 (s, 1H, H-7), 4.09–3.54 (2 × br s, 5H, H-2′, H-3′, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.35 (s, 3H, 2-CH₃), 2.27 (s, 3H, 6-CH₃), 2.08 (s, 3H, COCH₃), 2.08–1.97 (m, 1H, H-4′a), 1.84 (dq, J = 12.9, 6.6 Hz, 1H, H4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.1 (CO), 151.3 (C-3), 148.4 (C-3″), 148.3 (C-4″), 143.0 (p-Ph), 141.0 (C-8), 136.6 (C-2), 134.8 (i-Ph), 131.7 (C-9), 127.8 (o-Ph), 123.8 (C-3), 122.2 (C-1″), 122.0 (C-6″), 118.8 (m-Ph), 113.2 (C-2″), 111.8 (C-5″), 93.7 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.0 (C-2′), 52.4 (C-3′), 47.3* (C-5′), 30.8 (C-4′), 24.3 (COCH₃), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₈H₃₃N₆O₅S m/z: 565.2228 (M + H)⁺, found 565.2225.

(R)-4-Chloro-N-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-benzenesulfonamide (70).

Yield: 173 mg (80%). Chromatography: CHCl₃–acetone 22:3, crystallization from ethyl acetate–acetone. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.20 (s, 1H, NH), 7.90–7.77 (m, 2H, H-arom), 7.72–7.63 (m, 2H, H-arom), 7.27 (d, J = 2.0 Hz, 1H, H2″), 7.16 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.5 Hz, 1H, H-5″), 4.05–3.50 (2 × br s and m, 5H, H-2′, H-3′, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.37 (s, 3H, 2-CH₃), 2.27 (s, 3H, 6-CH₃), 2.11–2.00 (m, 1H, H-4′a), 1.92–1.80 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.3 (C-6), 148.4 (C-3″), 148.3 (C-4″), 140.9 (C-8), 140.2, 137.5, 136.5 (i-Ph, p-Ph, C-2), 131.7 (C-9), 129.7, 128.6 (o-, m-Ph), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 93.8 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.0 (C-2′), 52.6 (C-3′), 47.2 (C-5′), 30.7 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₆H₂₉ClN₅O₄S m/z: 542.1623 (M + H)⁺, found 542.1608.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-4-(trifluoromethyl)benzenesulfonamide (71).

Yield: 173 mg (75%) Chromatography: CHCl₃–acetone 8:1, crystallization from ethyl acetate. [α]_D²⁰ +27.9 (c 0.208, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.38 (s, 1H, NH), 8.13–8.04 (m, 2H, H-arom), 8.01–7.97 (m, 2H, H-arom), 7.26 (d, J = 2.0 Hz, 1H, H-2″), 7.15 (dd, J = 8.3, 2.0 Hz, 1H′, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.72 (s, 1H, H-7), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 3.52–4.04 (2 × br s and m, 5H, H-2′, H-3′, H-5′), 2.35 (s, 3H, 2-CH₃), 2.26 (s, 3H, 6-CH₃), 2.12–2.00 (m, 1H, H-4′a), 1.93–1.83 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.3 (C-2), 148.4 (C-3″), 148.3 (C-4″), 145.3 (i-Ph), 140.9 (C-8), 136.5 (C-2), 132.4 (q, J = 32.3 Hz, p-Ph), 131.7 (C-9), 127.6 (o-Ph), 126.7 (q, J = 3.9 Hz, m-Ph), 123.9 (C-3), 123.7 (q, J = 273 Hz, CF₃), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 93.8 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.0 (C-2′), 52.6 (C-3′), 47.3 (C-5′), 30.8 (C-4′), 21.6 (6-CH₃), 14.9 (2-CH₃). HRMS calcd for C₂₇H₂₉F₃N₅O₄S m/z: 576.1877 (M + H)⁺, found 542.1859.

(R)-4-Acetyl-N-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)benzenesulfonamide (72).

Yield: 167 mg (76%). Chromatography: CHCl₃–acetone 21:4, crystallization from ethyl acetate. [α]_D²⁰ +34.2 (c 0.263, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.30 (s, 1H, NH), 8.16–8.06 (m, 2H, H-arom), 8.01–7.86 (m, 2H, H-arom), 7.27 (d, J = 2.0 Hz, 1H, H-2″), 7.15 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.71 (s, 1H, H-7), 3.99–3.57 (2 × br s and m, 5H, H-2′, H-3′, H-5′), 3.82 (s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.60 (s, 3H, COCH₃), 2.34 (s, 3H, 2-CH₃), 2.27 (s, 3H, 6-CH₃), 2.12–2.00 (m, 1H, H-4′a), 1.94–1.83 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 197.3 (CO), 151.3 (C-6), 148.4 (C-3″), 148.3 (C-4″), 145.1 (C-arom), 140.8 (C-8), 139.5 (C-arom), 136.5 (C-2), 131.7 (C-9), 129.2 and 126.9 (o-, m-Ph), 123.8 (C-3), 122.1 (C-1″), 121.9 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 93.8 (C-7), 54.9 (3″-OCH₃, 4″-OCH₃), 55.0 (C-2′), 52.7 (C-3′), 47.2 (C-5′), 30.7 (C-4′), 27.1 (COCH₃), 21.7 (6-CH₃), 14.9 (2-CH₃). HRMS calcd for C₂₈H₃₁N₅O₅SNa m/z: 572.1938 (M + Na)⁺, found 572.1939.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)thiophene-2-sulfonamide (73).

Yield: 143 mg (70%). Chromatography: CHCl₃–acetone 21:3, crystallization from ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.33–8.26 (m, 1H, NH), 7.97 (dd, J = 5.0, 1.3 Hz, 1H, H-thienyl-4), 7.67 (dd, J = 3.7, 1.3 Hz, 1H, H-thienyl-2), 7.27 (d, J = 2.0 Hz, 1H, H-2″), 7.21 (dd, J = 5.0, 3.7 Hz, 1H, H-thienyl-3), 7.16 (dd, J = 8.5, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.5 Hz, 1H, H-5″), 5.74 (s, 1H, H-7), 4.04 (br s, 1H) and 3.98–3.90 (m, 1H) and 3.81 (s, 3H, H-2′, H-3′, OCH₃), 3.78 (s and br s, 6H, OCH₃, H-5′,) 2.38 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6CH₃), 2.14–2.03 (m, 1H, H-4′a), 1.94–1.83 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.3 (C-6), 148.4 (C-3″), 148.3 (C-4″), 142.0 (C-thienyl-1), 140.9 (C-8), 136.6 (C-2), 132.9 (C-thienyl), 132.0 (C-thienyl), 131.8 (C-9), 127.9 (C-thienyl), 123.9 (C3), 122.2 (C-1″), 122.0 (C-6″), 113.2 (C-2″), 111.8 (C-5″), 93.8 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.1 (C-2′), 52.8 (C-3′), 47.3 (C-5′), 30.7 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₄H₂₈N₅O₄S₂ m/z: 514.1577 (M + H)⁺, found 514.1578.

(R)-4-Cyano-N-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)benzenesulfonamide (74).

Yield: 160 mg (75%). Chromatography: CHCl₃–acetone 21:4, crystallization from ethyl acetate. [α]_D²⁰ +34.2 (c 0.263, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.40 (s, 1H, NH), 8.09–8.02 (m, 2H, H-arom), 8.02–7.97 (m, 2H, H-arom), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.16 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.72 (s, 1H, H-7), 4.05–3.52 (m, 11H, H2′, H-3′, H-5′, 3″-OCH₃, 4″-OCH₃), 2.36 (s, 3H, 2-CH₃), 2.27 (s, 3H, 6-CH₃), 2.14–2.00 (m, 1H, H-4′a), 1.88 (dd, J = 12.0, 6.3 Hz, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.3 (C-6), 148.4 (C-3″), 148.3 (C-4‴), 145.5 (C-arom), 140.8 (C-8), 136.5 (C-2), 133.6 (C-arom), 131.6 (C-9), 127.3 (C-arom), 123.9 (C-3), 122.1 (C-1″), 122.0 (C-6″), 117.9 and 115.0 (CN and C-arom), 113.3 (C-2″), 111.8 (C-5″), 93.8 (C-7), 55.7 (3‴-OCH₃, 4″-OCH₃), 54.9 (C-2′), 52.7 (C-3′), 47.1 (C-5′), 30.7 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₇H₂₉N₆O₄S m/z: 533.1966 (M + H)⁺, found 533.1965.

