Structure-Activity Relationships of Replacements for the Triazolopyridazine of Anti-Cryptosporidium Lead SLU-2633

Abstract

Cryptosporidiosis is a diarrheal disease particularly harmful to children and immunocompromised people. Infection is caused by the parasite Cryptosporidium and leads to dehydration, malnutrition, and death in severe cases. Nitazoxanide is the only FDA approved drug but is only modestly effective in children and ineffective in immunocompromised patients. To address this unmet medical need, we previously identified triazolopyridazine SLU-2633 as potent against Cryptosporidium parvum, with an EC₅₀ of 0.17 μM. In the present study, we develop structure-activity relationships (SAR) for the replacement of the triazolopyridazine head group by exploring different heteroaryl groups with the aim of maintaining potency while reducing affinity for the hERG channel. 64 new analogs of SLU-2633 were synthesized and assayed for potency versus C. parvum. The most potent compound, 7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazine 17a, was found to have a Cp EC₅₀ of 1.2 μM, 7-fold less potent than SLU-2633 but has an improved lipophilic efficiency (LipE) score. 17a was found to decrease inhibition in an hERG patch-clamp assay by about two-fold relative to SLU-2633 at 10 μM despite having similar inhibition in a [³H]-dofetilide competitive binding assay. While most other heterocycles were significantly less potent than the lead, some analogs such as azabenzothiazole 31b, have promising potency in the low micromolar range, similar to the drug nitazoxanide, and represent potential new leads for optimization. Overall, this work highlights the important role of the terminal heterocyclic head group and represents a significant extension of the understanding of the SAR for this class of anti-Cryptosporidium compounds.

Introduction

Diarrheal diseases predominantly impact immunocompromised people and, in low- and middle-income countries, children. For example, in sub-Saharan Africa, diarrheal diseases cause ~10% of mortality and morbidity in children under 5 years old.¹ Amongst infectious causes of diarrhea, cryptosporidiosis is particularly concerning because of high prevalence and a strong association with mortality in children and immunocompromised people.^{2, 3} Notable amongst its impacts are the ~220,000 deaths amongst children under 2 years in Sub-Saharan and South-Asian countries that were reported in a study published in 2018, and Cryptosporidium’s ability to cause large waterborne outbreaks even in high-income countries, such as a 1993 outbreak that affected over 400,000 Milwaukee residents.^{4, 5} Cryptosporidiosis is a zoonotic disease mainly transmitted through intake of food and water contaminated by Cryptosporidium spp., with C. parvum and C. hominis causing human disease.⁶ Nitazoxanide (NTZ; Fig. 1) is the only approved drug for treatment by the FDA but has only modest potency in vitro (EC₅₀ 3.8 μM), and lacks efficacy for immunocompromised patients and children under 12 months of age.^{7, 8} There have been other agents tested for AIDS patients with cryptosporidiosis, such as paromomycin and clofazimine. Unfortunately, these drugs have proven ineffective.^{7, 9}

Figure 1. Medicinal chemistry strategy.

The structures of nitazoxanide (NTZ), MMV665917 (1) and SLU-2633 (2a) are shown. Generic structure (3) illustrates our strategy for developing SAR for the heterocyclic head group.

Increased recognition of the importance of cryptosporidiosis as a cause of life-threatening diarrhea in young children has stimulated multiple drug development efforts and discovery of new drug leads, including the discovery of triazolopyridazinyl hit, MMV665917 (1), and subsequent lead, SLU-2633 (2a).^{6, 10} 1 was found to be modestly potent in vitro (EC₅₀ 2.1 μM) but has oral efficacy in multiple in vivo models, including acute and chronic C. parvum mouse infection¹¹ models, a dairy calf C. parvum infection model, and gnotobiotic piglet C. hominis infection model.^{6, 12} Despite the promising pharmacology of 1, its modest potency and potential cardiotoxicity via inhibition of the hERG ion channel tempers the enthusiasm for development as a drug.^{6, 12, 13} For this reason, we have previously optimized the linker and aryl tail regions of 1 for potency against Cryptosporidium while retaining the triazolopyrazine heteroaryl head leading to the discovery of 2a (Fig. 1).¹⁰ 2a was found to be an order of magnitude more potent in vitro (Cp EC₅₀ = 0.17 μM), non-cytotoxic (CC₅₀ >100 μM), and efficacious in vivo. Although both 1 and 2a showed inhibition of the hERG ion channel at higher concentrations (10 μM), the hERG IC₅₀/Cp EC₅₀ selectivity profile of 2a was significantly improved relative to 1 due to its superior potency, making 2a a better drug candidate than 1.¹⁰ 2a and 1 retain the[1,2,4]triazolo[4,3-b]pyridazine head group, which led to the present study to examine the relevance of the heteroaryl head group (triazolo[4,3-b]pyridazine) to the anti-Cryptosporidium activity of this compound class.

Herein, we explore the different heteroaryl head groups to replace the triazolopyridazine ring in 2a via chemical synthesis. Using phenotypic drug design, this work mainly focuses on the exploration of monocyclic 5- and 6-membered heteroaromatic rings, and 6,5-bicyclics as well as 6,6-bicyclic heteroaryl rings. Different substituents on these cyclic heteroaryls are explored as well. The significant drug–target interactions of these heteroaryl scaffolds were not considered in this work since we have not yet established the biological target of these compounds. The Cryptosporidium life cycle includes three rounds of asexual replication, after which gametocyte development and fertilization are required for continued propagation.¹⁴ Phenotypic studies using 1 and 2a show that it is parasiticidal and that it inhibits development of macrogamonts (female gametocytes) but has almost no effect on the first cycle of asexual replication.^{6, 15, 16}

We hypothesized that studying the SAR of the triazolopyridazyl scaffold with these heteroaryl substrates would lead to discovery of other leads and improve both in vitro potency and in vivo efficacy, while reducing hERG inhibition. We also hypothesized that this approach could influence the physio-chemical properties including the conformational profile, pKa, lipophilicity, dipole moment, and aqueous solubility. Parameters such as attenuation of pKa (reducing basicity), reducing lipophilicity (logP), formation of zwitterions, and even discrete structural modifications are known to affect hERG channel inhibition and represent common strategies towards mitigation of hERG affinity.¹⁷

Results and Discussion

The conformational significance of the piperazine, 2-(3,4-dichlorophenyl, and the methylene (-CH₂) between them in the structure of the lead compound 2 were established in prior work; hence these structural features were maintained throughout this systematic SAR interrogation of the heteroaryl scaffold (Fig. 1).^{6, 10} Below we describe SAR of the heterocyclic head group, effects on the hERG ion channel, and chemical syntheses of 64 new analogs.

In vitro Cryptosporidium parvum-infected HCT-8 Cell Assay (Cp HCT-8).

Newly synthesized compounds were assayed in vitro using a previously established high-content microscopy assay that measures asexual development of C. parvum, the only readily available Cryptosporidium species, within the colon carcinoma cell line HCT-8.¹⁸ HCT-8 cell monolayers were grown to near confluence in clear-bottomed microtiter plates and infected with C. parvum oocysts. Test compounds were added after allowing 3 hours for host cell invasion, followed by incubation for 48 hours, and then staining for parasite and host cells and enumeration by automated fluorescence microscopy.

C. parvum Structure Activity Relationships.

Results from the Cp HCT-8 assay are summarized in Figures 2 and 3 (for tabular data with 95% confidence intervals, see Table S1, Supporting Information). Specific compound SAR relative to lead compound 2a is presented in Figure 2. To establish the consistency in the significance of the −CH₂ being preferred in the X position, we synthesized a few urea analogs with -NH at the X position to compare to their acetamide counterparts which make up most of the SAR in this study.

Figure 2. Triazolopyridazine SAR.

EC₅₀ values are calculated from average potency in the Cp HCT-8 assay from at least two 9-point dose-response experiments (n=8 technical replicates per dose). The most potent analogs identified are colored in blue font and have EC₅₀ values ranging from 1.2 to 7.8 μM. In this assay, control compounds NTZ and MMV-665917 (1) have EC₅₀ values of 3.7 and 2.1 μM, respectively.

Figure 3. Plot of all compounds comparing lipophilicity, basicity, potency and affinity for the hERG channel.

LogP represents an estimate of lipophilicity and is the log of the partition coefficient. LogP was calculated in CDD Vault (www.collaborativedrug.com) using the method described by Gedeck et al.^{22, 23} pKa (Basic) was calculated in CDD Vault (www.collaborativedrug.com) using the method described by Lu et al.^{24, 25} Chemotypes are represented as amides (circles) and ureas (squares). (A) Plot of logP versus basic pKa to illustrate range covered by tested compounds. Individual compounds are color-coded by average EC₅₀ in the Cp HCT-8 assay with the most potent compounds being colored blue, moderately potent compounds green and weakly potent compounds orange and red. (B) Plot of logP versus basic pKa to illustrate hERG binding affinity. Individual compounds are color-coded by percent inhibition (%inh) of binding of [³H]-dofetilide to the hERG channel with low-affinity compounds being colored blue, moderate-affinity compounds green and high-affinity compounds orange and red. (B) LipE plot of logP versus pEC₅₀. LipE is the pEC₅₀ minus the logP to attempt normalize potency for lipophilicity. LipE lines are provided as diagonal lines. More drug-like compounds are expected to have high LipE values and be situated in the upper lefthand corner of the chart. Compound 17a represents an improvement in LipE versus lead compound 2a. Compounds are color coded for basic pKa with blue being the most basic and red the least basic.

Direct replacements of the heteroaryl group validated its importance: H, Boc, Ph for 4, 5, 6a-b, respectively, led to significant losses in potency (EC₅₀ ≥20 μM), more than 40-fold vs 2a-b. We also evaluated other monocyclic heterocycles such as pyridines, pyrimidines, pyridazines, and even the nitrothiazole found in the NTZ structure, in order to determine the necessity of the fused triazole ring in the triazolopyridazine. However, none of these compounds (7–13) retained potency in the assay except for weakly potent nitrothiazole 13.

We explored direct substitution of the triazolopyridazine ring (14a-l, 15a-b, and 16a-b). Simple additions of methyl, Cl, CF₃ or NH₂ to the 3- (14a-g) and methyl to the 7- (15a-b) and 8-positions (16a-b) were not tolerated, losing 50- to 100-fold potency versus H in these positions (2a-b). However, substitutions with larger groups in the 3-postion such as phenyl and benzyl (14i-j) retained potency similar to methyl. Interestingly, the 3-(2-CF₃-4-Pyr) analog 14l retained modest potency (6.1 μM) while unsubstituted pyridine 14k and morpholine 14h analogs did not. There were a few urea versions of the 3-substituted analogs which were subtly more potent than their acetamide counterparts, namely Cl and CF₃ (14c-f).

Reduced triazolopyridazinyl scaffold analog 17a gave the best potency relative to any other compounds in this study. With an EC₅₀ of 1.2 μM, 17a has 7-fold less potency compared to that of unsaturated 2a. The urea variant 17b was less potent with an EC₅₀ of 13 μM, ~20-fold potency reduction compared to urea 2b.

We also evaluated 6,5-bicyclic heterocycles to determine the importance of positioning of the N atoms around the ring system (18a-b, 19–24). Strikingly, the single atom exchange of N with CH in the 2-position drastically reduced the potency in the cases of both the amide 18a and urea 18b. Other heterocycles, including pyrazolopyrimidine, imidazolopyrazine, benzopyrazole, indazole, benzimidazole, and benzoxadiazole (19–24) also led to significant reductions in potency with EC₅₀ values ranging from 20–30 micromolar. Similarly, 6,6-bicyclic heterocycles including quinoline and naphthyridines (25–27) did not yield better potency, nor did 5,6-bicyclic benzimidazole 28, benzoxazole 29a or benzothiazole 29b.

Given the interesting dependence of potency on N in the 2-position (18a vs. 2a), we hypothesized that there may be a nearby hydrogen bond donor that we might be able to engage by extension off this position with amino and carbonyl groups (30a-f). However, none of these compounds showed a significant improvement in potency relative to 18a. In contrast, azabenzothiazoles 31a-d with 2-subsitution exhibited potency comparable to the reduced triazolopyridazine 17a and original hit 1. Simple amino analog 31b (EC₅₀ 3.2 μM) was more 5-fold potent than methyl analog 31a. Further extension as amides 31c and 31d yielded similar potency and represents a future opportunity to modulate potency. Benzothiazoles 32a-d were made as comparators to ascertain the importance of the nitrogen in the 5-position. 32b and 32c indicate this nitrogen provides at least 5-fold potency enhancement relative to 31b and 31c.

1H-pyrazolo[4,3-b]pyridine derivatives 33a-c were disappointingly not particularly potent. However, regioisomeric 34 with a 2-benzyl group retained potency (EC₅₀ 7.8 μM) comparable to azabenzothiazole analogs 31b-d. This indicates that there is likely some room to extend off the 2-position of 6,5-bicyclic heterocycles.

The compounds we have prepared represent a range of lipophilicity (logP) and basicity (pKa), both factors that can influence binding to the hERG channel (Fig. 3; Supplementary Table S1).¹⁷ Despite covering a broad range, we found very few head groups that retain potency comparable to lead amide 2a or urea 2b (the two blue symbols in Fig. 3A). There were a few head groups that resulted in modest potencies (green symbols in Fig. 3A) which have similar calculated basic pKa values around 6, comparable 2a and 2b. The lone exceptions to this trend are 17a and 17b, which have EC₅₀ values of 1.2 μM and 13 μM, respectively, and less basic pKa values of 4.3–4.8 and logP 1.8–2.3. Plotting the pEC₅₀ (pEC50 = −log(EC₅₀) where the EC₅₀ is given in M; a pEC₅₀ = 6 is 1 μM) versus logP illustrates that the most potent compounds (leads 2a and 2b) have log P values between 3–4 (Fig. 3C). However, when factoring in lipophilicity, 17a has a superior LipE (lipophilic efficiency or ligand-lipophilic efficiency, LLE)^19–21 value (4.13 vs. 3.3 for 2a) and could represent a superior headgroup with lower basicity.

Affinity for the hERG channel.

A motivation for exploration for potential replacements for the triazolopyridazine head group was the potential safety concern with this series given the modest hERG affinity and effect in a cell-based hERG patch clamp electrophysiology assay reported for 1 and 2a-b.^{10, 13} While most of the analogs evaluated in this study were not worth follow up studies due to weak potency, we were interested to determine if changes made in this series impacted effects on hERG. Select compounds were evaluated in a [³H]-dofetilide competitive binding assay and in a cell-based hERG patch clamp electrophysiology assay (Table 1; Fig. 3B).^{26, 27} Compounds were selected to represent a range of pKa (basic; 3.0 to 7.7), cLogP (1.8 to 6.1), phenyl (6a), other heterocycles (18b, 31b, 34), reduced heterocycles (17a), and extended moieties (14l, 31c, 34) that could physically restrict access to the hERG binding site.

Table 1.

In vitro Safety Profiling Data

					hERG bindingc	hERG-CHO automated patch clamp, % inhibitiond
Compound	Headgroup	Class	cLogPa	pKa (basic)b	%inh, 10 μM	1 μM	10 μM	30 μM
2a		amide	3.3	5.9	11e	19e	67e	-
2b		urea	3.9	5.9	56e	41e	87e	-
6a		amide	4.3	3.0	2	-	-	-
14l		amide	4.9	6.0	95	-	-	-
17a		amide	1.8	4.4	15	8.7	34	61
18b		urea	4.4	6.4	77	-	-	-
31b		amide	4.5	5.9	34	-	-	-
31c		amide	6.1	7.7	47	-	-	-
34		amide	5.7	6.2	80	-	-	-

^aLogP was calculated in CDD Vault (www.collaborativedrug.com) using the method described by Gedeck et al.^{22, 23}

^bpKa (Basic) was calculated in CDD Vault (www.collaborativedrug.com) using the method described by Lu et al.^{24, 25}

^cCompound binding was calculated as a % inhibition of the binding of [³H]-dofetilide to the human potassium channel hERG (Eurofins, Saint Charles, MO, USA);

^dFunctional hERG cell-based electrophysiology automated patch clamp assay; Compounds were evaluated in duplicate with average % inhibition of tail current reported at each concentration.²⁷ (Eurofins, Saint Charles, MO, USA);

^eData published previously.¹⁰

For reference, lead compound 2a only weakly binds the hERG channel at 10 μM but displays 67% inhibition in the patch clamp assay, and we have observed a general trend where the urea class tends to have higher affinity for hERG versus the acetamides as illustrated by comparison of 2a versus 2b.¹⁰ In the hERG binding assay, phenyl analog 6a does not exhibit significant effect at 10 μM. However, a number of the other heterocycles exhibited low (17a, 15%) to high (14l, 95%) affinity for hERG. Of the compounds tested, only those with basic pKa values below 5 had the lowest affinity for hERG (Fig. 3B). We then tested our more potent analog, 17a, the reduced triazolopyridazine, in the patch clamp assay at concentrations of 1, 10 and 30 μM to determine functional relevance and compare to unsaturated lead analog 2a. 17a has a lower effect in this more physiologically relevant assay than 2a with 34% inhibition at 10 μM versus 67% for 2a. This may be due to 17a having lower calculated pKa and log P than 2a.

Synthesis of heteroaryl analogs (Scheme 1).

Scheme 1.

Reagents and conditions: (a) Boc-piperazine, K₂CO₃ or Cs₂CO₃, DMF or acetonitrile, rt-100°C, 1–24h; (b) Boc-piperazine, DIEA, EtOH or NMP, 100–150°C, 4–24h, 60–90%; (c) Boc-piperazine, nBuOH, 130°C, 64%; (d) Boc-piperazine, Pd₂(dba)₃, RuPhos, NaO^tBu, dioxane, 100°C, overnight, 35–94%; (e) 4M HCl in dioxane, 2h, 98%; (f) 2-(3,4-dichlorophenyl)acetic acid, HOBt, EDC-HCl, TEA, DMF, overnight, 10–70%; (g) 2-(3,4-dichlorophenyl)acetyl chloride, TEA, DCM, 0°C to RT, 12–67%; (h) 2-(3,4-dichlorophenyl)isocyanate, TEA, DMF, 15–68%.

The general synthetic scheme used to synthesize heteroaryl analogs is illustrated in Scheme 1. Heteroaryl halide precursors (35a-35y) were either purchased or synthesized (described below). Boc-piperazine intermediates 36a-aw were synthesized by nucleophilic substitution or by Buchwald – Hartwig amination, the choice of the reaction depended on the yield as well as the purity.²⁸ Most of the intermediates 36a-aw synthesized by nucleophilic substitution gave higher purity 85–95% after water-ethyl acetate extraction, hence were carried to the next step without purification. Boc deprotection of 36a-aw gave piperazine intermediates 37a-aw with crude yields mostly > 95%. Amide coupling of the crude amine HCl salts with HOBt and EDC-HCl as the coupling reagents with 2-(3,4-dichlorophenyl)acetic acid, was used to synthesize the final compounds. Most of the final compounds (2–34) were extracted with water and ethyl acetate, followed by reverse-phase chromatography and sometimes followed by normal phase chromatography or vice versa to achieve HPLC purity ≥95%.

Some final compounds were synthesized via the reversed sequence (Scheme 2). The amide and urea syntheses were completed first to give late-stage intermediates 4 and 40, respectively. Subsequent nucleophilic substitution with the corresponding heteroaryl halides to obtain their final compounds (10b, 11b, 14a, 14d-f, 14i, 15b, 16b, 20). Although this sequence is more efficient for analog generation, reaction products were generally more difficult to purify using this route versus the route in Scheme 1.

Scheme 2.

Reagents and Conditions: (a) Boc-piperazine, TEA, DMF, 15–68%; (b) Boc-piperazine, HOBt, EDC-HCl, TEA, DMF, overnight, 10–70%; (c) 4M HCl in dioxane, 2h, 98%; (d) HetAr-X, DIEA, EtOH, 100°C, 2h, 13–35%.

Synthesis of substituted [1,2,4]triazolo[4,3-b]pyridazyl chlorides 35a-j (Scheme 2).

Hydrazine intermediates 42a-b were obtained via nucleophilic substitution of 41a-b with hydrazine in EtOH.^{10, 29} Methyl hydrazinopyridazine 42b was obtained and used as a mixture of isomers. Condensation directly with the corresponding carboxylic acid (R¹ = H, CH₃, CF₃) or POCl₃ assisted cyclization (R¹ = 2-CF₃-4-Pyr, 4-Pyr) gave heterocyclic intermediates 35a-j which were then converted to final analogs as outlined in Schemes 1 and 2.^{10, 29}

Synthesis of 7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazyl intermediate (Scheme 4).

Scheme 4.

Reagents and conditions: (a) H₂, Pd/C, MeOH, 60 psi, 48h, 83%.

Intermediate 36e was synthesized as previously described. Intermediate 36h was obtained by hydrogenation of 36e over Pd/C using a modification of the procedure by Bradbury et al.³⁰ 36h was then converted to the final analogs as illustrated in Scheme 1.

Synthesis of 3-substituted [1,2,4]triazolo[4,3-b]pyridazyl intermediates 36i-j (Scheme 5).

Scheme 5.

Reagents and conditions: (a) Boc-piperazine, DIEA, DMF, rt, 16h, 98%; (b) morpholine, 160°C, overnight, 87%.

Boc-piperazine intermediate 36i was obtained from commercially available dichloropyridazine 35k via nucleophilic substitution at room temperature with Boc-piperazine in DMF. 36i was converted to 36j via a neat reaction in morpholine at 160°C.^{10, 31, 32} 36i-j were converted to the final compounds as described in Scheme 1.

Synthesis of 2-substituted imidazo[1,2-b]pyridazinyl analogs 30b-f (Scheme 6).

Scheme 6.

Reagents and conditions. (a) ROH, SOCl₂, refluxed, overnight, 70–95%; (b) EDC-HCl, HOBt, TEA, DMF, overnight, 20–89%; (c) Boc-piperazine, DIEA, NMP, 150°C, overnight, 80–89%; (d) HCl, 2h, 98–99% then EDC-HCl, HOBt, TEA, DMF, overnight, 20–70%; (e) LiOH, MeOH, overnight, 60%.

Thionyl chloride acid-esterification of carboxylate 43 with EtOH and n-butanol led to the synthesis of intermediates of 35l-m, respectively.^{32, 33} Via amide coupling, 43 was converted to intermediates 35n-o. These intermediates 35l-o were converted to final compounds by nucleophilic substitution reaction, Boc-deprotection, and amide coupling sequentially as described above (Scheme 1). Ethyl ester 30b was hydrolyzed to give carboxylic acid analog 30d.

Synthesis of 2-substituted thiazolo[4,5-b]pyridyl and benzo[d]thiazol-2-amine intermediates (Schemes 7 and 8).

Scheme 7. Reagents and Conditions.

(a) DCM, rt, overnight, 57%; (b) CuI, K₂CO₃, L-proline, overnight, 77%; (c) Boc-piperazine, Pd₂(dba)₃, RuPhos, NaO^tBu, dioxane, 100°C, overnight, 78%; (d) HCl, 98%; (e) acetyl chloride, TEA, DCM, overnight, 86%; (f) Lawesson’s reagent, DCM, rt, overnight, 70%; (g) 1,10-phenanthroline, KO^tBu, MeOH, 3h, 98%.

Scheme 8. Reagents and Conditions:

(a) Boc₂O, DMAP, DCM, overnight; (b) RCOCl, TEA, DCM, overnight, 50%; (c) ArCO₂H, HOBT, EDC-HCl, TEA, DMF, overnight, 76%.

Iodoaminopyridine 44 was converted to Cbz-protected thiourea 46 using intermediate 45 using the procedure described by Jefferson and coworkers.³⁴ 46 was converted to thiazolo[4,5-b]pyridine 35p under Ullman-type conditions.³⁵ Nucleophilic displacement with Boc-piperazine gave intermediate 36o which was deprotected with anhydrous HCl to give 37o. Piperazine 37o was converted to final analog 31b as described in Scheme 1.

Iodoaminopyridine 44 was also acylated followed by thionation with Lawesson’s reagent to give thioacyl 47b. S-C bond formation via a K^tOBu assisted S-C coupling reaction³⁶ was utilized to obtain 35q from 47b. Intermediate 35q was converted to final compounds by Buchwald-Hartwig amination as described in Scheme 1.³⁷

Thiazolo[4,5-b]pyridyl intermediate 48a (Scheme 8) was synthesized as previously described by Molette.³⁵ Benzo[d]thiazol-2-amine 48b was purchased and Boc-protected to give 35t. Amide coupling was used to convert intermediates 48a-b to intermediates 35s-u. Intermediates 35s-u were converted to final compounds as described in Scheme 1.

Synthesis of 5-chloro-1H-pyrazolo[4,3-b]pyridine derivatives (Scheme 9).

Scheme 9. Reagents and conditions:

(a) BnBr/MeI, K₂CO₃, DMF, 0–90°C, 2h, 81–85%; (b) MeI, K₂CO₃, DMF, 0°C to rt, 81%.

A procedure reported by Vechorkin et al³⁸ was used to convert 49 to benzyl alkylated intermediates 35v-x. The mixture was then worked up and proceeded to the Buchwald-Hartwig amination using the Boc-piperazine (Scheme 1). The addition of the Boc-protected piperazine provided enough lipophilicity to be able separate the isomers by normal phase chromatography. The methylated intermediate 35w was synthesized by similar procedure except at a temperature of 0°C and allowed to warm to room temperature for 2 h. The mixture was worked up and proceeded to Buchwald amination except that the isomers were inseparable, so they were further proceeded to the final step to isolate isomeric final compound 33b. The assignment of the position of the benzyl and methyl groups was determined by NOESY.

Synthesis of imidazo[1,2-b]pyridazin-2-amine analog 30 (Scheme 10).

Scheme 10. Reagents and conditions.

(a) hexane-2,5-dione, malonic acid hydrate, EtOH, 80°C, 2h, 73%; (b) Boc-piperazine, DIEA, NMP, 150°C, overnight, 31%; (c) HCl, 2h, 96%; (d) 2-(3,4-dichlorophenyl)acetic acid, EDC-HCl, HOBT, TEA, DMF, overnight, 30%; (e) NH₂OH-HCl, EtOH, H₂O, 120°C, 48h, 80%.