(R)-N-[1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl]sulfuric Diamide (75).

To a mixture of chlorosulfonyl isocyanate (105 μL, 1.2 mmol) in dry dichloromethane (3 mL) was added tert-butanol (116 μL, 1.2 mmol) at room temperature, and the resulting solution was stirred for 30 min. This solution was then transferred to a mixture of 50 (160 mg, 0.4 mmol) and triethylamine (0.28 mL, 2 mmol) in dichloromethane (5 mL). The reaction mixture was stirred overnight at room temperature, and the solvent was then evaporated. The residue was absorbed on silica gel (from chloroform) and filtered through a plug of silica gel (50 g, chloroform–ethanol 20:1). Bocylated intermediate was dissolved in a mixture of H₂O/DMF (13 mL, 10:3) and heated to 100 °C for an hour. The reaction mixture was then evaporated, and the product was isolated by column chromatography (100 g, chloroform–ethanol 10:1). An amount of 89 mg (50%) of the product was obtained, which was recrystallized from acetone. [α]_D²⁰ +19.8 (c 0.325, DMSO). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.27 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 6.98 (d, J = 6.4 Hz, 1H, NH), 6.68 (s, 2H, NH₂), 5.77 (s, 1H, C-7), 4.13 (br s, 1H) and 4.03–3.94 (m, 1H) and 3.81 (s and br s, 6H) and 3.78 (s, 3H, H-2′, H-3′, H-5′, 3″-OCH₃, 4-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.27–2.16 (m, 1H, H-4′a), 2.08–1.94 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.1 (C-8), 136.6 (C-2), 131.9 (C-9), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 111.8 (C-2″), 113.3 (C-5″), 93.8 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.1 (C-2′), 52.3 (C-3′), 47.8 (C-5′), 31.1 (C-5′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₀H₂₇N₆O₄S m/z: 447.1809 (M + H)⁺, found 447.1811.

Procedure for Introduction of Amine Substituents in Position 8 (76a–84a).

Compound 33 (500 mg, 1.53 mmol), appropriate amine (2 mmol), DIPEA (3 mmol for amines, 4 mmol for amine hydrochlorides) were dissolved in acetonitrile (10 mL). The reaction mixture was heated at 85 °C for 16 h and cooled down. Products were separated by column chromatography or directly as precipitated solids were filtered off and washed with acetonitrile.

6-Chloro-3-iodo-2-methyl-8-(pyrrolidin-1-yl)imidazo[1,2-b]pyridazine (76a).

Yield: 79%. Chromatography: toluene–ethyl acetate 20:1. UPLC–MS: t = 5.28 (M + H, 363.1/365.1).

4-(6-Chloro-3-iodo-2-methylimidazo[1,2-b]pyridazin-8-yl)morpholine (77a).

Yield: 81%. Chromatography: cyclohexane–ethyl acetate 10:1. UPLC–MS: t = 5.06 (M + H, 379.1/381.1).

N-(2-((6-Chloro-3-iodo-2-methylimidazo[1,2-b]pyridazin-8-yl)(ethyl)amino)ethyl)acetamide (78a).

Yield: 73%. Chromatography: CHCl₃–acetone 7:1. UPLC–MS: t = 4.46 (M + H, 422.2/424.1).

6-Chloro-8-(2,5-dihydro-1H-pyrrol-1-yl)-3-iodo-2-methylimidazo[1,2-b]pyridazine (79a).

Yield: 94%. Chromatography: toluene–ethyl acetate 40:1. UPLC–MS: t = 5.23 (M + H, 361.1/363.0).

(R)-6-Chloro-8-(3-fluoropyrrolidin-1-yl)-3-iodo-2-methylimidazo[1,2-b]pyridazine (80a).

Yield: 97%. Chromatography: toluene–ethyl acetate 15:1. UPLC–MS: t = 4.95 (M + H, 381.1/383.3).

6-Chloro-8-(3,3-difluoropyrrolidin-1-yl)-3-iodo-2-methylimidazo[1,2-b]pyridazine (81a).

Yield: 88%. Chromatography: cyclohexane–ethyl acetate 15:1. UPLC–MS: t = 5.15 (M + H, 399.1/401.3).

(R)-1-(6-Chloro-3-iodo-2-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-ol (82a).

Yield: 93%, precipitated solid was filtered off and used without further purification. UPLC–MS: t = 4.26 (M + H, 379.0/381.0).

(S)-1-(6-Chloro-3-iodo-2-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-ol (83a).

Yield: 89%, precipitated solid was filtered off and used without further purification. UPLC–MS: t = 4.26 (M + H, 379.0/381.0).

tert-Butyl 3-(6-Chloro-3-iodo-2-methylimidazo[1,2-b]pyridazin-8-yl)imidazolidine-1-carboxylate (84a).

Yield: 64%. Chromatography: cyclohexane–ethyl acetate 3:1. UPLC–MS: t = 5.43 (M + H, 463.3/466.2).

General method B with 3,4-dimethoxyphenylboronic acid as a coupling agent was used.

6-Chloro-3-(3,4-dimethoxyphenyl)-2-methyl-8-(pyrrolidin-1-yl)imidazo[1,2-b]pyridazine (76b).

Yield: 65%. Chromatography: CHCl₃–acetone 40:1. ¹H NMR (400 MHz, CDCl₃, 25 °C) δ 7.26 (s, 1H, covered by CDCl₃), 7.20 (dd, J = 8.4, 2.1 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 5.77 (s, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.93 (br s, 4H), 2.12–2.00 (m, 4H). ¹³C NMR (101 MHz, CDCl₃, 25 °C) δ 148.8, 147.3, 142.5, 137.9, 132.2, 122.4, 121.9, 112.9, 111.3, 93.3, 56.1, 15.1. UPLC–MS: t = 5.03 (M + H, 373.2/375.2).

4-(6-Chloro-3-(3,4-dimethoxyphenyl)-2-methylimidazo[1,2-b]pyridazin-8-yl)morpholine (77b).

Yield: 83%. Chromatography: cyclohexane–ethyl acetate 3:2. UPLC–MS: t = 5.64 (M + H, 389.3/391.3. ¹H NMR (401 MHz, CDCl₃, 25 °C) δ 7.23 (d, J = 2.0 Hz, 1H), 7.19 (dd, J = 8.3, 2.0 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.09 (s, 1H), 4.04–3.99 (m, 4H), 3.94 (s, 3H) and 3.92 (s, 3H) and 3.96–3.90 (m, 4H), 2.50 (s, 3H). ¹³C NMR (101 MHz, CDCl₃, 25 °C) δ 149.0, 148.9, 147.1, 138.1, 132.0, 125.5, 122.5, 121.4, 112.8, 111.3, 96.4, 66.7, 56.1, 48.2, 15.0.

N-(2-((6-Chloro-3-(3,4-dimethoxyphenyl)-2-methylimidazo-[1,2-b]pyridazin-8-yl)(ethyl)amino)ethyl)acetamide (78b).

Yield: 65%. Chromatography: CH₂Cl₂–ethanol 25:1. UPLC–MS: t = 4.34 (M + H, 432.4/434.3). ¹H NMR (400 MHz, DMSO-d₆, 25 °C): δ 8.06 (t, J = 5.8 Hz, 1H), 6.96–7.31 (m, 3H), 6.26 (s, 1H), 3.68 (2 × br m + 2s, 10H), 3.40–3.33 (m, 2H), 2.40 (s, 3H), 1.80 (s, 3H), 1.21 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.87, 148.77, 148.55, 146.58, 142.96, 137.10, 130.81, 124.77, 122.28, 121.06, 113.33, 111.90, 93.41, 55.80, 49.60, 46.42, 36.81, 22.80, 14.83, 13.20.