Pyrrole protection of the commercially available 6-chloroimidazo[1,2-b]pyridazin-2-amine 50 via Paal-Knorr type condensation with hexane-2,5-dione yielded pyrroled intermediate 35y.³⁹ Pyrrole-protection was chosen because the Boc and Cbz protected substrate did not give the desired product in the subsequent nucleophilic substitution. This was followed by nucleophilic substitution with Boc-piperazine, Boc-deprotection, and amide coupling to give pyrrole intermediate 51. Hydrolysis of the pyrrole protected amine to primary amine gave final compound 30a.^{10, 40}

Conclusion

In conclusion, we synthesized and assayed 64 analogs in an attempt to substitute and replace the triazolopydazine head group of lead compound 2a. From the SAR, the triazolopydazine was remarkably intolerant to substitution by even simple methyl groups or replacement with other heterocycles. All heterocyclic head groups explored yielded less potent compounds. As we had previously seen, the amide analogs were generally, but not universally, more potent than the corresponding urea analogs.

Reduced triazolopyridazine 17a was the most potent replacement with an EC₅₀ of 1.2 μM, ~7-fold less potent than 2a. 17a has a two-fold reduced effect at 10 μM in hERG patch-clamp assay relative to 2a, even though it had similar inhibition in the [³H]-dofetilide competitive binding assay. It is possible that reduced lipophilicity led to the difference in the hERG inhibition.

Other heteroaryl groups, including some azabenzothiazoles derivatives such as 31b, were found to be modestly potent, only two- to three-fold less than 17a and comparable to nitazoxanide. Such compounds may represent starting points for future analog development.

Experimental Section

General.

Commercially available reagents and solvents were used without further purification unless stated otherwise. Reactions that were heated under microwave irradiation were conducted using a Biotage Inititiator+ Robot8 microwave. HPLC and LC-MS analyses were performed on an Agilent 1100 HPLC/MSD electrospray mass spectrometer in positive ion mode with scan range was 100–1000 Da or an Agilent 1260 HPLC/MSD electrospray mass spectrometer in positive and negative ion modes with a scan range of 100–1000 Da. Preparative normal phase chromatography was performed on a CombiFlash Rf+ (Teledyne Isco) with SiliaFlash F60 40–63 μm (230–400 mesh) silica gel (SiliCycle Inc.). Preparative reverse phase HPLC was performed on a ACCQPrep HP150 (Teledyne Isco) equipped with 20×250mm or 30×250mm C18 RediSep Prep HPLC columns or a CombiFlash Rf+ (Teledyne Isco) with RediSep Rf Gold pre-packed C18 cartridges and an acetonitrile/water/0.1% formic acid gradient. NMR spectra were recorded on Bruker 400 MHz and 700 MHz spectrometers. Chemical shifts (δ) are given in ppm and are referenced to residual not fully deuterated solvent signal. Coupling constants (J) are given in Hz. HRMS spectra were recorded on an ABSciex 5600+ instrument. All final compounds were purified to ≥95% as determined by HPLC UV absorbance unless noted otherwise.

General Method A. 2-(3,4-dichlorophenyl)-1-(piperazin-1-yl)ethanone hydrochloride (4).

Boc piperazinyl compound 5 (1.57 g, 4.21 mmol) was suspended in dioxane (10 mL) and treated with 4M HCl in dioxane (20 mL). Everything dissolved and a white precipitate soon formed. The reaction mixture was stirred overnight. LCMS showed the reaction was complete. The mixture was concentrated to dryness under vacuo to give the product as white solid and washed with diethyl ether and dried again under vacuo to obtain desire product 4. (1.20 g, 97%), LCMS m/z 274 (MH)⁺.

General Method B. tert-Butyl 4-(8-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36f) and tert-Butyl 4-(7-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36g).⁴¹

A dry pressure vial was with stirring bar was charged with 6-chloro-7-methyl-[1,2,4]triazolo[4,3-b]pyridazine 35i and 6-chloro-8-methyl-[1,2,4]triazolo[4,3-b]pyridazine 35j isomeric mixture (300 mg, 1.78 mmol), Boc-piperazine (496 mg, 2.67 mmol). The mixture was dissolved with NMP (6 mL), DIEA (0.616 mL, 3.56 mmol) was added and stirred at 150°C overnight. The mixture is then cooled to room temperature and extracted with water and EtOAc, dried with Na₂SO₄. The dried organic was then concentrated to give brown oily compound as the product. (500 mg, 88%), LCMS m/z 319 (MH)⁺.

General Method C. tert-Butyl 4-(2-(benzyloxy)carbonyl)amino)thiazolo[4,5-b]pyridin-5-yl)piperazine-1-carboxylate (36o).

A well-dried round bottom flask with a stirring bar was charged with 35p (218 mg, 0.682 mmol), Boc-piperazine (190 mg, 1.02 mmol), RuPhos (6.36 mg, 0.0136 mmol), Pd₂(dba)₃ (6.24 mg, 0.00682 mmol), and NaO^tBu (327 mg, 3.41 mmol) were weighed into a round bottom flask. The round bottom flask was then capped and purged with Nitrogen gas for 5 mins after which 10 ml of dioxane was added and purged for another 5 mins. The mixture was then allowed to heat overnight. The reaction mixture was then allowed to cool, then filtered and purified by flash chromatography using normal phase with 0→100% EtOAc/Hex to obtain a brown solid compound as intermediate 36o. (250 mg, 78%), LCMS m/z 470 (MH)⁺.³⁷

General Method D. tert-Butyl 4-([1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36e).

To a microwave vial was added 6-chloro[1,2,4]triazolo[4,3-b]pyridazine 35h (1.54 g, 9.96 mmol), N-Boc-piperazine (2.04 g, 11.0 mmol), and DIEA (2.22 mL, 13.0 mmol). The starting materials were dissolved in ethanol. The vail was sealed and heated to 100 °C in the microwave reactor for 2 hours. Ethanol was removed via vacuo; water was added, and the crude product was extracted with dichloromethane three times. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to give 2.16 g (74%) of crude tert-butyl 4-{[1,2,4]triazolo[4,3-b]pyridazin-6-yl}piperazine-1-carboxylate. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.50 (s, 9H), 3.52 – 3.63 (m, 8H), 6.94 (d, J=10.2 Hz, 1H) 7.91 (d, J=10.2 Hz, 1H), 8.8 (s, 1H). LC-MS: m/z 305.1 (MH)⁺.

General Method E. 2-(3,4-dichlorophenyl)-1-(4-(7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)ethanone (17a).

A suspension of the 37h (110 mg, 0.454 mmol) and 2-(3,4-dichlorophenyl)acetic acid (140 mg, 0.683 mmol), HOBt (72.0 mg, 0.533 mmol) and EDC-HCl (122 mg, 0.639 mmol) was treated with TEA (70 μL) in DMF (6 mL) at room temperature. The reaction was allowed to stir at room temperature for 16h. The reaction mixture was then stirred overnight, after which the mixture was extracted using ethyl acetate and water three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo. The crude compound was purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) and further purified by silica gel chromatography with 0→20% MeOH/EtOAc to obtain a white solid as the title compound (60.0 mg, 38%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.52 (s, 1H), 7.44 – 7.62 (m, 2H), 7.23 (d, J = 8.1 Hz, 1H), 3.80 (s, 2H), 3.51 – 3.65 (m, 4H), 3.47 (br. s., 4H), 2.96 – 3.11 (m, 2H), 2.76 – 2.88 (m, 2H). ¹³C NMR (176 MHz, DMSO-d₆) δ 168.2, 159.6, 142.0, 139.2, 136.9, 131.2, 130.3, 130.0, 129.6, 128.8, 44.4, 44.2, 44.0, 40.4, 37.8, 20.6, 16.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₈Cl₂N₆O 393.0997; found 393.09869.

General Method F. tert-Butyl 4-((3,4-dichlorophenyl)carbamoyl)piperazine-1-carboxylate (39).

1,2-dichloro-4-isocyanatobenzene (2.00 g, 10.6 mmol) was added to a solution of Boc-piperazine (2.30 g, 12.7 mmol), and DIEA (3.60 mL, 21.2 mmol) in DCM (40 mL) and stirred at 0°C. The ice bath was removed, and the reaction mixture was allowed to warm to room temperature. The reaction was complete in 45 mins. When checked by the LCMS. After 3h, the reaction solution was concentrated to a white solid. The white solid was recrystallized from EtOAc. (1.94 g, 49%), LCMS m/z 375 (MH)⁺.

6-chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazine (35a).

3-chloro-6-hydrazinopyridazine (100 mg, 0.7 mmol) was weighed into a 20-mL microwave vial equipped with a stirring bar, followed by addition of acetic acid (5 mL), sealed, and heated to 100 °C in the microwave reactor for 2 h. The reaction mixture was neutralized with satd. NaHCO₃. The aqueous layer was then extracted three times with dichloromethane. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to give 107 mg of the crude title compound. LC-MS: m/z 168.0 (MH⁺).

6-chloro-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazine (35b).

To a microwave vial was added intermediate 42a (100 mg, 0.700 mmol). The starting material was dissolved in 5 mL trifluoroacetic acid, sealed, then heated to 100 °C in the microwave reactor for 2 h. The reaction mixture was neutralized with satd. NaHCO₃. The aqueous layer was then extracted three times with dichloromethane. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to give 94.0 mg of the crude title compound. LC-MS: m/z 222.0, 224.0 (MH⁺).

6-chloro-3-phenyl-[1,2,4]triazolo[4,3-b]pyridazine (35c).

To a round bottom flask was added 3-42a (100 mg, 0.700 mmol) and phenylacetaldehyde (0.100 mL, 0.760 mmol). The mixture was dissolved in ethanol, heated to 60 °C, and stirred for 30 minutes. Me₄NBr (21.0 mg, 0.140 mmol) and Oxone (158 mg, 1.04 mmol) were added. The reaction mixture was then stirred overnight at 60 °C. The crude mixture was diluted with water and extracted 3 times with DCM. The organic layers were dried over magnesium sulfate, filtered, and concentrated to give 67.0 mg of the crude product. LC-MS: m/z 230.0 (MH⁺).

3-benzyl-6-chloro-[1,2,4]triazolo[4,3-b]pyridazine (35d).

To a round bottom flask was added 3-chloro-6-hydrazinopyridazine (500 mg, 2.75 mmol), phenylacetic acid (415 mg, 3.05 mmol), EDC-HCl (1.07 g, 5.55 mmol), HOBt-H₂O (750 mg, 5.55 mmol), and triethylamine (1.25 mL, 8.35 mL). The mixture was dissolved in acetonitrile and stirred overnight at room temperature. The acetonitrile was removed in vacuo and the remaining mixture was dissolved in toluene and equipped with a Dean-Stark trap. The reaction mixture was heated to 200 °C overnight. The solvent was removed in vacuo and the product was extracted in ethyl acetate, rinsing twice with water and once with brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated to give the title compound (138 mg, 20%). LC-MS: m/z 244.1 (MH⁺).

(Z)-3-chloro-6-hydrazono-1,6-dihydropyridazine hydrochloride (42a).⁴⁰

3,6-dichloropyridazine (1.00 g, 7.22 mmol) was dissolved in ethanol (5 mL) and treat with hydrazine monohydrate (0.500 mL, 0.3 mmol) at temperature in a round bottom flask equipped with a stirring bar. The reaction mixture was reflux for 2.5h to completion, which was monitored using the LCMS. The mixture was then concentrated under vacuo to give a white solid as the product, intermediate 42a. (1.02 g, 98%), LC-MS: m/z 145 (MH)⁺.

(Z)-3-chloro-6-hydrazono-5-methyl-1,6-dihydropyridazine hydrochloride (42b) and (Z)-3-chloro-6-hydrazono-4-methyl-1,6-dihydropyridazine hydrochloride (42c).⁴⁰

General Method A was followed using 3,6-dichloro-4-methylpyridazine (1.00 g, 6.13 mmol) and hydrazine (3 mL, 60.0 mmol), which was refluxed in EtOH (30 mL) overnight. The reaction mixture was concentrated under vacuo to give a white solid as products, mixture of 2b and 2c. (1.10 g, 92%). LC-MS: m/z 159 (MH)⁺.

N’-(6-chloropyridazin-3-yl)isonicotinohydrazide (42d).⁴²

A suspension of intermediate 42a (1 g, 5.54 mmol) and isonicotinoyl chloride hydrochloride (1.19 g, 6.65 mmol) in DCM (15 mL) was treated with TEA (1.4 mL, 10.0 mmol). The reaction mixture was then stirred overnight, after which the mixture was extracted using ethyl acetate and water three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo to obtain a yellow solid as the product. (1.2 g, 87%), LCMS m/z 250 (MH)⁺.

6-chloro-3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazine (35e).

(a) A dried 50 mL round bottom flask was charged with 42d (1.00 g, 4.00 mmol), followed by 10 mL of POCl₃. The mixture is then refluxed for 30h, after which the cooled mixture was concentrated and quenched with ice and NaHCO₃. The mixture was extracted with water and EtOAc, dried with Na₂SO₄. The dried organic was then concentrated to give yellow solid as the product. (560 mg, 60%), LCMS m/z 232 (MH)⁺.

6-chloro-3-(2-(trifluoromethyl)pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazine (35f).⁴³

A round bottom flask was charged with intermediate 42a (1.00 g, 6.94 mmol), 2-(trifluoromethyl)isonicotinic acid (1.50 g, 7.85 mmol) and 6 mL of POCl₃. The mixture was refluxed for 30h after which the cooled mixture was concentrated and quenched with ice and NaHCO₃. The mixture was extracted with water and EtOAc, dried with Na₂SO₄. The dried organic was then concentrated to give white solid as the product. (1.30 g, 60%), LCMS m/z 301 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.22 (d, J = 0.7 Hz, 1H), 8.1 (s, 1H), 7.37 (d, J = 10.3 Hz, 1H), 7.22 (d, J = 9.5 Hz, 4H), 3.76 (s, 2H), 3.62 (br. s., 4H), 3.49 – 3.58 (m, 6H), 3.44 – 3.49 (m, 2H), 3.42 (s, 2H), 2.33 (d, J = 3.9 Hz, 4H).

6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-amine (35g).

Procedure by Kawano et al⁴⁴ was utilized to prepare intermediate 35g, using Intermediate 42a (2.00 g, 11.0 mmol) was added to a cleaned, dried round bottom flask, and added 96 mL of EtOH and 30 mL of water. Cyanogen bromide (15 mL) was added to the mixture and allowed to stir overnight to completion. The reaction mixture was then neutralized with 2M NaOH and allowed to stand overnight. The suspension was filtered and washed with water. The yellow residue was allowed to air dry and further lyophilized as well to obtain a yellow solid as the product, 35g. (885 mg, 47%), LCMS m/z 170 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.11 (d, J = 9.8 Hz, 1H), 7.11 (d, J = 9.5 Hz, 1H), 6.69 (s, 2H).

6-chloro-[1,2,4]triazolo[4,3-b]pyridazine (35h).

Intermediate 42a (1.02 g, 6.60 mmol) was weighed into microwave vail with a stirring bar. 4 mL of formic acid was added and the vail was capped and microwaved for 30 minutes. The cooled mixture was then concentrated, quenched with NaHCO_3, and extracted with DCM (3×30 mL) with water (100 mL). The organic extract was dried with sodium sulfate and concentrated to give an off-white solid as the product, 35h. (708 mg, 65%), LC-MS: m/z 155 (MH)⁺. ¹H NMR (400 MHz, D₂O) δ ppm 7.43 – 7.49 (m, 1H), 8.21 – 8.27 (m, 1H), 9.30 – 9.34 (m, 1H). LC-MS: m/z 155.0, 157.0 (MH)⁺.

6-chloro-8-methyl-[1,2,4]triazolo[4,3-b]pyridazine (35i) and 6-chloro-7-methyl-[1,2,4]triazolo[4,3-b]pyridazine (35j).

Procedure for 35h was repeated using 42b and 42c mixture (1.10 g, 5.66 mmol) to yield a white solid product, 35i and 35j. (800 mg, 84%), LC-MS: m/z 169 (MH)⁺.

Ethyl 6-chloroimidazo[1,2-b]pyridazine-2-carboxylate (35l).

An oven-dried 50 mL round bottom flask with a stirring bar was charged with intermediate 43 (400 mg, 2.02 mmol), and 15 mL of ethanol. Thionyl chloride (2 mL) was added in droplets to the stirring mixture. The mixture was refluxed at 130 °C overnight and cooled to room temperature. The mixture is then concentrated, washed with pentane, and dried under vacuo to give a white solid product confirmed by p-NMR. (400 mg, 88%), LCMS m/z 226 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.52 (s, 1H), 7.16 – 7.30 (m, 2H), 3.82 (s, 2H), 3.45 – 3.70 (m, 8H), 2.53(s, 3H).

Butyl 6-chloroimidazo[1,2-b]pyridazine-2-carboxylate (35m).

An oven-dried 50 mL round bottom flask with a stirring bar was charged with intermediate 43 (400 mg, 2.02 mmol), and 15 mL of 1-butanol. Thionyl chloride (2 mL) was added in droplets to the stirring mixture. The mixture was refluxed at 130 °C overnight and cooled to room temperature. The suspension was filtered under vacuo while being washed with ethyl ether to obtain brown crystalline solid confirmed by the LCMS. (300 mg, 58%), LCMS m/z 254 (MH)⁺.

6-chloro-N,N-dimethylimidazo[1,2-b]pyridazine-2-carboxamide (35n).

A suspension of dimethylamine hydrochloride (635 mg, 7.60 mmol) and intermediate 43 (1 g, 5.06 mmol) was treated with TEA (1.4 mL, 10.0 mmol), HOBT (683 mg, 5.06 mmol) and EDC-HCl (1.16 g, 6.07 mmol) at room temperature. The reaction mixture was then stirred overnight, after which the mixture was extracted using ethyl acetate (3×25 mL) and water three times. After which, the organic portion was then dried over Na₂SO₄ and concentrated under vacuo to obtain brown solid as the product. (1.01 g, 89%), LCMS m/z 225 (MH)⁺.

(6-chloroimidazo[1,2-b]pyridazin-2-yl)(morpholino)methanone (35o).

A suspension of (6-chloroimidazo[1,2-b]pyridazin-2-yl)(morpholino)methanone (0.53 mL, 6.14 mmol) and 6-chloroimidazo[1,2-b]pyridazine-2-carboxylic acid, intermediate 43 (1.00 g, 5.06 mmol) was treated with TEA (1.4 mL, 10.0 mmol), HOBT (683 mg, 5.06 mmol) and EDC-HCl (1.16 g, 6.07 mmol)) at room temperature. The reaction mixture was then stirred overnight, after which the mixture was extracted using ethyl acetate (3×25 mL) and water three times. After which, the organic portion was then dried over Na₂SO₄ and concentrated under vacuo. The crude product is purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as product. (276 mg, 20%), LCMS m/z 267 (MH)⁺.

N-(6-chloro-3-iodopyridin-2-yl)acetamide (47a).

An oven-dried 50 mL round bottom flask was charged with 6-chloro-3-iodopyridin-2-amine (44, 300 mg, 1.18 mmol) and dissolved in DCM (6 mL). Acetyl chloride (0.3 mL, 4.20 mmol) was added dropwise to the mixture placed in an ice bath while stirring. The reaction mixture was stirred overnight, after which the mixture was extracted using ethyl acetate (3×25 mL) and water three times. The organic fraction was then dried over Na₂SO₄ and concentrated under vacuo and purified by normal phase using amine column (0→5% MeOH in EtOAc) to obtain a white solid as the product. (300 mg, 86%), LCMS m/z 297 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 10.27 (s, 1H), 8.31 (d, J = 8.3 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 1.88 – 2.16 (m, 3H).

N-(6-chloro-3-iodopyridin-2-yl)ethanethioamide (47b).

An oven-dried 50 mL round bottom flask with stirring bar was charged with N-(6-chloro-3-iodopyridin-2-yl)acetamide (100 mg, 0.340 mmol) and Lawesson’s reagent. The mixture was dissolved with 4 mL of DCM. The reaction mixture was allowed to stir overnight, after which the mixture was concentrated and purified by normal phase using amine column (0→5% MeOH in EtOAc) to obtain a white crystalline solid as the product. (73 mg, 70%), LCMS m/z 313 (MH)⁺.

O-benzyl carbonisothiocyanatidate (45).

A clean, dry round bottom flask with stirring bar was added potassium isothiocyanate (3.00 g) in 30 mL of ethyl acetate under nitrogen was cooled to 0 °C and 4 mL (19.2 mmol) 0f benzyl chloroformate (4 mL) was added dropwise. The reaction mixture was allowed to warm to room temperature and then stirred under nitrogen overnight. The reaction mixture was filtered through Celite and evaporated to dryness to give a crude yellow oil (1.50 g, 41%), LCMS m/z 194 (MH)⁺.

1-(6-chloro-3-iodopyridin-2-yl)thiourea carbamate (46).

Cleaned and dried RBF was charged with 6-chloro-3-iodopyridin-2-amine 45 (1.00 g, 3.93 mmol), and O-benzyl carbonisothiocyanatidate (1.50 g, 7.76 mmol) in DCM (30 mL). The mixture was stirred for 48 hours, after which was concentrated, and recrystallized in EtOAc to give white solid as product. (963 mg, 57%), LCMS m/z 448 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 11.63 (s, 1H), 11.41 (s, 1H), 8.35 (d, J = 8.3 Hz, 1H), 7.33 – 7.53 (m, 5H), 7.28 (d, J = 8.3 Hz, 1H), 5.27 (s, 2H).

Benzyl (5-chlorothiazolo[4,5-b]pyridin-2-yl)carbamate (35p).

An oven dried round bottom flask with stirring bar was charged with intermediate 46 (400 mg, 0.893 mmol), L-proline (10.3 mg, 0.0895 mmol), Copper iodide (34.0 mg, 0.178), and potassium carbonate (247 mg, 1.79 mmol) in dioxane (10 mL). The mixture was then heated at 100°C overnight. The mixture was cooled to room temperature and added to it was satd. NH₄Cl solution for 1h. The mixture was filtered, washed with water, and dried to obtain a brown compound. (219 mg, 77%), LCMS m/z 320 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.62 (s, 1H), 8.47 (d, J = 8.6 Hz, 1H), 7.28 – 7.54 (m, 6H), 5.30 (s, 2H).

5-chloro-2-methylthiazolo[4,5-b]pyridine (35q).

Procedure by Nandy et al was used to synthesize 35q.³⁶ An oven-dried 50 mL round bottom flask with stirring bar was charged with N-(6-chloro-3-iodopyridin-2-yl)ethanethioamide 47b (73.0 mg, 0.234 mmol), 1,10-phenanthroline (8.40 μg, 0.0466 μmol) and potassium tert-butoxide. The mixture was stirred in MeOH (2 mL), for 3h after which the reaction was concentrated and purified by normal phase using amine column (0→5% MeOH in EtOAc) to obtain a brown oily compound as the product. (42.0 mg, 98%), LCMS m/z 185 (MH)⁺. ¹H NMR (400 MHz, CDCl₃) 8.11 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 2.90 (s, 3H).

5-chlorothiazolo[4,5-b]pyridin-2-amine (48a).³⁵

Procedure by Molette et al was followed to synthesize intermediate 48a. A suspension of 35r (350 mg, 1.21 mmol) in 10 mL of conc. H₂SO₄ in a 50 mL round bottom was refluxed for 16h. The reaction mixture was cooled to room temperature followed by neutralizing with 2M NaOH. The mixture was extracted using EtOAc (3 × 30 mL) and water three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo to obtain dark-brown solid as product. (219 mg, 98%), LCMS m/z 185 (MH)⁺.

N-(5-chlorothiazolo[4,5-b]pyridin-2-yl)benzamide (35r).

Procedure described by Molette et al was used in the synthesis of 35r.³⁵ An oven dried round bottom flask with stirring bar was charged N-((6-chloro-3-iodopyridin-2-yl)carbamothioyl)benzamide (1.08 g, 2.59 mmol), L-proline (64.0 mg, 0.556 mmol), copper iodide (60 mg, 0.315), and potassium carbonate (700 mg, 5.07 mmol) in dioxane (15 mL). The mixture was then heated at 100°C overnight. The mixture was cooled to room temperature and added to it was satd. NH₄Cl solution for 1h. The mixture was filtered, washed with water, and dried to obtain a brown compound. (740 mg, 99%), LCMS m/z 290 (MH)⁺.

N-(5-chlorothiazolo[4,5-b]pyridin-2-yl)isonicotinamide (35s).

A clean, dry round bottom flask with magnetic stirrer was charged with 5-chlorothiazolo[4,5-b]pyridin-2-amine 48a (90.0 mg, 0.485 mmol), isonicotinoyl chloride hydrochloride (86.0 mg, 0.483 mmol). The mixture was dissolved in DCM (6 mL), and treated with DIEA (170 μL, 0.979 mmol). The reaction mixture was then stirred overnight, after which the mixture was extracted using ethyl acetate and water three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo to obtain brown solid as product. (70.0 mg, 50%), LCMS m/z 291 (MH)⁺.

tert-Butyl (5-bromobenzo[d]thiazol-2-yl)carbamate (35t).⁴⁵

A well-dried round bottom flask with a stirring bar was charged with 5-bromobenzo[d]thiazol-2-amine 48b (100 mg, 0.436 mmol), Boc anhydride (143 mg, 0.655), and DMAP (0.18 mg, 0.00147 mmol). The mixture was dissolved in 6 mL of DCM and allowed to stir in RT for 16h then concentrated by vacuo. The solid residue was then suspended in 2M NH₃ in MeOH and stirred for 2h after which was concentrated by vacuo. The residue was then suspended in water and filtered to obtain white solid as product. (130 mg, 95%), LCMS m/z 330 (MH)⁺.

N-(5-chlorobenzo[d]thiazol-2-yl)benzamide (35u).

A suspension of 5-bromobenzo[d]thiazol-2-amine 48b (250 mg, 1.35 mmol) and benzoic acid (330 mg, 2.70 mmol) was treated with TEA (0.2 mL, 1.43 mmol), HOBT (183 mg, 1.36 mmol) and EDC-HCl (323 mg, 1.70 mmol) at room temperature. The reaction mixture was then stirred overnight, after which the mixture was extracted using ethyl acetate and water three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo to obtain white solid as product. 340 mg, 76%), LCMS m/z 333 (MH)⁺.

Isomeric mixture of 1-benzyl-5-chloro-1H-pyrazolo[4,3-b]pyridine (35v) and 2-benzyl-5-chloro-2H-pyrazolo[4,3-b]pyridine (35x).³⁸

A well-dried round bottom flask with a stirring bar was charged with 5-chloro-1H-pyrazolo[4,3-b]pyridine (500 mg, 3.26 mmol), Cs₂CO₃ (2.12 g, 6.51 mmol) and allowed to stir for ~5 mins in DMF (15 mL), then benzyl bromide (864 mg, 5.05 mmol) is added and allowed to stir at RT for 10 mins, then further heated to 90 °C for 2h. The reaction mixture was then cooled to room temperature and extracted three times with water and EtOAc, after which the organic extracts was dried with sodium sulfate and concentrated to give white solid as the isomeric mixture, a white solid. (675 mg, 85%), LCMS m/z 244 (MH)⁺.