6-Chloro-8-(2,5-dihydro-1H-pyrrol-1-yl)-3-(3,4-dimethoxyphenyl)-2-methylimidazo[1,2-b]pyridazine (79b).

Yield: 65% (85% purity). Chromatography: CHCl₃–acetone 40:1. UPLC–MS: t = 4.85 (M + H, 370.2/372.1).

(R)-6-Chloro-3-(3,4-dimethoxyphenyl)-8-(3-fluoropyrrolidin-1-yl)-2-methylimidazo[1,2-b]pyridazine (80b).

Yield: 92%. Chromatography: CHCl₃–ethyl acetate 15:1. UPLC–MS: t = 4.85 (M + H, 390.1/393.2). 1H NMR (401 MHz, DMSO-d₆, 25 °C) δ 7.20 (d, J = 1.9 Hz, 1H), 7.16 (dd, J = 8.3, 1.9 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.05 (s, 1H), 5.50 (dt, J = 53.0, 3.4 Hz, 1H), 3.82 (s, 2H), 3.79 (s, 2H), 2.41 (s, 2H), 2.36–2.08 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 148.8, 148.8, 146.5, 142.0, 137.6, 131.2, 125.0, 122.2, 121.0, 113.23, 111.9, 93.3, 55.8, 55.7, 14.8 (carbon signals of pyrrolidine ring were not detected due to a signal broadening).

6-Chloro-8-(3,3-difluoropyrrolidin-1-yl)-3-(3,4-dimethoxyphenyl)-2-methylimidazo[1,2-b]pyridazine (81b).

Yield: 87%. Chromatography: cyclohexane–ethyl acetate 4:1. UPLC–MS: t = 5.05 (M + H, 409.3/411.2). ¹H NMR (400 MHz, DMSO-d₆, 25 °C): δ 2.41 (s, 3H), 2.53–2.66 (m, 2H), 3.78 (s, 3H), 3.82 (s, 3H), 4.08 (br s, 2H), 4.40 (br s, 2H), 6.13 (s, 1H), 7.07–7.23 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 148.8, 148.6, 146.5, 142.0, 137.6, 131.2, 128.2 (t, J = 245.7 Hz), 125.0, 122.2, 121.0, 113.2, 111.9, 93.3, 55.8, 55.7, 47.7, 32.7 14.8 (one CH₂ carbon on pyrrolidine ring was not detected due to a signal broadening).

(R)-1-(6-Chloro-3-(3,4-dimethoxyphenyl)-2-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl Acetate (82b).

Residue after Suzuki coupling was not purified on silica gel column but was immediately acetylated (Ac₂O (1.2 equiv), Et₃N (2 equiv), DMAP (cat.), CH₃CN) to afford acetyl derivative. Yield: 89% (two steps). Chromatography: toluene–ethyl acetate 3:1. UPLC–MS: t = 4.77 (M + H, 431.3/433.3). ¹H NMR (400 MHz, DMSO-d₆, 25 °C): δ 7.20 (d, J = 1.9 Hz, 1H), 7.16 (dd, J = 8.4, 2.0 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.03 (s, 1H), 5.38 (tt, J = 4.4, 2.0 Hz, 1H), 4.91–3.48 (2 × br s, 4H), 3.83 (s, 3H), 3.79 (s, 3H), 2.40 (s, 3H), 2.32–2.30 (m, 1H), 2.19–2.11 (m, 1H), 2.03 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 170.3, 148.8, 148.5, 146.5, 142.0, 137.6, 131.2, 124.9, 122.2, 121.0, 113.2, 111.9, 93.2, 55.8, 55.7, 21.1, 14.8 8 (carbons on pyrrolidine ring were not detected due to a signal broadening).

(S)-1-(6-Chloro-3-(3,4-dimethoxyphenyl)-2-methylimidazo-[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl Acetate (83b).

Residue after Suzuki coupling was not purified on silica gel column but was immediately acetylated (Ac₂O (1.2 equiv), Et₃N (2 equiv), DMAP (cat.), CH₃CN) to afford acetyl derivative. Yield: 89% (two steps). Chromatography: toluene–ethyl acetate 3:1. UPLC–MS: t = 4.77 (M + H, 431.3/433.3). The NMR spectra were identical to 82b.

tert-Butyl 3-(6-Chloro-3-(3,4-dimethoxyphenyl)-2-methylimidazo[1,2-b]pyridazin-8-yl)imidazolidine-1-carboxylate (84b).

Yield: 92%. Chromatography: cyclohexane–ethyl acetate 2:1. UPLC–MS: t = 5.30 (M + H, 474.4/476.5). ¹H NMR (400 MHz, DMSO-d₆, 25 °C): δ 7.20 (d, J = 1.9 Hz, 1H), 7.16 (dd, J = 8.4, 1.9 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.12 (s, 1H), 5.24 (br s, 2H), 3.99 (br s, 2H), 3.82 (s, 3H), 3.79 (s, 3H), 3.66 (dd, J = 7.6, 6.0 Hz, 2H), 2.41 (s, 3H), 1.46 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 152.5, 148.8, 148.6, 146.5, 140.6, 138.0, 130.8, 125.1, 122.2, 120.8, 113.2, 111.9, 78.0, 55.8, 55.7, 28.2, 14.8 (carbons on imidazolidine ring were not detected due to a signal broadening).

General Procedure for Methylation of Position 6 (Method D).

To a solution of DABCO (98 mg, 0.88 mmol) in 3 mL freshly distilled THF, AlMe₃ (2 M in hexane, 0.89 mL, 1.78 mmol) was added dropwise, and the mixture was stirred at room temperature for 30 min under argon atmosphere. A solution of chloro derivative (1 mmol), Pd₂(dba)₃ (50 mg, 0.06 mmol), and X-Phos (57 mg, 0.12 mmol) in 15 mL of freshly distilled THF was subsequently added to the solution, and the reaction mixture was stirred at 75 °C overnight under argon atmosphere. The mixture was cooled to 0 °C and quenched with sat. NH₄Cl (4 mL), diluted with acetone and ethyl acetate, and filtered through Celite. The Celite pad was thoroughly washed with acetone and ethyl acetate. The filtrate was evaporated, and the residue was purified by silica gel column chromatography (120 g). Solids were then crystallized from the appropriate solvent.

3-(3,4-Dimethoxyphenyl)-2,6-dimethyl-8-(pyrrolidin-1-yl)imidazo[1,2-b]pyridazine (76c).

Yield: 73%. Chromatography: CHCl₃–acetone 30:1. Crystallization: ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.29 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″) 5.76 (s, 1H, H-7), 3.80 (br s, 4H, H-2′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″OCH₃), 2.39 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6-CH₃), 1.91–2.01 (m, 4H, H-3′). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.3 (C-6), 148.4 (C-3″), 148.2 (C-4″), 141.1 (C-8), 136.4 (C-2), 132.0 (C-9), 123.8 (C-3), 122.3 (C-1″), 122.0 (C-6″), 113.3 (C-2″), 111.8 (C-5″), 93.7 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 49.6 (C-2′), 25.1 (C-3′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₀H₂₅N₄O₂ m/z: 353.1972 (M + H)⁺, found 353.1972.

4-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)morpholine (77c).

Yield: 93%. Chromatography: CHCl₃–acetone 20:1. Crystallization: ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.26 (d, J = 2.0 Hz, 1H, H-2″), 7.16 (dd, J = 8.4, 2.0 Hz, 1H, H-6″)), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 6.24 (s, 1H, H-7), 3.91 (t, J = 4.7 Hz, 4H, NCH₂), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 3.79–3.74 (m, 4H, OCH₂), 2.39 (s, 1H, 2-CH₃), 2.33 (s, 1H, 6-CH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.5 (C-6), 148.5 (C-3″), 148.4 (C-4″), 143.4 (C-8), 136.5 (C-2), 132.0 (C-9), 123.9 (C-3), 122.1 (C-6″), 121.9 (C-1″), 113.3 (C-2″), 111.8 (C-5″), 97.1 (C-7), 66.1 (OCH₂), 55.7 (3″-OCH₃, 4″-OCH₃), 47.9 (NCH₂), 21.8 (6-CH₃), 14.8 (2-CH₃). HRMS calcd for C₂₀H₂₅N₄O₃ m/z: 369.1921 (M + H)⁺, found 369.1921.