5-chloro-1-methyl-1H-pyrazolo[4,3-b]pyridine (35x) and 5-chloro-2-methyl-2H-pyrazolo[4,3-b]pyridine (35w).³⁸

A well-dried round bottom flask with a stirring bar was charged 5-chloro-1H-pyrazolo[4,3-b]pyridine (500 mg, 3.26 mmol), Cs₂CO₃ (2.12 g, 6.51 mmol) and allowed to stir for ~5 mins, then MeI (0.300 mL, 4.88 mmol) is added and allowed to stir at RTfor 2h in DMF (15 mL). The reaction mixture was then cooled to room temperature and extracted three times with water and EtOAc, after which the organic extracts was dried with sodium sulfate and concentrated to give white solid as a mixture of regioisomers (white solid, 445 mg, 81%). LCMS m/z 168 (MH)⁺.

6-chloro-2-(3,4-dimethyl-1H-pyrrol-1-yl)imidazo[1,2-b]pyridazine (35y).³⁹

A vial with stirring bar is charged with 6-chloroimidazo[1,2-b]pyridazin-2-amine 50 (500 mg, 2.97 mmol), malonic acid (62.0 mg, 0.593) dissolved in ethanol (10 mL) and hexane-2,5-dione (0.42 mL, 3.58 mmol). The mixture was stirred for 2h at 80 °C, after which the crude mixture was cooled to room temperature. The mixture is then concentrated and purified by normal phase using flash column (0→100% EtOAc in Hexane) to obtain a colorless greasy compound as the product. (350 mg, 49%), LCMS m/z 247 (MH)⁺.

tert-Butyl 4-(2-(3,4-dichlorophenyl)acetyl)piperazine-1-carboxylate (5).

The procedure for intermediate 3n was followed 2-(3,4-dichlorophenyl)acetic acid (1.00 g, 4.90 mmol) and N-Boc-piperazine (1.09 g, 5.88 mmol) to give the title compound (1.57 g, 86%), LCMS m/z 374 (MH)⁺.

N-(3,4-dichlorophenyl)piperazine-1-carboxamide hydrochloride (40).

Intermediate 39 was suspended in dioxane (10 mL) and treated with 4M HCl in dioxane (7.00 mL). Everything dissolved and a white precipitate soon formed. The reaction mixture was stirred overnight. LCMS showed the reaction was complete. The mixture was concentrated to dryness under vacuo to give the product as white solid and washed with diethyl ether and dried again under vacuo to obtain desire product. (894 mg, 99%), LCMS m/z 275 (MH)⁺.

tert-butyl 4-(3-benzyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36a).

3-benzyl-6-chloro-[1,2,4]triazolo[4,3-b]pyridazine 35d (138 mg, 0.570 mmol) was added to a microwave vial with N-Boc-piperazine (116 mg, 0.620 mmol) and DIEA (0.2 mL, 1.13 mmol), then dissolved in 5 mL of ethanol. The vial was sealed and microwaved at 100 °C for 4h. The solvent was removed in vacuo, and the remaining solid was purified via Reverse Phase Flash Chromatography (5→95% CH₃CN/H₂O) to give the title compound (189 mg, 84%). LC-MS m/z 395 (M)⁺.

3-benzyl-6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazine hydrochloride (37a).

General Method A was followed using the whole portion of 36a to give intermediate, 37a. (157 mg, 99%), LCMS m/z 295 (MH)⁺.

tert-Butyl 4-(3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36b).

A dried round bottom flask with a stirring bar was charged with pyridazine 35e (200 mg, 0.860 mmol), tert-Butyl piperazine-1-carboxylate (241 mg, 1.30 mmol), 0.07 ml of DIEA (0.3 mL, 1.71 mmol) and 6 mL of EtOH. The mixture was allowed to heat at 100°C for 2h and cooled to room temperature. The mixture was concentrated by vacuo then extracted with water and 3 portions of 25 mL of EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a yellow solid compound as the product. (300 mg, 91%), LCMS m/z 382 (MH)⁺.

6-(piperazin-1-yl)-3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazine hydrochloride (37b).

General Method A was then followed using the whole portion of 36b to give a brown solid as intermediate 37b. (270 mg, 99%), LCMS m/z 282 (MH)⁺.

tert-Butyl 4-(3-(2-(trifluoromethyl)pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36c).

A dried round bottom flask with a stirring bar was charged with 6-chloro-3-(2-(trifluoromethyl)pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazine 35f (100 mg, 0.334 mmol), tert-Butyl piperazine-1-carboxylate (68.0 mg, 0.366 mmol), 0.07 ml of DIEA (0.409 mmol) and 6 mL of EtOH. The mixture was allowed to heat at 100°C for 2h and cooled to room temperature. The mixture was concentrated by vacuo then extracted with water and 3 portions of 25 mL of EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a white solid compound as the product. (85.0 mg, 80%), LCMS m/z 450 (MH)⁺.

6-(piperazin-1-yl)-3-(2-(trifluoromethyl)pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazine hydrochloride (37c).

General Method A was followed using the whole portion of 36c to give a white solid as intermediate 37c. (71 mg, 97%), LCMS m/z 350 (MH)⁺.

tert-Butyl 4-(3-amino-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36d).⁴⁶

An oven dried round bottom flask with stirring bar was charged with 6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-amine 35g (100 mg, 0.590 mmol), Boc-piperazine (200 mg, 1.08 mmol), dissolved in 6 mL of n-butanol and refluxed for 48h at 130°C. The mixture was then cooled to room temperature and extracted using ethyl acetate and water three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo to obtain brown solid as the product with 75% purity. (120 mg, 64%), LCMS m/z 320 (MH)⁺.

6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-amine hydrochloride (37d).

General Method A was then followed using the whole portion of 36d to give a brown solid as intermediate 37d. (95.0 mg, 99%), LCMS m/z 220 (MH)⁺.

6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazine (37e).

General Method A was then followed using the whole portion of 36e to give a brown solid as intermediate 37e. (1.65 g, 97%), LCMS m/z 205 (MH)⁺.

8-methyl-6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazine hydrochloride (37f) and 7-methyl-6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazine hydrochloride (37g).

General Method A was then followed using the whole portion of 36f and 36g to give a brown solid as intermediate 37f and 37g. (395 mg, 99%), LCMS m/z 219 (MH)⁺.

tert-Butyl 4-(7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36h).³⁰

The hydrogenation reaction vessel with a stirring bar was charged with intermediate 36e (120 mg, 0.395 mmol) and Pd/C (12.0 mg, 10% mol) was dissolved in ~100 mL of MeOH. The reaction mixture was hydrogenated by a pressure of 60 psi of H₂(g) for 72h. The reaction mixture was filtered through celite, the filtrate is concentrated and purified by flash chromatography using normal phase with 0→100% EtOAc in hexane to give the product as white solid. (100 mg, 83%). LCMS m/z 306 (MH)⁺.

6-(piperazin-1-yl)-7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazine hydrochloride (37h).

The full portion of Intermediate 36h was suspended in 4M HCl in dioxane (10 mL) in in 5 ml of MeOH in separate round bottom flask with stirring bar. The reaction crude was allowed to stir overnight to ensure completion after which the mixture was concentrated and washed three times with Et₂O and concentrated via vacuo to give white solid as intermediate 37h. (78.0 mg, 99%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.45 (s, 1H), 3.71 – 3.90 (m, 4H), 3.10 – 3.27 (m, 6H), 2.88 – 2.99 (m, 2H). LCMS m/z 206 (MH)⁺.

tert-Butyl 4-(3-chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36i).⁴⁷

A dried round bottom flask was charged with 3,6-dichloro-[1,2,4]triazolo[4,3-b]pyridazine (1.00 g, 5.29 mmol), tert-Butyl piperazine-1-carboxylate (1.40 g, 7.53 mmol), and DIEA (1.4 mL, 8.10 mmol) and 10 mL of DMF. The mixture was stirred under nitrogen overnight, after which the mixture was washed with water, filtered, and dried under vacuo to obtain white solid. (1.30 g, 73%), LCMS m/z 339 (MH)⁺.

3-chloro-6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazine hydrochloride (37i).

General Method A was followed using a portion of intermediate 36i (200 mg, 0.592 mmol) to give a white solid as intermediate 37i. (160 mg, 99%), LCMS m/z 239 (MH)⁺.

tert-Butyl 4-(3-morpholino-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate (36j).³¹

A clean round bottom flask was charged with tert-Butyl 4-(3-chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxylate 36i (200 mg, 0.590 mmol), and morpholine (76 μL, 0.886 mmol). The mixture was then heated at 160 °C for 30h. The reaction mixture was cooled to room temperature and was extracted using ethyl acetate and water three times. After which, the organic fraction was dried over Na₂SO₄ and concentrated under vacuo to obtain brown solid as the product. (210 mg, 91%), LCMS m/z 390 (MH)⁺.

4-(6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)morpholine hydrochloride (37j).

General Method A was then followed using intermediate 36j (210 mg, 0.538 mmol) to give a brown solid as intermediate 37j. (170 mg, 97%), LCMS m/z 290 (MH)⁺.

Ethyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)imidazo[1,2-b]pyridazine-2-carboxylate (36k).

General Method B was followed using intermediate 35l (200 mg, 0.533 mmol), Boc-piperazine (330 mg, 1.77 mmol), and treated with DIEA (0.300 mL, 1.73 mmol) in NMP (10 mL) to obtain a brown oily compound as intermediate 36k. (250 mg, 75%), LCMS m/z 376 (MH)⁺.

Ethyl 6-(piperazin-1-yl)imidazo[1,2-b]pyridazine-2-carboxylate hydrochloride (37k).

General Method A was then followed to give a brown solid as intermediate 37k, (200 mg, 96%), LCMS m/z 276 (MH)⁺.

Butyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)imidazo[1,2-b]pyridazine-2-carboxylate (36l).

General Method B was followed using intermediate 35m (200 mg, 0.788 mmol), Boc-piperazine (220 mg, 1.18 mmol), and treated with DIEA (0.203 mL, 1.17 mmol) in NMP (10 mL) to obtain a brown oily compound as intermediate 36l. (270 mg, 85%), LCMS m/z 404 (MH)⁺.

Butyl 6-(piperazin-1-yl)imidazo[1,2-b]pyridazine-2-carboxylate hydrochloride (37l).

General Method A was followed using 36l to synthesize brown solid compound as the desired product, 37l. (225 mg, 99%), LCMS m/z 304 (MH)⁺.

tert-Butyl 4-(2-(dimethylcarbamoyl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate (36m).

General Method B was followed using intermediate 35n (200 mg, 0.890 mmol), Boc-piperazine (248 mg, 1.33 mmol), and treated with DIEA (0.231 mL, 1.33 mmol) in NMP (6.00 mL) to obtain a brown compound as intermediate 36m. (250 mg, 75%), LCMS m/z 375 (MH)⁺, N,N-dimethyl-6-(piperazin-1-yl)imidazo[1,2-b]pyridazine-2-carboxamide hydrochloride (37m), General Method A was then followed using the whole portion of 36m to brown solid compound, 37m. (200 mg, 96%), LCMS m/z 275 (MH)⁺.

tert-Butyl 4-(2-(morpholine-4-carbonyl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate (36n).

General Method B was followed using intermediate 35o (200 mg, 0.750 mmol), Boc-piperazine (209 mg, 1.25 mmol), and treated with DIEA (0.195 mL, 1.25 mmol) in NMP (10 mL) to obtain a brown compound as intermediate 36n. (300 mg, 96%), LCMS m/z 417 (MH)⁺.

Morpholino(6-(piperazin-1-yl)imidazo[1,2-b]pyridazin-2-yl)methanone hydrochloride (37n).

General Method A was then followed using the whole portion of 36n to obtain brown solid compound, (250 mg, 98%), LCMS m/z 317 (MH)⁺.

5-(piperazin-1-yl)thiazolo[4,5-b]pyridin-2-amine hydrochloride (37o).

General Method A was then followed using the whole portion of 36o in MeOH to obtain double-deprotected brown solid compound, 37o. (140 mg, 97%), LCMS m/z 236 (MH)⁺.

tert-Butyl 4-(2-methylbenzo[d]thiazol-5-yl)piperazine-1-carboxylate (36p).

General Method B was followed using intermediate 35q (42.0 mg, 0.244 mmol), Boc-piperazine (181 mg, 0.975 mmol), and treated with DIEA (0.17 mL, 10.5 mmol) in NMP (10 mL) to obtain a brown oily compound as intermediate 36p. (35.0 mg, 46%), LCMS m/z 335 (MH)⁺.

2-methyl-5-(piperazin-1-yl)thiazolo[4,5-b]pyridine hydrochloride (37p).

General Method A was then followed using the whole portion of 36p obtain brown solid compound. (27.0 mg, 96%), LCMS m/z 235 (MH)⁺.

tert-Butyl 4-(2-((tert-butoxycarbonyl)amino)benzo[d]thiazol-5-yl)piperazine-1-carboxylate (36q).

General Method C was followed using 35t (125 mg, 0.380 mmol), tert-Butyl piperazine-1-carboxylate (212 mg, 1.14 mmol), sodium tert-butoxide (213 mg, 1.90 mmol), Pd₂(dba)₃ (35 mg, 0.0383 mmol) and RuPhos (35.0 mg, 0.0751 mmol) in 10 ml of dioxane to give the product as yellowish solid. (150 mg, 94%), LCMS m/z 435 (MH)⁺.

5-(piperazin-1-yl)benzo[d]thiazol-2-amine hydrochloride (37q).

General Method A was then followed using the whole portion of 36q to give a brown solid as intermediate 37q. (93.0 mg, 99%), LCMS m/z 235 (MH)⁺.

tert-Butyl 4-(2-benzamidothiazolo[4,5-b]pyridin-5-yl)piperazine-1-carboxylate (36r).²⁸

General Method C was followed using 35r (200 mg, 0.692 mmol), tert-Butyl piperazine-1-carboxylate (193 mg, 1.04 mmol), sodium tert-butoxide (386 mg, 3.44 mmol), Pd₂(dba)₃ (6.00 mg, 0.00656 mmol) and RuPhos (6.40 mg, 0.0137 mmol) in 10 ml of dioxane to give the product as white solid to yellow solid. (111 mg, 37%), LCMS m/z 440 (MH)⁺.

N-(5-(piperazin-1-yl)thiazolo[4,5-b]pyridin-2-yl)benzamide hydrochloride (37r).

General Method A was then followed using the whole portion of 36r to give a yellow solid as intermediate 37r. (91.0 mg, 96%), LCMS m/z 340 (MH)⁺.

tert-Butyl 4-(2-benzamidobenzo[d]thiazol-5-yl)piperazine-1-carboxylate (36s).

General Method C was followed using 35u (250 mg, 0.754 mmol), tert-Butyl piperazine-1-carboxylate (421 mg, 2.26 mmol), sodium tert-butoxide (422 mg, 4.39 mmol), Pd₂(dba)₃ (7.00 mg, 0.0076 mmol) and RuPhos (7.00 mg, 0.0150 mmol) in 6 ml of dioxane to give the preferred product as brown solid and yellow crystalline solid respectively. (300 mg, 91%), LCMS m/z 439 (MH)⁺.

N-(5-(piperazin-1-yl)benzo[d]thiazol-2-yl)benzamide hydrochloride (37s).

General Method A was then followed using the whole portion of 36s to give a brown solid as intermediate 37s. (250 mg, 98%), LCMS m/z 339 (MH)⁺.

tert-Butyl 4-(2-(isonicotinamido)thiazolo[4,5-b]pyridin-5-yl)piperazine-1-carboxylate (36t).

General Method C was followed using 35s (70.0 mg, 0.241 mmol), Boc-piperazine (134 mg, 0.720 mmol), RuPhos (3.00 mg, 0.00644 mmol), Pd₂(dba)₃ (3.00 mg, 0.00328 mmol) and NaO^tBu (135 mg, 1.41 mmol) in 4 mL of dioxane to obtain a brown solid compound as intermediate 36t. (75.0 mg, 71%), LCMS m/z 441 (MH)⁺.

N-(5-(piperazin-1-yl)thiazolo[4,5-b]pyridin-2-yl)isonicotinamide hydrochloride (37t).

General Method A was then followed using the whole portion of 36t to obtain brown solid compound, 37t. (60.0 mg, 94%), LCMS m/z 341 (MH)⁺.

tert-Butyl 4-(2-(3,4-dimethyl-1H-pyrrol-1-yl)imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate (36u).

General Method B was followed using intermediate 35y (350 mg, 1.42 mmol), Boc-piperazine (395 mg, 2.12 mmol), and treated with DIEA (0.368 mL, 2.13 mmol) in NMP (10 mL) to obtain a brown oily compound as intermediate 36u. (350 mg, 62%), LCMS m/z 397 (MH)⁺.

2-(3,4-dimethyl-1H-pyrrol-1-yl)-6-(piperazin-1-yl)imidazo[1,2-b]pyridazine hydrochloride (37u).

General Method A was followed to obtain brown solid compound, 37u (295 mg, 95%), LCMS m/z 297 (MH)⁺.

tert-Butyl 4-(imidazo[1,2-a]pyrazin-6-yl)piperazine-1-carboxylate (36v).

General Method B was followed using 6-chloroimidazo[1,2-a]pyrazine (2 00mg, 1.30 mmol), tert-butyl-piperazine-1-carboxylate (290.5mg, 1.56mmol), DIEA (0.28 mL, 1.69 mmol), in ~2mL NMP, and purified via normal phase (0→20% MeOH/EtOAc), concentrated to obtain a yellow solid compound as intermediate 37v. (23.0 mg, 6%), LCMS m/z 304 (MH)⁺.

6-(piperazin-1-yl)imidazo[1,2-a]pyrazine hydrochloride (37v).

General Method A was then followed using the whole portion of 36v to obtain a white solid compound, 37v (15.4 mg, 84%), LCMS m/z 204 (MH)⁺.

tert-Butyl 4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate (36w).

Following General Method D, 2-chloro-5-(trifluoromethyl)pyrimidine (0.176 mL, 1.36 mmol) and Boc-piperazine (500 mg, 2.72 mmol) were dissolved in EtOH (10 mL) and treated with DIEA (0.445 mL, 2.72 mmol). The reaction was heated at 100°C for 1h. The mixture was then cooled, washed with water, and filtered to give white solid as desired product as residue which then dried. (792 mg, 670%), LCMS m/z 333 (MH)⁺.

2-(piperazin-1-yl)-5-(trifluoromethyl)pyrimidine (37w).

General Method A was then followed using the whole portion of 36w to give intermediate 37w. (408 mg, 97%), LCMS m/z 233 (MH)⁺.

tert-Butyl 4-(1-benzyl-1H-pyrazolo[4,3-b]pyridin-5-yl)piperazine-1-carboxylate (36x) and tert-Butyl 4-(2-benzyl-2H-pyrazolo[4,3-b]pyridin-5-yl)piperazine-1-carboxylate (36y).

General Method C was followed using isomeric mixture of 35w and 35x (678 mg, 2.79 mmol), tert-Butyl piperazine-1-carboxylate (2.00 g, 10.8 mmol), sodium tert-butoxide (1.60 g, 14.3 mmol), Pd₂(dba)₃ (26.0 mg, 0.0284 mmol) and RuPhos (26.0 mg, 0.0558 mmol) in 10 ml of dioxane. The two isomeric products were separated by silica gel chromatography with 0→100% EtOAc, with the first product eluting to be 36y and followed by the second product being 36x, all eluting between 40→100% EtOAc/Hexane. tert-Butyl 4-(1-benzyl-1H-pyrazolo[4,3-b]pyridine-5-yl)piperazine-1-carboxylate (36x) was isolated as white solid (454 mg, 41%), LCMS m/z 394 (MH)⁺, and tert-Butyl 4-(2-benzyl-2H-pyrazolo[4,3-b]pyridin-5-yl)piperazine-1-carboxylate (36y) as a yellow crystalline solid respectively (204 mg, 18%), LCMS m/z 394 (MH)⁺.

1-benzyl-5-(piperazin-1-yl)-1H-pyrazolo[4,3-b]pyridine hydrochloride (37x).

General Method A was then followed using the whole portion of 36x to give a white solid (377 mg, 99%), LCMS m/z 294 (MH)⁺.

2-benzyl-5-(piperazin-1-yl)-2H-pyrazolo[4,3-b]pyridine hydrochloride (37y).

General Method A was then followed using the whole portion of 36y to give a white solid (170 mg, 99%), LCMS m/z 294 (MH)⁺.

tert-Butyl 4-(1-methyl-1H-pyrazolo[4,3-b]pyridin-5-yl)piperazine-1-carboxylate (36z).

General Method C was followed using 35y (218 mg, 1.30 mmol), tert-Butyl piperazine-1-carboxylate (968 mg, 5.20 mmol), sodium tert-butoxide (728 mg, 6.49 mmol), Pd₂(dba)₃ (12.0 mg, 0.0131 mmol) and RuPhos (12.0 mg, 0.0258 mmol) in 10 ml of dioxane to give the named isomer as white solid. (230 mg, 56%), LCMS m/z 318 (MH)⁺.

1-methyl-5-(piperazin-1-yl)-1H-pyrazolo[4,3-b]pyridine hydrochloride (37z).

General Method A was then followed using the whole portion of 36z to give a white solid as intermediate 37z. (180 mg, 98%), LCMS m/z 218 (MH)⁺.

tert-Butyl 4-(2-oxo-2,3-dihydrobenzo[d]thiazol-6-yl)piperazine-1-carboxylate (36aa).

General Method C was followed using 6-bromobenzo[d]thiazol-2(3H)-one (200 mg, 0.869 mmol), tert-Butyl piperazine-1-carboxylate (486 mg, 2.61 mmol), sodium tert-butoxide (mg, 4.35 mmol), Pd₂(dba)₃ (80.0 mg, 0.0874 mmol) and RuPhos (82.0 mg, 0.176 mmol) in 10 ml of dioxane to give the product as brown solid as product, 36aa. (119 mg, 90%), LCMS m/z 336 (MH)⁺.

6-(piperazin-1-yl)benzo[d]thiazol-2(3H)-one hydrochloride (37aa).

General Method A was then followed using the whole portion of 36aa to give a brown solid as intermediate 37aa. (95.0 mg, 99%), LCMS m/z 236 (MH)⁺.

tert-Butyl 4-(6-methylpyridin-2-yl)piperazine-1-carboxylate (36ab).

General Method C was followed using 2-chloro-6-methylpyridine (200 mg, 1.57 mmol), tert-Butyl piperazine-1-carboxylate (876 mg, 4.71 mmol), sodium tert-butoxide (879 mg, 7.84 mmol), Pd₂(dba)₃ (144 mg, 0.157 mmol) and RuPhos (146 mg, 0.313 mmol) in 10 ml of dioxane to give the product as brown solid. (430 mg, 98%), LCMS m/z 278 (MH)⁺.

1-(6-methylpyridin-2-yl)piperazine hydrochloride (37ab).

General Method A was then followed using the whole portion of 36ab to give a brown solid as intermediate 37ab. (329 mg, 99%), LCMS m/z 178 (MH)⁺.

tert-Butyl 4-(pyrimidin-2-yl)piperazine-1-carboxylate (36ac).

A cleaned and dry pressure vial was charged with stirring bar was charged 2-chloropyrimidine (200 mg, 1.75 mmol), Boc-piperazine (390 mg, 2.09 mmol). The mixture was dissolved with EtOH (6 mL), DIEA (0.400 mL, 2.34 mmol) was added and stirred at 100°C for 2h. The mixture is then cooled to room temperature, concentrated, and extracted with water and EtOAc, dried with Na₂SO₄. The dried organic was then concentrated to give white solid compound as the desired product. (350 mg, 76%), LCMS m/z 265 (MH)⁺.

2-(piperazin-1-yl)pyrimidine hydrochloride (37ac).

General Method A was then followed using the whole portion of 36ac to give a white solid as intermediate 37ac. (250 mg, 94%), LCMS m/z 165 (MH)⁺.

tert-Butyl 4-(pyridin-2-yl)piperazine-1-carboxylate (36ad).

General Method C was followed using 2-bromopyridine (200 mg, 1.27 mmol), tert-Butyl piperazine-1-carboxylate (707 mg, 3.80 mmol), sodium tert-butoxide (710 mg, 6.33 mmol), Pd₂(dba)₃ (115 mg, 0.127 mmol) and RuPhos (116 mg, 0.253 mmol) in 10 ml of dioxane to give the product as light-yellow solid. (486 mg, 80%), LCMS m/z 264 (MH)⁺.

1-(pyridin-2-yl)piperazine hydrochloride (37ad).

General Method A was then followed using the whole portion of 36ad to give a cream white solid as intermediate 37ad. (367 mg, 99%), LCMS m/z 164 (MH)⁺.

tert-Butyl 4-(1H-indazol-6-yl)piperazine-1-carboxylate (36ae).

General Method C was followed using 6-chloro-1H-indazole (150 mg, 0.983 mmol), tert-Butyl piperazine-1-carboxylate (549 mg, 2.95 mmol), sodium tert-butoxide (552 mg, 4.92 mmol), Pd₂(dba)₃ (90.0 mg, 0.0984 mmol) and RuPhos (92.0 mg, 0.197 mmol) in10 ml of dioxane to give the product as brown solid. (280 mg, 94%), LCMS m/z 303 (MH)⁺.

6-(piperazin-1-yl)-1H-indazole hydrochloride (37ae).

General Method A was then followed using the whole portion of 36ae to give a brown solid as intermediate 37ae. (219 mg, 99%), LCMS m/z 203(MH)⁺.

tert-Butyl 4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate (36af).

General Method C was followed using 2-chloro-4-(trifluoromethyl)pyrimidine (200 mg, 1.10 mmol), tert-Butyl piperazine-1-carboxylate (611 mg, 3.28 mmol), sodium tert-butoxide (615 mg, 5.48 mmol), Pd₂(dba)₃ (100 mg, 0.109 mmol) and RuPhos (102 mg, 0.219 mmol) in 10 ml of dioxane to give the product as brown solid. (305 mg, 84%), LCMS m/z 333 (MH)⁺.

2-(piperazin-1-yl)-4-(trifluoromethyl)pyrimidine hydrochloride (37af).