N-(2-((3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)(ethyl)amino)ethyl)acetamide (78c).

Yield: 86%. Chromatography: (1) CH₂Cl₂–ethanol 25:1, (2) CH₂Cl₂–acetone 2:1. Crystallization: The product was isolated as a foam. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.09 (t, J = 5.7 Hz, 1H, NH), 7.27 (d, J = 1.9 Hz, 1H, H-2″), 7.16 (dd, J = 8.4, 1.9 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 6.08 (s, 1H, H-7), 3.97 (q, J = 6.5 Hz, 2H, H4′), 3.85 (t, J = 6.5 Hz, 2H, H-2′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″OCH₃, 4″-OCH₃), 3.40–3.33 (m, 2H, H-3′), 2.40 (s, 3H, 2-CH₃), 2.00 (s, 3H, 6-CH₃), 1.80 (s, 3H, COCH₃), 1.19 (t, J = 6.9 Hz, 3H, CH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.7 (CO), 151.2 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.7 (C-8), 136.2 (C-2), 131.5 (C-9), 123.8 (C-3), 122.1 (C-1″), 122.0 ((C-6″), 113.4 (C-2″), 111.8 (C-5″), 94.6 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 49.3 (C-2′), 45.8 (C-4′), 37.1 (C-3′), 22.8 (COCH₃), 21.7 (6-CH₃), 15.0 (2-CH₃), 13.1 (CH₃). HRMS calcd for C₂₂H₃₀N₅O₃ m/z: 412.2343 (M + H)⁺, found 412.2327.

8-(2,5-Dihydro-1H-pyrrol-1-yl)-3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazine (79c).

Yield: 40%. Chromatography: (1) CHCl₃–acetone 30:1, (2) reverse phase flash chromatography (C18, 50 g, water/methanol 30% to 100%). Crystallization: ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.29 (d, J = 2.0 Hz, 1H, H-2″), 7.18 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.4 Hz, 1H, H-5″), 6.07 (s, 2H, –CH═CH–), 5.77 (s, 1H, H-7), 4.60 (br s, 4H, CH₂NCH₂), 2.40 (s, 3H, 2-CH₃), 2.30 (s, 3H, 6-CH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.5 (C-6), 148.4 (C-3″), 148.3 (C-4″), 140.5 (C-8), 136.8 (C-2), 131.8 (C-9), 125.9* (–CH═CH–), 124.0 (C-3), 122.2 (C-1″), 122.0 (C-6″), 113.3 (C-2″), 111.8 (C-5″), 93.8 (C-7), 56.5* (CH₂NCH₂), 55.7 (3″-OCH₃, 4″-OCH₃), 21.7 (2-CH₃), 15.0 (6-CH₃). HRMS calcd for C₂₀H₂₄N₄O₂ m/ z: 351.1816 (M + H)⁺, found 351.1813.

(R)-3-(3,4-Dimethoxyphenyl)-8-(3-fluoropyrrolidin-1-yl)-2,6-dimethylimidazo[1,2-b]pyridazine (80c).

Yield: 68%. Chromatography: CHCl₃–acetone 15:1. Crystallization: ethyl acetate. [α]_D²⁰ –19.5 (c 0.303, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.4 Hz, 1H, H-5″), 5.86 (s, 1H, H-7), 5.48 (dt, J = 53.4, 3.6 Hz, 1H, H-3′), 4.52–4.23 (br s, 1H) and 4.15–3.86 (m, 2H) and 3.85–3.63 (m, 7H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 2.40 (s, 3H, 2-CH₃), 2.35–2.08 (s + m, 5H, H-4′, 6-CH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.0 (C-8), 136.7 (C-2), 131.8 (C-9), 124.0 (C-3), 122.1 (C-1″), 122.0 (C-6″), 113.3 (C-2″), 111.8 (C-5″), 94.3 (c-7), 93.0 (d, J = 172.0 Hz, C-3′), 56.3 (d, J = 23.2 Hz, C-2′), 55.7 (3″-OCH₃, 4″-OCH₃), 47.1 (C-5′), 31.4 (d, J = 20.8 Hz, C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₀H₂₄FN₄O₂ m/z: 371.1878 (M + H)⁺, found 371.1895.

8-(3,3-Difluoropyrrolidin-1-yl)-3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazine (81c).

Yield: 60%. Chromatography: cyclohexane–ethyl acetate 5:2. Crystallization: ethyl acetate (freezer). [α]_D²⁰ +2.4 (c 0.333, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.4 Hz, 1H, H-5″), 5.94 (s, 1H, H-7), 4.37 (t, J = 13.1 Hz, 2H) and 4.05–3.94 (m, 2H, H-2′, H-5′), 3.82 (s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.68–2.51 (m, 2H, H-4′), 2.41 (s, 3H, 2-CH₃), 2.32 (s, 3H, 6-CH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.5 (C-6), 148.5 (C-3″), 148.4 (C-4″), 140.7 (C-8), 137.0 (C-2), 131.5 (C-9), 128.5 (t, J = 245.7 Hz, C-3′), 124.1 (C-3), 122.0 (C-6″), 121.9 (C-1″), 113.2 (C-2″), 111.8 (C-5″), 94.8 (C-7), 56.0 (t, J = 32.4 Hz, C2′), 55.7 (3″-OCH₃, 4″-OCH₃), 47.2 (C-5′), 32.9 (t, J = 23.3 Hz, C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₀H₂₃F₂N₄O₂ m/z: 389.1784 (M + H)⁺, found 389.1720.

(R)-1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-ol (82c).

During the methylation step, partial deacetylation occurred. After filtration through Celite, the residue was then completely deacetylated by potassium carbonate (1 equiv) in methanol. Yield: 79%. Chromatography: CHCl₃–ethanol 20:1. Crystallization: ethyl acetate. [α]_D²⁰ –11.2 (c 0.277, DMSO). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.29 (d, J = 2.0 Hz, 1H,H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.77 (s, 1H, H-7), 5.01 (d, J = 3.4 Hz, 1H, OH), 4.45–4.38 (m, 1H, H-3′), 4.13–3.58 (br s + 2s, 10H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6-CH₃), 2.09–1.98 (m, 1H, H-4′a), 1.96–1.88 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.3 (C-2), 148.4 (C-3″), 148.2 (C-4″), 141.3 (C-8), 136.5 (C-2), 132.0 (C-9), 123.8 (C-3), 122.3 (C-1″), 122.0 (C-6″), 113.3 (C-2″), 111.8 (C-6″), 93.6 (C-7), 69.1 (C-3′), 58.2 (C-2′), 55.7 (3″-OCH₃, 4″-OCH₃), 47.5 (C-5′), 33.3 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₀H₂₅N₄O₃ m/z: 369.1921 (M + H)⁺, found 369.1932.

(S)-1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-ol (83c).

During the methylation step, partial deacetylation occurred. Residue after filtration through Celite was then completely deacetylated by potassium carbonate (1 equiv) in methanol. Yield: 74%. Chromatography: CHCl₃–ethanol 20:1. Crystallization: ethyl acetate. Physical and spectral properties are identical to those for compound 82c. [α]_D²⁰ +13.5 (c 0.325, DMSO). HRMS calcd for C₂₀H₂₅N₄O₃ m/z: 369.1921 (M + H)⁺, found 369.1932.

tert-Butyl 3-(3-(3, 4-Dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl)imidazolidine-1-carboxylate (84c).