General Method A was then followed using the whole portion of 36af to give a brown solid as intermediate 37af. (240 mg, 97%), LCMS m/z 233 (MH)⁺.

tert-Butyl 4-(1H-benzo[d]imidazol-6-yl)piperazine-1-carboxylate (36ag).

General Method C was followed using 6-chloro-1H-benzo[d]imidazole (200 mg, 1.310 mmol), tert-Butyl piperazine-1-carboxylate (731 mg, 3.93 mmol), sodium tert-butoxide (735 mg, 6.56 mmol), Pd₂(dba)₃ (12 mg, 0.0131 mmol) and RuPhos (12 mg, 0.0257 mmol) in 10 ml of dioxane to give the product as brown solid, 36ag. (310 mg, 78%), LCMS m/z 303 (MH)⁺.

6-(piperazin-1-yl)-1H-benzo[d]imidazole hydrochloride (37ag).

General Method A was then followed using the whole portion of 36ag to give a brown solid as intermediate 37ag. (241 mg, 99%), LCMS m/z 203 (MH)⁺.

tert-Butyl 4-(benzo[c][1,2,5]oxadiazol-5-yl)piperazine-1-carboxylate (36ah).

General Method C was followed using 5-bromobenzo[c][1,2,5]oxadiazole (50.0 mg, 0.251 mmol), tert-Butyl piperazine-1-carboxylate (70.0 mg, 0.376 mmol), sodium tert-butoxide (124 mg, 1.11 mmol), Pd₂(dba)₃ (10.0 mg, 0.0109 mmol) and RuPhos (20.0 mg, 0.0429 mmol) in 10 ml of dioxane to give the product as brown solid. (50.0 mg, 51%), LCMS m/z 304 (MH)⁺.

5-(piperazin-1-yl)benzo[c][1,2,5]oxadiazole hydrochloride (37ah).

General Method A was then followed using the whole portion of 36ah to give a brown solid as intermediate 37ah. (37.7 mg, 95%), LCMS m/z 204 (MH)⁺.

tert-Butyl 4-(benzo[d]thiazol-2-yl)piperazine-1-carboxylate (36ai).

General Method C was followed 2-bromobenzo[d]thiazole (200 mg, 0.934 mmol), tert-Butyl piperazine-1-carboxylate (522 mg, 2.81 mmol), sodium tert-butoxide (524 mg, 4.67 mmol), Pd₂(dba)₃ (86 mg, 0.0941 mmol) and RuPhos (88.0 mg, 0.189 mmol) in 10 ml of dioxane to give the product as brown solid. (223 mg, 95%), LCMS m/z 320 (MH)⁺.

2-(piperazin-1-yl)benzo[d]thiazole hydrochloride (37ai).

General Method A was then followed using the whole portion of 36ai to give a brown solid as intermediate 37ai. (176 mg, 98%), LCMS m/z 220 (MH)⁺.

tert-Butyl 4-(6-methoxypyridazin-3-yl)piperazine-1-carboxylate (36aj).

General Method C was followed using 3-chloro-6-methoxypyridazine (200 mg, 1.38 mmol), tert-Butyl piperazine-1-carboxylate (386 mg, 2.07 mmol), sodium tert-butoxide (844 mg, 7.53 mmol), Pd₂(dba)₃ (13.0 mg, 0.0142 mmol) and RuPhos (14 mg, 0.0300 mmol) in 10 ml of dioxane to give the product as brown solid. (300 mg, 74%), LCMS m/z 295 (MH)⁺.

3-methoxy-6-(piperazin-1-yl)pyridazine hydrochloride (37aj).

General Method A was then followed using the whole portion of 36aj to give a brown solid as intermediate 37aj. (230 mg, 98%), LCMS m/z 195 (MH)⁺.

tert-Butyl 4-(1H-pyrazolo[4,3-b]pyridin-5-yl)piperazine-1-carboxylate (36ak).

General Method C was followed using 5-chloro-1H-pyrazolo[4,3-b]pyridine 49 (100 mg, 0.651 mmol), tert-butyl piperazine-1-carboxylate (485 mg, 2.61 mmol), sodium tert-butoxide (365 mg, 3.26 mmol), Pd₂(dba)₃ (6.00 mg, 0.00656 mmol) and RuPhos (6 mg, 0.0129 mmol) in 10 ml of dioxane to give the product as white solid. (190 mg, 96%), LCMS m/z 304 (MH)⁺.

5-(piperazin-1-yl)-1H-pyrazolo[4,3-b]pyridine hydrochloride (37ak).

General Method A was then followed using the whole portion of 36ak to give a white solid as intermediate 37ak. (145 mg, 97%), LCMS m/z 204 (MH)⁺.

tert-Butyl 4-(2-methylbenzo[d]thiazol-5-yl)piperazine-1-carboxylate (36al).

General Method C was followed using 5-bromo-2-methylbenzo[d]thiazole (200 mg, 0.877 mmol), tert-Butyl piperazine-1-carboxylate (490 mg, 2.63 mmol), sodium tert-butoxide (492 mg, 5.13 mmol), Pd₂(dba)₃ (81.0 mg, 0.0885 mmol) and RuPhos (82.0 mg, 0.176 mmol) in 10 ml of dioxane to obtain a brown solid as intermediate 36al. (110 mg, 72%), LCMS m/z 303 (MH)⁺.

2-Methyl-5-(piperazin-1-yl)benzo[d]thiazole hydrochloride (37al).

General Method A was then followed using the whole portion of 36al to obtain a brown solid compound, 37al. (84.0 mg, 97%), LCMS m/z 203 (MH)⁺.

tert-Butyl 4-(pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (36am).

The procedure by Yingzhi was followed to synthesize 36am.⁴⁸ Suspension of 5-chloropyrazolo[1,5-a]pyrimidine (200 mg, 1.30 mmol) and Boc-piperazine (291 mg, 1.56 mmol) in 2-propanol (15 mL) was treated with DIEA (280 μL, 1.61 mmol). The mixture was stirred at 65 °C for 16 h. The reaction was then allowed to cool to RTand concentrated as well as extracted using ethyl acetate (30 × 25 mL) and water three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo to obtain the title compound. (185 mg, 94%), LCMS m/z 304 (MH)⁺.

5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine hydrochloride (37am).

General Method A was then followed using the whole portion of 36am to give a brown solid as intermediate 37am. (140 mg, 96%), LCMS m/z 204 (MH)⁺.

tert-Butyl 4-(1H-pyrrolo[2,3-b]pyridin-6-yl)piperazine-1-carboxylate (36an).

General Method C was followed using 6-bromo-1H-pyrrolo[2,3-b]pyridine (77.5 mg, 0.508 mmol), tert-Butyl piperazine-1-carboxylate (142 mg, 0.765 mmol), sodium tert-butoxide (245 mg, 2.55 mmol), Pd₂(dba)₃ (4.66 mg, 0.00510 mmol) and RuPhos (4.75 mg, 0.0102 mmol) in 10 ml of dioxane to obtain a brown solid as intermediate 36an. (110 mg, 72%), LCMS m/z 303 (MH)⁺.

6-(piperazin-1-yl)-1H-pyrrolo[2,3-b]pyridine hydrochloride (37an).

General Method A was then followed using the whole portion of 36an to give a brown solid compound, 37an. (84.0 mg, 97%), LCMS m/z 203 (MH)⁺.

tert-Butyl 4-(6-(trifluoromethyl)pyridazin-3-yl)piperazine-1-carboxylate (36ao).⁴⁹

A suspension of 3-chloro-6-(trifluoromethyl)pyridazine (334 mg, 1.83 mmol), Boc-piperazine (1. 00 g, 5.38 mmol) in 10 mL of acetonitrile. The mixture was treated with DIEA (1.00 mL, 5.76 mmol) and heated at 180°C for 30 mins in the microwave. The reaction mixture was then allowed to cool and concentrated under vacuo. The crude mixture was then extracted three times with water and 3 portions of 25 mL EtOAc. The organic extract was dried with sodium sulfate and concentrated to a white solid as the product, 36ao. (600 mg, 99%), LCMS m/z 333 (MH)⁺.

3-(piperazin-1-yl)-6-(trifluoromethyl)pyridazine hydrochloride (37ao).

General Method A was then followed using the whole portion of 36ao to give a white solid as intermediate 37ao. (230 mg, 98%), LCMS m/z 233 (MH)⁺.

tert-Butyl 4-(imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate (36ap).

A dry microwave vail with a stirring bar was charged with 6-chloroimidazo[1,2-b]pyridazine (100 mg, 0.651 mmol), tert-Butyl piperazine-1-carboxylate (363 mg, 1.95 mmol). The mixture was then dissolved with 4 mL NMP, the vial was then capped and heated in the MW at 130°C for 8h. The mixture was then allowed to cool and then extracted three times with water and 3 portions of 25 mL EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a crude brown solid, which was proceeded to the next step by adding 6 mL of 4M HCl in dioxane and stirring for overnight at room temperature to obtain a brown solid as desired product. (131 mg, 66%), LCMS m/z 304 (MH)⁺.

6-(piperazin-1-yl)imidazo[1,2-b]pyridazine hydrochloride (37ap).

General Method A was then followed using the whole portion of 36ap to give a brown solid as intermediate 37ap. (102 mg, 98%), LCMS m/z 204 (MH)⁺.

tert-Butyl 4-(5-nitrothiazol-2-yl)piperazine-1-carboxylate (36aq).⁵⁰

A round bottom flask with a stirring bar was then charged with 2-bromo-5-nitrothiazole (100 mg, 0.478 mmol), tert-Butyl piperazine-1-carboxylate (178 mg, 0.957 mmol), and K₂CO₃ (132 mg, 0.955 mmol). 5 mL of acetonitrile was added to the mixture and allowed to stir for 4h while capped. The crude mixture was concentrated via vacuo then extracted three times with water and 3 portions of 25 mL EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a yellow oily solid compound as the desired product. (140 mg, 94%), LCMS m/z 314 (MH)⁺.

5-nitro-2-(piperazin-1-yl)thiazole hydrochloride (37aq).

General Method A was then followed using the whole portion of 36aq to give a yellow solid as intermediate 37aq. (110 mg, 99%), LCMS m/z 214 (MH)⁺.

tert-Butyl 4-(2-methylpyrimidin-4-yl)piperazine-1-carboxylate (36ar).

A dried round bottom flask with a stirring bar was charged with 4-chloro-2-methylpyrimidine (521 mg, 0.334 mmol), tert-Butyl piperazine-1-carboxylate (715 mg, 0.366 mmol), 1.00 ml of DIEA (0.409 mmol) and 15 mL of dioxane. The mixture was allowed to heat at 150°C for 30 mins and cooled to room temperature. The mixture was concentrated by vacuo then extracted with water and 3 portions of 25 mL of EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a white solid compound as the product. (1.01 g, 90%), LCMS m/z 279 (MH)⁺.

2-methyl-4-(piperazin-1-yl)pyrimidine hydrochloride (37ar).

General Method A was then followed using the whole portion of 36ar to give a white solid as intermediate 37ar. (770 mg, 99%), LCMS m/z 179 (MH)⁺.

tert-Butyl 4-(2,7-naphthyridin-3-yl)piperazine-1-carboxylate (36as).

A dry microwave vail with a stirring bar was charged with 3-chloro-2,7-naphthyridine (100 mg, 0.608 mmol), tert-Butyl piperazine-1-carboxylate (170 mg, 0.914 mmol). The mixture was then dissolved with 4 mL NMP, the vial was then capped and heated in the MW at 130°C for 30 mins. The mixture was then allowed to cool and then extracted three times with water and 3 portions of 25 mL EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a crude brown solid, which was proceeded to the next step by adding 6 mL of 4M HCl in dioxane and stirring for overnight at room temperature to obtain a brown solid as product. (111 mg, 85%), LCMS m/z 315 (MH)⁺.

3-(piperazin-1-yl)-2,7-naphthyridine hydrochloride (37as).

General Method A was then followed using the whole portion of 36as to give a brown solid as intermediate 37as. (70.0 mg, 99%), LCMS m/z 215 (MH)⁺.

tert-Butyl 4-(1,5-naphthyridin-2-yl)piperazine-1-carboxylate (36at).

A dry microwave vail with a stirring bar was charged with 3-chloro-2,7-naphthyridine (100 mg, 0.608 mmol), tert-Butyl piperazine-1-carboxylate (170 mg, 0.914 mmol). The mixture was then dissolved with 4 mL NMP, the vial was then capped and heated in the MW at 130°C for 30 mins. The mixture was then allowed to cool and then extracted three times with water and 3 portions of 25 mL EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a crude brown solid as the product. (153 mg, 80%), LCMS m/z 315 (MH)⁺.

2-(piperazin-1-yl)-1,5-naphthyridine hydrochloride (37at).

General Method A was then followed using the whole portion of 36at to give a brown solid as intermediate 37at. (120 mg, 98%), LCMS m/z 215 (MH)⁺.

tert-Butyl 4-(quinolin-2-yl)piperazine-1-carboxylate (36au).⁵¹

2-chloroquinoline (300 mg, 1.83 mmol), Boc-piperazine (409 mg, 2.20 mmol) and K₂CO₃ were weighed into a clean, dry round bottom flask with a stirring bar. The mixture was dissolved with acetonitrile and refluxed for 16h. The reaction mixture was allowed to cool to room temperature, concentrated and extracted three times with water and 3 portions of 25 mL EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a crude brown solid as the product. (500 mg, 87%), LCMS m/z 314 (MH)⁺.