Yield: 75%. Chromatography: cyclohexane–ethyl acetate 1:1. Crystallization: ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.4 Hz, 1H, H-5″), 5.95 (s, 1H, H-7), 5.17 (br s, 2H, H-2), 3.97 (br s, 2H) and 3.65 (dd, J = 7.6, 6.0 Hz, 2H, H-4′, H5′), 3.82 (s, 3H) and 3.79 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.41 (s, 3H, 2-CH₃), 2.32 (s, 3H, 6-CH₃), 1.47 (s, 9H, C(CH₃)₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 152.6 (CO), 151.6 (C-6), 148.5 (C-3″), 148.4 (C-4″), 139.6 (C-8), 137.1 (C-2), 131.4 (C-9), 124.2 (C-3), 122.0 (C-6″), 121.9 (C-1″), 113.2 (C-2″), 111.8 (C-5″), 95.4 (C-7), 79.8 (C(CH₃)₃), 55.7 (3″-OCH₃, 4″-OCH₃), 43.6 (C-2′or C-5′), 28.2 (C(CH₃)₃), 21.6 (6-CH₃), 15.0 (2-CH₃) (two CH₂ carbons were not detected due to a signal broadening). HRMS calcd for C₂₄H₃₂N₅O₄ m/ z: 454.2449 (M + H)⁺, found 454.2491.

(R)-S-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo-[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl) Ethanethioate (85).

A solution of the starting material 83c (500 mg, 1.36 mmol), triphenylphosphine (712 mg, 2.72 mmol) in THF (14 mL) was cooled to 0 °C, and DIAD (0.54 mL, 2.72 mmol) was dropwise added followed by dropwise addition of thioacetic acid (194 μL, 2.72 mL). The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was cooled to 0 °C, and the same amount of the reagents was added again. The reaction mixture was stirred at room temperature overnight, and the solvent was then evaporated. The residue was chromatographed on silica gel (200 g, CHCl₃–acetone 30:1). Fractions containing the product were evaporated and subjected to reverse-phase flash chromatography (C18, 100 g, water/acetonitrile 10% to 100%). An amount of 156 mg (27%) of the product was obtained. [α]_D²⁰ +46.8 (c 0.265, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.27 (d, J = 2.0 Hz, 1H, H-2″), 7.16 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.3 Hz, 1H, H-5″), 5.82 (s, 1H, H-7), 4.32 (br s, 1H) and 4.03–4.14 (m, 1H) and 3.74 (m, 9H, H-2′, H-3′, H-5′, 3″-OCH₃, 4″-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.47–2.39 (m, 1H, H-4′a), 2.37 (s, 3H, COCH₃), 2.29 (s, 3H, 6-CH₃), 2.05–1.95 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 195.4 (CO), 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 140.8 (C-8), 136.7 (C-2), 131.7 (C-9), 124.0 (C-3), 122.1 (C-1″), 122.0 (C-6″), 113.3 (C-2″), 111.9 (C-5″), 94.2 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.4 (C-2′), 48.3 (C-5′), 41.1 (C-3′), 30.8 (C-4′), 30.7 (COCH₃), 21.6 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₂H₂₇N₄O₃S m/z: 427.1798 (M + H)⁺, found 427.1824.

(R)-8-(3-Azidopyrrolidin-1-yl)-3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazine (86).

To a solution of compound 83c (500 mg, 1.37 mmol), triphenylphosphine (538 mg, 2.05 mmol) in THF (20 mL) was added DIAD (0.4 mL, 2.05 mmol) at 0 °C followed by dropwise addition of diphenylphosphoryl azide (0.59 mL, 2.72 mmol) at 0 °C. The reaction mixture was slowly allowed to room temperature and stirred overnight. The solvent was then evaporated. The residue was purified on flash chromatography (120 g, CHCl₃–ethyl acetate 10:1). Fractions containing the product were evaporated and subjected to reverse-phase flash chromatography (C18, 100 g, water/acetonitrile 10% to 100%). An amount of 425 mg (79%) of the product was obtained. [α]_D²⁰ –53.5 (c 0.325, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.84 (s, 1H, H-7), 4.56–4.49 (m, 1H, H-3′), 4.20 (br s, 1H) and 3.99 (br s, 1H) and 3.65 (m, 8H, H-2′, H-5′, 3″-OCH₃, 4″-OCH₃), 2.40 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.28–2.19 (m, 1H, H-4′a), 2.15–2.06 (m, 1H, H4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.4 (C-6), 148.4 (C-3″), 148.3 (C-4″), 140.8 (C-8), 136.7 (C-2), 131.7 (C-9), 124.0 (C-3), 122.1 (C-1″), 122.0 (C-6″), 111.8 (C-2″), 113.3 (C-5″), 94.2 (C-7), 60.2 (C-3′), 55.7 (3″-OCH₃, 4″-OCH₃), 54.7 (C-2′), 47.4 (C-5′), 30.3 (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₀H₂₄N₇O₂ m/z: 394.1986 (M + H)⁺, found 394.2022.

(R)-3-(3,4-Dimethoxyphenyl)-2,6-dimethyl-8-(3-(4-phenyl-1H−1,2,3-triazol-1-yl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazine (87).

To a solution of azide 86 (200 mg, 0.56 mmol) and phenylacetylene (92 μL, 0.84 mmol) in THF/H₂O (9 mL, 2:1) were added CuSO₄·5H₂0 (7 mg, 0.03 mmol) and sodium ascorbate (11 mg, 0.06 mmol) at room temperature under an argon atmosphere. The reaction mixture was heated at 55 °C for 14 h. Resulting solution was evaporated and chromatographed on silica gel (150 g, CHCl₃–acetone 8:1). An amount of 191 mg (69%) of the solid was obtained, which was recrystallized from ethyl acetate/acetone. [α]_D²⁰ –106.3 (c 0.223, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.74 (s, 1H, Htriazole), 7.92–7.75 (m, 2H, o-Ph), 7.44 (t, J = 7.6 Hz, 2H, m-Ph), 7.37–7.29 (m, 1H, p-Ph), 7.28 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.3, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H,H-5″), 5.91 (s, 1H, H-7), 5.55–5.32 (m, 1H, H-3′), 4.59 (br s, 1H) and 4.45 (br s, 1H, H-2′), 3.99 (br s, 2H, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.71–2.54 (m, 2H, H-4′), 2.39 (s, 3H, 2-CH₃), 2.31 (s, 3H, 6-CH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.5 (C-6), 148.4 (C-3″), 148.3 (C-4″), 146.7 (i-Ph), 140.8 (C-8), 136.7 (C-2), 131.8 (C-9), 130.8 (C-triazole-quart.), 128.1 (m-Ph), 129.0 (p-Ph), 125.3 (o-Ph), 124.0 (C-3), 122.1 (C-1″), 122.0 (C-6″), 120.7 (C-5-triazole), 111.8 (C-2″), 113.3 (C-5″), 94.5 (C-7), 59.2 (C-3′), 55.7 (3″-OCH₃, 4″-OCH₃), 55.3 (C-2′), 47.9 (C-5′), 30.8 (C-4′), 21.7 (6-CH₃), 14.9 (2-CH₃). HRMS calcd for C₂₈H₃₀N₇O₂ m/z: 496.2455 (M + H)⁺, found 496.2461.

(R)-4-(1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)morpholine (88).

Hydrochloride 50 (320 mg, 0.73 mmol) was combined with sodium iodide (55 mg, 0.36 mmol), DIPEA (1 mL, 5.8 mmol), 2,2′-bischlorodiethyl ether (130 μL, 1.1 mmol) in DMF (4 mL) at room temperature, and the resulting mixture was heated to 100 °C for 24 h. Reaction mixture was cooled down. DIPEA (0.5 mL, 2.9 mmol) and 2,2′-bischlorodiethyl ether (130 μL, 1.1 mmol) were added, and then heating was continued for 5 h at 110 °C. Solvents were evaporated and the residue was chromatographed on silica gel (150 g, CHCl₃–acetone 1:1). Final purification of the product (101 mg, 31%) was achieved by reverse-phase flash chromatography (C18, 50 g, water/acetonitrile 10% to 100%). [α]_D²⁰ +28.8 (c 0.250, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.27 (d, J = 2.0 Hz, 1H, H-2″), 7.16 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.80 (s, 1H, H-7), 4.12 (br s, 2H, H-2′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 3.70 (br s, 1H, H-5′), 3.61 (t, J = 4.6 Hz, 4H, OCH₂), 3.55 (br s, 1H, H-5′), 2.97–2.87 (m, 1H, H-3′), 2.50–2.40 (m, 4H, NCH₂), 2.39 (s, 3H, 2-CH₃), 2.28 (s, 3H, 6-CH₃), 2.23–2.14 (m, 1H, H-4′a), 1.92–1.76 (m, 1H, H-4′b). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 151.4 (C-6), 148.4 (C-3), 148.3 (C-4″), 141.0 (C-8), 136.6 (C-2), 131.8 (C-9), 123.9 (C-3), 122.2 (C-1″), 122.0 (C-6″), 111.8 (C-2″), 113.3 (C-5″), 93.8 (C-7), 66.3 (OCH₂), 63.9 (C-3′), 55.7 (3″-OCH₃, 4″-OCH₃), 53.1 (C-2′), 51.9 (NCH₂), 48.4* (C-5′), 28.4* (C-4′), 21.7 (6-CH₃), 15.0 (2-CH₃). HRMS calcd for C₂₄H₃₂N₅O₃ m/z: 438.2500 (M + H)⁺, found 438.2503.

(R)-1-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl Acetate (89).

To a mixture of starting material 82c (221 mg, 0.6 mmol), triethylamine (0.22 mL, 1.6 mmol), and DMAP (cat. amount) was added acetanhydride (0.1 mL, 1.1 mmol) at room temperature. The reaction mixture was stirred for 2 h, and the solvent was then evaporated. The residue was chromatographed on a silica gel column (50 g, toluene–ethyl acetate 3:1) to afford 220 mg (89%) of the product. Analytical sample was then recrystallized from ethyl acetate. [α]_D²⁰ –8.7 (c 0.333, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.27 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.84 (s, 1H, H-7), 5.40–5.32 (m, 1H, H-3′), 4.28–3.63 (m, 10H, H-2′, H5′, 3″-OCH₃, 4″-OCH₃), 2.39 (s, 3H, 2-CH₃), 2.29 (s, 3H, 6-CH₃), 2.28–2.18 (m, 1H, H-4′a), 2.18–2.07 (m, 1H, H-4′b), 2.01 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 170.4 (CO), 151.5 (C-6), 148.4 (C-3″), 148.3 (C-4″), 141.0 (C-8), 136.7 (C-2), 131.8 (C-9), 124.0 (C-3), 122.2 (C-1″), 122.0 (C-6″), 111.8 (C-2″), 113.3 (C-5″), 94.2 (C-7), 73.4 (C-3′), 55.8 (3″-OCH₃, 4″-OCH₃), 55.5 (C-2′), 47.4 (C-5′), 30.5 (C-4′), 21.7 (6-CH₃), 21.1 (COCH₃), 15.0 (2-CH₃). HRMS calcd for C₂₇H₂₇N₄O₄ m/z: 411.2027 (M + H)⁺, found 411.2029.

Cyclohexyl(4-(3-(3, 4-dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl)piperazin-1-yl)methanone (91).

Compound 90 (200 mg, 0.43 mmol) was dissolved in a mixture of trifluoroacetic acid and dichloromethane (8 mL, 1:7, v/ v). The reaction mixture was stirred at room temperature for 2 h, evaporated, coevaporated twice with acetonitrile (2 × 10 mL), and redissolved in acetonitrile (15 mL). Et₃N (0.3 mL, 2.15 mmol), catalytic amount of DMAP, and cyclohexanecarbonyl chloride (88 μL, 0.65 mmol) were sequentially added to this solution. The reaction mixture was stirred at room temperature for 2 h, then methanol (1 mL) was added and the mixture was evaporated. The residue was purified by column chromatography (75 g, chloroform–acetone 5:1) to obtain 154 mg (75%) of the product. Obtained solid was recrystallized from ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.26 (d, J = 2.0 Hz, 1H, H-2″), 7.16 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.4 Hz, 1H, H-5″), 6.24 (s, 1H, H-7), 4.05 (br s, 2H, CH₂-piperazine), 3.84 (br s, 2H, CH₂-piperazine), 3.82 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 3.74–3.57 (m, 4H, CH₂-piperazine), 2.64 (q, J = 10.3, 7.4 Hz, 1H, COCH), 2.40 (s, 3H, 2-CH₃), 2.33 (s, 3H, 6-CH₃), 1.79–1.59 (m, 4H, cyclohexyl-CH₂), 1.33 (dt, J = 11.4, 8.1 Hz, 4H, cyclohexyl-CH₂), 1.27–1.09 (m, 1H, cyclohexyl-CH₂). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 173.8 (CO), 151.5 (C-6), 148.5 (C-3″), 148.4 (C-4″), 143.0 (C-8), 136.4 (C-2), 131.9 (C-9), 123.9 (C-3), 122.1 (C-6″), 121.9 (C-1″), 113.3 (C-2″), 111.8 (C-5″), 97.3 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 48.0 (CH₂-piperazine), 47.1 (CH₂-piperazine), 44.5 (CH₂-piperazine), 40.7 (CH₂-piperazine), 39.2 (COCH), 29.3 (cyclohexyl-CH₂), 25.8 (cyclohexyl-CH₂), 25.3 (cyclohexyl-CH₂), 21.8 (6-CH₃), 14.9 (2-CH₃). HRMS calcd for C₂₇H₃₆O₄N₅ (M + H)⁺ 478.2813; found, 478.2806.

1-(4-(3-(3,4-Dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)piperazin-1-yl)ethan-1-one (92).

Compound 90 (281 mg, 0.6 mmol) was dissolved in a mixture of trifluoroacetic acid and dichloromethane (12 mL, 1:5, v/v). The reaction mixture was stirred at room temperature for 2 h, evaporated, coevaporated twice with acetonitrile (2 × 15 mL), and redissolved in acetonitrile (14 mL). Et₃N (0.4 mL, 2.86 mmol), catalytic amount of DMAP, and acetic anhydride (100 μL, 1.06 mmol) were sequentially added to this solution. The reaction mixture was stirred at room temperature for 2 h, and then methanol (1 mL) was added and the mixture was evaporated. The residue was purified by column chromatography (100 g, chloroform–ethanol 20:1) to obtain 221 mg (90%) of the product. Obtained solid was recrystallized from ethyl acetate. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 7.26 (d, J = 2.0 Hz, 1H,H-2″), 7.16 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.08 (d, J = 8.4 Hz, 1H, H-5″), 6.25 (s, 1H, h-7), 4.07 (t, J = 5.2 Hz, 2H, CH₂-piperazine), 3.91–3.84 (m, 2H, CH₂-piperazine), 3.82 (s, 3H) and3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 3.63 (dt, J = 6.4, 2.8 Hz, 4H, CH₂-piperazine), 2.41 (s, 3H, 6-CH₃), 2.34 (s, 3H, 2-CH₃), 2.06 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 168.6 (CO), 151.5 (C-6), 148.5 (C-3″), 148.4 (C-4″), 142.9 (C-8), 136.4 (C-2), 131.9 (C-9), 123.9 (C-3), 122.1 (C-6″), 121.9 (C-1″), 113.4 (C-2″), 111.8 (C-5″), 97.2 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 47.5 (CH₂-piperazine), 47.0 (CH₂-piperazine), 45.4 (CH₂-piperazine), 40.5 (CH₂-piperazine), 21.7 (COCH₃), 21.4 (6-CH₃), 14.9 (2-CH₃). HRMS calcd for C₂₂H₂₅N₅O₃ m/z: 410.2187 (M + H)⁺, found 410.2188.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-2-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (93).

Compound 49 (200 mg, 0.47 mmol) was dissolved in a mixture of methanol (100 mL), DMF (20 mL) and Et₃N (1 mL), and then Pd/C (200 mg) was added. The flask was purged with argon (three times) and then with hydrogen (three times). The reaction mixture was stirred for 16 h, filtered through Celite, and the filtrate was evaporated. The residue was chromatographed on a silica gel column (100 g, ethyl acetate–toluene–acetone–ethanol 17:4:3:1) to obtain 113 mg (61%) of the product. 41 mg of starting material 49 was recovered. The obtained solid was recrystallized from ethyl acetate. [α]_D²⁰ +9.6 (c 0.312, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.19 (d, J = 6.6 Hz, 1H, NH), 7.92 (d, J = 5.6 Hz, 1H, H-6), 7.22 (d, J = 2.0 Hz, 1H, H-2″), 7.17 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.07 (d, J = 8.4 Hz, 1H, H-5″), 5.87 (d, J = 5.6 Hz, 1H, H-7), 4.37 (p, J = 5.7 Hz, 1H, H-3′), 4.11 (br s, 1H) and 3.81 (brs, 3H, H-2′, H-5′), 3.81 (s, 3H) and 3.78 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.41 (s, 3H, 2-CH₃), 2.18 (dq, J = 13.7, 7.3 Hz, 1H, H-4′a), 1.96–1.87 (m, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (CO), 148.5 (C-3″), 148.4 (C-4″), 143.5 (C-6), 141.6 (C-8), 136.9 (C-2), 132.8 (C-9), 124.2 (C-3), 122.2 (C-6″), 122.0 (C-1″), 113.3 (C-2″), 111.8 (C-5″), 92.7 (C-7), 55.8 and 55.7 (3″-OCH₃, 4″-OCH₃), 55.4 (C-2′), 48.8 (C-3′), 47.9 (C-5′), 30.5 (C-4′), 22.8 (COCH₃), 14.9 (2-CH₃). HRMS calcd for C₂₁H₂₆N₅O₃ m/z: 396.2030 (M + H)⁺, found 396.2031.

3-Bromo-6,8-dichloroimidazo[1,2-b]pyridazine (95).

A suspension of compound 94 (820 mg, 3.9 mmol) in isopropanol (11 mL) and aqueous solution of chloroacetaldehyde (1.5 mL, 50%) was heated at 95 °C (bath) for 24 h. The reaction mixture was evaporated, and the residue was taken into ethyl acetate (200 mL). Organic phase was washed with saturated aqueous NaHCO₃ (2 × 100 mL). The organic phase was dried over Na₂SO₄ and evaporated. The residue was filtered through a silica gel plug (40 g, cyclohexane–ethyl acetate 1:1). The obtained solid (400 mg) was used in the next step without further purification. The solid was dissolved in chloroform (11 mL), and NBS (575 mg, 3.2 mmol) was added at room temperature. Then the reaction mixture was heated to 70 °C (bath) for 16 h. Consequently, a second portion of NBS (575 mg, 3.2 mmol) was added at room temperature, and then heating at 70 °C (bath) was continued for another 5 h. The reaction mixture was evaporated, and the residue was taken into ethyl acetate (200 mL). Organic phase was washed with saturated aqueous NaHCO₃ (2 × 100 mL). The organic phase was dried over Na₂SO₄ and evaporated. Product was isolated by flash chromatography (80 g, cyclohexane–ethyl acetate 0 → 15%) as an off-white solid (472 mg, 45%). ¹H NMR (401 MHz, DMSO-d₆, 25 °C) δ 8.03 (s, 1H), 7.91 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 146.8, 136.7, 134.9, 134.3, 118.9, 102.9. UPLC–MS: t = 4.55 (M + H, 265.9/267.8/269.8).

(R)-N-(1-(3-Bromo-6-chloroimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (96).

A mixture of starting material 95 (400 mg, 1.5 mmol), (R)-N-(pyrrolidin-3-yl)acetamide 11(R) (211 mg, 1.65 mmol), DIPEA (0.35 mL, 1.95 mmol) in acetonitrile (10 mL) was heated at 85 °C for 16 h, then cooled down, and evaporated. The residue was purified by column chromatography on silica gel (100 g, ethyl acetate → ethyl acetate–ethanol 10:1) to yield 520 mg (97%). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.17 (s, 1H), 7.69 (s, 1H), 6.08 (s, 1H), 4.35 and 4.22 (2 × br s, 3H), 3.65 and 3.52 (2 × br s, 2H), 2.16 (dq, J = 14.0, 7.6 Hz, 1H), 2.00–1.85 (m, 1H), 1.80 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4, 148.3, 142.6, 133.6, 131.0, 101.0, 93.6, 49.1*, 47.5*, 29.2 *, 22.7. UPLC–MS: t = 3.30 (M + H, 358.2/360.2/360.2).

(R)-N-(1-(6-Chloro-3-(3,4-dimethoxyphenyl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (97).

A suspension of starting material 96 (480 mg, 1.29 mmol), 3,4-dimethoxyphenylboronic acid (284 mg, 1.55 mmol), sodium carbonate (207 mg, 1.94 mmol) in a mixture of dioxane and water (20 mL, 4:1) was three times purged with argon. Then Pd(dppf)₂Cl₂ (53 mg, 0.065 mmol) was added, and again the flask was purged with argon. The reaction mixture was then heated to 95 °C (bath) overnight, cooled down, and diluted with ethyl acetate (300 mL). Organic phase was dried over Na₂SO₄ and evaporated. The residue was chromatographed on a silica gel column (200 g, dichloromethane–ethanol 15:1) to obtain 469 mg (86%) of compound 97. ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.19 (d, J = 6.4 Hz, 1H), 7.93 (s, 1H), 7.65 (dd, J = 8.4, 2.0 Hz, 1H), 7.61 (d, J = 2.0 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 6.04 (s, 1H), 4.52–4.25 (m, 1H), 4.30 (br s, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.55 (br s, 2H), 2.18 (dq, J = 13.8, 7.6 Hz, 1H), 1.94 (dp, J = 11.9, 6.0 Hz, 1H), 1.82 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4, 148.8, 147.1, 142.8, 133.5, 129.3, 128.0, 121.2, 119.5, 112.1, 110.8, 92.8, 55.8, 48.8*, 30.1*, 22.8 (two CH₂ groups were not detected). UPLC–MS: t = 3.55 (M + H, 416.3/418.3).

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)-6-methylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (98).

General method for methylation of position 6 was used starting from compound 97 (250 mg, 0.6 mmol). Yield: 73%. Chromatography: CHCl₃–ethanol 15:1. Crystallization: acetone. [α]_D²⁰ –8.2 (c 0.305, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.19 (d, J = 6.5 Hz, 1H, NH), 7.85 (s, 1H, H-2), 7.78 (d, J = 2.0 Hz, 1H, H-2″), 7.71 (dd, J = 8.4, 2.1 Hz, 1H, H-6″), 7.05 (d, J = 8.4 Hz, 1H, H-5″), 5.85 (s, 1H, H-7), 4.35 (h, J = 5.5 Hz, 1H, H-3′), 4.05 (br s, 1H) and 3.83 (br s, 3H, H-2′, H-5′), 3.83 (s, 3H) and 3.80 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.38 (s, 3H, 6-CH₃), 2.17 (dq, J = 13.7, 7.4 Hz, 1H, H-4′a), 1.92 (dq, J = 12.3, 6.2 Hz, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (CO), 152.2 (C-6), 148.7 (C-3″), 148.3 (C-4″), 141.8 (C-8), 134.1 (C-9), 128.7 (C-2), 127.1 (C-3), 122.4 (C-1″), 119.1 (C-6″), 112.0 (C-2″), 110.5 (C-5″), 93.7 (C-7), 55.7 (3″-OCH₃, 4″-OCH₃), 55.2* (C-2′), 48.7* (C-3′), 48.0* (C-5′), 30.4* (C-4′), 22.8 (COCH₃), 21.9 (6-CH₃). HRMS calcd for C₂₁H₂₅N₅O₃Na m/z: 418.1850 (M + Na)⁺, found 418.1849.

(R)-N-(1-(3-(3,4-Dimethoxyphenyl)imidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)acetamide (99).

Compound 97 (250 mg, 0.6 mmol) was dissolved in a mixture of methanol (15 mL), THF (15 mL), DMF (15 mL), and Et₃N (1 mL), and then Pd/C (75 mg) was added. The flask was purged with argon (three times) and then with hydrogen (three times). The reaction mixture was stirred for 16 h, filtered through Celite, and the filtrate was evaporated. The residue was chromatographed on a silica gel column (100 g, dichloromethane–ethanol 15:1) to obtain 195 mg (85%) of the product. The obtained solid was recrystallized from ethyl acetate. [α]_D²⁰ +16.6 (c 0.301, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆, 25 °C) δ 8.19 (d, J = 6.6 Hz, 1H, NH), 8.07 (d, J = 5.7 Hz, 1H, H-8), 7.90 (s, 1H, H-2), 7.71 (dd, J = 8.4, 2.0 Hz, 1H, H-6″), 7.68 (d, J = 2.1 Hz, 1H, H-2″), 7.05 (d, J = 8.4 Hz, 1H, H-5″), 5.92 (d, J = 5.7 Hz, 1H, H-7), 4.38 (p, J = 5.4 Hz, 1H, H-3′), 3.90 (br s, 4H, H-2′, H-5′), 3.83 (s, 3H), and 3.80 (s, 3H, 3″-OCH₃, 4″-OCH₃), 2.18 (dq, J = 13.7, 7.4 Hz, 1H, H-4′a), 1.93 (dq, J = 12.2, 6.2 Hz, 1H, H-4′b), 1.82 (s, 3H, COCH₃). ¹³C NMR (101 MHz, DMSO-d₆, 25 °C) δ 169.4 (CO), 148.8 (C-3″), 148.5 (C-4″), 144.2 (C-6), 142.3 (C-8), 135.0 (C-9), 128.9 (C-2), 127.4 (C-3), 122.1 (C-1″), 119.4 (C-6″), 112.0 (C-2″), 110.8 (C-5″), 92.5 (C-7), 55.8 and 55.7 (3″-OCH₃, 4″-OCH₃), 55.4 (C-2′), 48.7 (C-3′), 48.0 (C-5′), 30.5 (C-4′), 22.5 (COCH₃). HRMS calcd for C₂₀H₂₄N₅O₃ m/z: 382.1874 (M + H)⁺, found 382.1872.

Screening Assay.

Preparation of human recombinant nSMase2 and the fluorescence-based assay to monitor its activity in the presence or absence of potential inhibitors has been described.¹⁹ In brief, lysates of HEK293 cells that have been transfected with full human length nSMase2 were used to catalyze the hydrolysis of sphingomyelin (SM) to ceramide and phosphorylcholine. Phosphorylcholine in the presence of the Amplex Red system generates the fluorescent molecule resorufin. Extent of fluorescence is directly proportional to the extent of SM hydrolysis. The assay showed high reliability (Z′ = 0.8–0.9). A counterscreen was concomitantly carried out to identify false positives resulting from inhibition of the coupling enzymes. For the counterscreen, the alkaline phosphatase, choline oxidase, and HRP reactions were carried out in the absence of human nSMase2 and initiated by the addition of phosphorylcholine (2 μM), the alkaline phosphatase substrate. Compounds that showed inhibition of the coupling enzymes were considered false positives and were not used further.

In Vitro Metabolic Stability Studies.

The metabolic stability was evaluated using mouse and human liver microsomes (Xenotech LLC, USA) as previously described.^38,39 For the cytochrome P450 (CYP)-mediated metabolism, the reaction was carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, 0.4 U/mL glucose 6-phosphate dehydrogenase, 50 μM sodium citrate). Reactions, in triplicate, were initiated by addition of the liver microsomes to the incubation mixture (compound final concentration was 1 μM; 0.2 mg/mL microsomes). Controls in the absence of cofactors were carried out to determine the specific cofactor-free degradation. After 60 min of incubation, aliquots of the mixture were removed, and the reaction was quenched by addition of three times the volume of ice-cold acetonitrile spiked with the internal standard. Compound disappearance was monitored over time using a liquid chromatography and tandem mass spectrometry (LC–MS/MS) method. Additionally, metabolite identification (compound 1) was performed by full scan analysis on Thermo Q Exactive Orbitrap mass spectrometer, and phase-I metabolites were monitored.

5XFAD Mice.

Hemizygous 5XFAD males, overexpressing human PS1 and APP695 driven by the mouse Thy1 promoter (Jackson Laboratories, MMRRC Stock 34840-JAX), were bred in-house to wild type B6SJLF1/J female mice (Jackson Laboratories, stock 100012). Resultant litters were genotyped for APP695 and PS1 (Transnetyx). Mice were housed in the Johns Hopkins Miller Research Building animal facility and maintained on a 12 h light–dark cycle. All animal studies were conducted according to protocols approved by the Johns Hopkins University Animal Care and Use Committee.

Pharmacokinetics and Bioanalysis of 1 (PDDC).

To confirm that the pharmacokinetic profile of 1 (PDDC) was similar in AD mice to that our laboratory recently reported in wild type mice,²¹ aged 11-month hemizygous 5XFAD mice received a terminal intraperitoneal dose of 10 mg/kg 1 (PDDC). 1 (PDDC) was formulated in NMP–Tween 80–saline (2.5:5:92.5 v/v/v) and dosed ip at a dosing volume of 10 mL/kg for pharmacokinetic studies. Blood and brain tissue were collected at 0.25, 0.5, 1, 3, and 6 h postdose (n = 3 per time point). Blood was obtained after terminal sacrifice via cardiac puncture, and plasma was harvested from blood by centrifugation at 3000g for 15 min and stored at –80 °C. Brain tissues were harvested following blood collection and immediately snap frozen in liquid nitrogen and stored at –80 °C. Bioanalysis was performed as we have described previously. Briefly, calibration standards were prepared using naive mouse plasma or brain spiked with 1 (PDDC). 1 (PDDC) standards and samples were extracted from plasma and brain by a one-step protein precipitation using acetonitrile (100% v/v) containing internal standard (losartan, 0.5 μM). The samples were vortex mixed for 30 s and centrifuged at 10 000g for 10 min at 4 °C. An aliquot of the supernatant (50 μL) was diluted with water (50 μL) and transferred to a 250 μL polypropylene vial sealed with a Teflon cap and analyzed via LC–MS/MS. Chromatographic analysis was performed using an Accela ultrahigh-performance system consisting of an analytical pump and an autosampler coupled with a TSQ Vantage mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). Separation of analyte was achieved at ambient temperature using Agilent Eclipse Plus column (100 mm × 2.1 mm i.d.) packed with a 1.8 μm C18 stationary phase. The mobile phase consisted of 0.1% formic acid in acetonitrile and 0.1% formic acid in water with gradient elution. The [M + H]⁺ ion transitions for MS-882 were m/z 488.050 → 394.249, 378.218, and for losartan (IS) they were m/z 423.130 → 207.090, 180.071.

Mean plasma and brain concentrations of 1 (PDDC) were analyzed using noncompartmental method as implemented in the computer software program Phoenix WinNonlin version 7.0 (Certara USA, Inc., Princeton, NJ). The maximum plasma and tissue concentration (C_max) and time to C_max (T_max) were the observed values. The area under the plasma and tissue concentration time curve (AUC) value was calculated up to 6 h (AUC₀–_t) by use of the log–linear trapezoidal rule. Brain to plasma ratio is calculated from mean total AUC_brain versus AUC_plasma.

Cognitive Tests in 5XFAD Mice.

Male and female hemizygous 5XFAD and WT mice were aged to 3 months, and intraperitoneal injections of vehicle (5% DMSO, 5% Tween 80, 90% PBS) or 1 (PDDC) 10 mg/kg body weight were delivered daily for 5.5 months, until the time of sacrifice (n = 13–16). At 8 months of age, mice were subjected to fear conditioning test as previously described.⁴⁰ Briefly, on the first day of testing, mice were placed in a fear conditioning chamber (Clever Sys Inc.) for 240 s and trained to associate a 30 s CS 1000 Hz tone with a 2 s US 0.5 mA footshock, via two tone-footshock pairings at 120 and 180 s. On the second day of testing, contextual fear memory was measured by placing mice back into the same fear conditioning chamber for 300 s. On the third day of testing, mice were placed in a novel wooden box container within the fear conditioning chamber for 240 s, and cued fear memory was measured by playing the CS 1000 Hz tone for the last 120 s of the test. Freezing behavior was recorded using FreezeScan software (Clever Sys Inc.).