2-(piperazin-1-yl)quinoline hydrochloride (37au).

General Method A was then followed using the whole portion of 36au to give a brown solid as intermediate 37au. (389 mg, 98%), LCMS m/z 214 (MH)⁺.

tert-Butyl 4-(benzo[d]oxazol-2-yl)piperazine-1-carboxylate (36av).

Procedure by Kumar et al was followed to synthesize this intermediate.⁵² A dry microwave vial with a stirring bar was charged with 2-chlorobenzo[d]oxazole (200 mg, 0.608 mmol), tert-Butyl piperazine-1-carboxylate (363 mg, 0.914 mmol). The vial was then capped and heated in the MW at 130°C for 30 mins. The mixture was then allowed to cool and then extracted three times with water and 3 portions of 25 mL EtOAc. The organic extract was dried with sodium sulfate and concentrated to give a crude brown solid as the product. (298 mg, 75%), LCMS m/z 304 (MH)⁺.

2-(piperazin-1-yl)benzo[d]oxazole hydrochloride (37av).

General Method A was then followed using whole portion of 36av to give a brown solid as intermediate 37av. (230 mg, 98%), LCMS m/z 204 (MH)⁺.

tert-Butyl 4-phenylpiperazine-1-carboxylate (36aw).

General Method C was followed using iodobenzene (0.490 mmol), Boc-piperazine (274 mg, 1.47 mmol), Pd₂(dba)₃ (45.0 mg, 0.0490 mmol), RuPhos (46.0 mg, 0.0986 mmol) and NaO^tBu (275 mg, 2.45 mmol) to give the title compound as a brown solid. (100 mg, 78%), LCMS m/z 263 (MH)⁺.

1-phenylpiperazine hydrochloride (37aw).

General Method A was followed using the whole portion of the 36aw to obtain brown solid compound, 37aw (75.0 mg, 94%), LCMS m/z 162 (MH)⁺.

1-(4-([1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (2a).

SLU-2633 was synthesized and characterized in our previous work.¹⁰

4-([1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-(3,4-dichlorophenyl)piperazine-1-carboxamide (2b) was synthesized and characterized in our previous work.¹⁰

2-(3,4-dichlorophenyl)-1-(4-phenylpiperazin-1-yl)ethanone (6a).

Following General Method E using 37aw (75.0 mg, 0.377 mmol) and 2-(3,4-dichlorophenyl)acetic acid (93.0 mg, 0.454 mmol), HOBt (51.0 mg, 0.377 mmol) and EDC-HCl (86.5 mg, 0.454 mmol) was treated with TEA (105 μL, 0.756 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (98.0 mg, 46%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.45 – 7.61 (m, 2H), 7.22 (t, J = 7.7 Hz, 3H), 6.95 (d, J = 8.1 Hz, 2H), 6.75 – 6.86 (m, 1H), 3.81 (s, 2H), 3.55 – 3.71 (m, 4H), 3.11 (br. s., 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₈Cl₂N₂O 349.0874; found 349.0865.

2-(3,4-dichlorophenyl)-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (7).

Following General Method E using 37ad (184 mg, 0.919 mmol), and 2-(3,4-dichlorophenyl)acetic acid (226 mg, 1.10 mmol), HOBt (149 mg, 0.919 mmol) and EDC-HCl (211 mg, 1.10 mmol) was treated with TEA (256 μL, 1.84 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (124 mg, 39%), HPLC purity 97%. (¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (dd, J=4.9, 1.5 Hz, 1 H) 7.49 – 7.61 (m, 3 H) 7.24 (dd, J=8.1, 2.0 Hz, 1 H) 6.85 (d, J=8.8 Hz, 1 H) 6.67 (dd, J=7.0, 5.0 Hz, 1 H) 3.82 (s, 2 H) 3.55 – 3.68 (m, 4 H) 3.44 – 3.55 (m, 4 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₇Cl₂N₃O 350.0827; found 350.0814.

2-(3,4-dichlorophenyl)-1-(4-(6-methylpyridin-2-yl)piperazin-1-yl)ethanone (8).

Following General Method E using 37ab (150 mg, 0.702 mmol) and 2-(3,4-dichlorophenyl)acetic acid (173 mg, 0.844 mmol), HOBt (94.7 mg, 0.702 mmol) and EDC-HCl (161 mg, 0.844 mmol) was treated with TEA (196 μL, 1.40 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (133 mg, 52%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.56 (d, J=8.1 Hz, 1 H) 7.52 (d, J=2.0 Hz, 1 H) 7.43 (dd, J=8.4, 7.2 Hz, 1 H) 7.23 (dd, J=8.2, 2.1 Hz, 1 H) 6.62 (d, J=8.3 Hz, 1 H) 6.53 (d, J=7.3 Hz, 1 H) 3.80 (s, 2 H) 3.59 (dt, J=17.7, 5.1 Hz, 4 H) 3.39 – 3.52 (m, 4 H) 2.30 (s, 3 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₉Cl₂N₃O 364.0983; found 364.0970.

2-(3,4-dichlorophenyl)-1-(4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)ethanone (9).

Following General Method E using 37af (100 mg, 0.372 mmol) and 2-(3,4-dichlorophenyl)acetic acid (91.5 mg, 0.446 mmol), HOBt (50.2 mg, 0.372 mmol) and EDC-HCl (85.3 mg, 0.446 mmol) was treated with TEA (104 μL, 0.744 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (52.0 mg, 33%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.72 (d, J=4.9 Hz, 1 H) 7.57 (d, J=8.3 Hz, 1 H) 7.52 (d, J=1.7 Hz, 1 H) 7.24 (dd, J=8.3, 2.0 Hz, 1 H) 7.07 (d, J=4.9 Hz, 1 H) 3.72 – 3.87 (m, 6 H) 3.52 – 3.69 (m, 4 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₅Cl₂F₃N₄O 419.0653; found 419.0638.

2-(3,4-dichlorophenyl)-1-[4-(pyrimidin-2-yl)piperazin-1-yl]ethan-1-one (10a).

Following General Method E using 37ac (100 mg, 0.197 mmol) and 2-(3,4-dichlorophenyl)acetic acid (313 mg, 0.237 mmol), HOBt (57.5 mg, 0.197 mmol) in DMF (6 mL) and was treated with DIEA (40.0 μL, 0.394 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (11.0 mg, 14%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 4.9 Hz, 2H), 7.54 – 7.60 (m, 1H), 7.50 – 7.53 (m, 1H), 7.21 – 7.28 (m, 1H), 6.63 – 6.70 (m, 1H), 3.81 (s, 2H), 3.68 – 3.77 (m, 4H), 3.52 – 3.64 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₆Cl₂N₄O 351.0779; found 351.0770.

N-(3,4-dichlorophenyl)-4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxamide (10b).

Following General Method F, using 37w (340 mg, 1.46 mmol), 1,2-dichloro-4-isocyanatobenzene (330 mg, 1.76 mmol) in DCM (2 mL) and treated with DIEA (0.752 mL, 4.39 mmol). The crude mixture was concentrated and purified by normal phase (0→15% MeOH/DCM) to obtain a white solid as desired product. (94.8 mg, 16%), HPLC purity 96%, LCMS m/z 420 (MH)⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.55 (d, J = 4.6 Hz, 1H), 7.62 (d, J = 2.7 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.23 (dd, J = 2.6, 8.7 Hz, 1H), 6.85 (d, J = 4.6 Hz, 1H), 6.41 (s, 1H), 3.90 – 4.08 (m, 4H), 3.56 – 3.66 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₄Cl₂F₃N₅O 420.0606; found 420.0596.

2-(3,4-dichlorophenyl)-1-(4-(2-methylpyrimidin-4-yl)piperazin-1-yl)ethanone (11a).

Following General Method E using 37ar (100 mg, 0.466 mmol) and 2-(3,4-dichlorophenyl)acetic acid (115 mg, 0.561 mmol), HOBt (62.9 mg, 0.466 mmol) and EDC-HCl (107 mg, 0.560 mmol) was treated with TEA (130 μL, 0.931 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain pale yellow solid as the desired product. (53.0 mg, 31%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.33 (d, J = 7.3 Hz, 1H), 7.57 (dd, J = 1.1, 8.2 Hz, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.23 (dd, J = 2.0, 8.3 Hz, 1H), 7.11 (d, J = 7.3 Hz, 1H), 3.76 – 4.00 (m, 6H), 3.54 – 3.75 (m, 4H), 2.52 – 2.56 (m, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₈Cl₂N₄O 365.0936; found 365.0924.

N-(3,4-dichlorophenyl)-4-(2-(methylamino)pyrimidin-4-yl)piperazine-1-carboxamide (11b).

General method D was followed using 4-chloro-N-methylpyrimidin-2-amine (34.6 mg, 0.241 mmol) and 40 (50.0 mg, 0.161 mmol) dissolved EtOH (1 mL) and treated with 0.08mL DIEA (80.0 μL, 0.483 mmol) and heated in microwave at 100 C for 3h. The crude mixture was then concentrated and purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain a white solid as desired product. (21.1 mg, 34%), HPLC purity 96%, LCMS m/z 381(MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.95 (s, 1H), 7.99 – 8.21 (m, 1H), 7.79 – 7.94 (m, 2H), 7.38 – 7.60 (m, 2H), 6.55 (d, J = 6.6 Hz, 1H), 3.70 – 4.03 (m, 4H), 3.60 (br. s., 4H), 2.88 (d, J = 4.4 Hz, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₈Cl₂N₆O 381.0997; found 381.0989.

2-(3,4-dichlorophenyl)-1-(4-(6-methoxypyridazin-3-yl)piperazin-1-yl)ethanone (12a).

Following General Method E using 37aj (230 mg, 0.997 mmol) and 2-(3,4-dichlorophenyl)acetic acid (245 mg, 1.20 mmol), HOBt (135 mg, 0.997 mmol) and EDC-HCl (228 mg, 1.20 mmol) was treated with TEA (278 μL, 1.99 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (264 mg, 65%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.57 (d, J = 8.3 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.43 (d, J = 9.5 Hz, 1H), 7.25 (dd, J = 2.0, 8.31 Hz, 1H), 7.07 (d, J = 9.5 Hz, 1H), 3.91 (s, 3H), 3.83 (s, 2H), 3.63 (td, J = 5.1, 19.7 Hz, 4H), 3.45 (td, J = 5.1, 13.3 Hz, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₈Cl₂N₄O₂ 381.0885; found 381.0872.

2-(3,4-dichlorophenyl)-1-(4-(6-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)ethanone (12b).

Following General Method E using 37ao (230 mg, 0.856 mmol) and 2-(3,4-dichlorophenyl)acetic acid (211 mg, 1.03 mmol), HOBt (116 mg, 0.859 mmol) and EDC-HCl (196 mg, 1.03 mmol) was treated with TEA (239 μL, 1.71 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (248 mg, 69%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.85 (d, J = 9.8 Hz, 1H), 7.51 – 7.65 (m, 2H), 7.43 (d, J = 9.5 Hz, 1H), 7.25 (dd, J = 2.1, 8.2 Hz, 1H), 3.82 – 3.88 (m, 2H), 3.60 – 3.82 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₅Cl₂F₃N₄O₂ 419.0653; found 419.0637.

2-(3,4-dichlorophenyl)-1-(4-(5-nitrothiazol-2-yl)piperazin-1-yl)ethanone (13).

Following General Method E using 37aq (110 mg, 0.439 mmol) and 2-(3,4-dichlorophenyl)acetic acid (108 mg, 0.527 mmol), HOBt (59.2 mg, 0.439mmol) and EDC-HCl (101 mg, 0.527 mmol) was treated with TEA (122 μL, 0.877 mmol) in DMF (6 mL) purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (30.0 mg, 17%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.43 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.50 (s, 1H), 7.22 (d, J = 9.8 Hz, 1H), 3.82 (s, 2H), 3.61 – 3.74 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₅H₁₄Cl₂N₄O₃S 401.0242; found 401.0229.

2-(3,4-dichlorophenyl)-1-(4-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)et han-1-one (14a).

General method D was repeated using intermediate 35a (90.0 mg, 0.610 mmol) and 4 (179 mg, 0.660 mmol) were dissolved in ethanol and placed in a microwave vial, to which DIEA (0.16 mL, 0.910 mmol) was added. The vail was sealed and heated to 100 °C for 4 hours. Solvent was removed in vacuo, and the crude product was extracted in ethyl acetate, rinsing twice with water and once with brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The crude product was purified via normal phase flash chromatography (0–20% MeOH/EtOAc) to give the title compound (32.0 mg, 13%). LC-MS: m/z 405 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, J=10.0 Hz, 1 H), 7.43 (d, J=8.2 Hz, 1 H), 7.39 (d, J=1.8 Hz, 1 H), 7.13 (dd, J=6.2, 1.8 Hz, 1 H), 6.88 (d, J=10.1 Hz, 1 H), 3.83 (br.s., 2H), 3.75 (s, 2H), 3.65 (br.s., 2H), 3.51–3.59 (m, 4H), 2.69 (s, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₈Cl₂N₆O 405.0997; found 405.0986.

N-(3,4-dichlorophenyl)-4-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxamide (14b).

Following General Method F, using intermediate 35a (255 mg, 1.51 mmol), 3,4-dichlorophenyl isocyanate (226 mg, 3.00 mmol) and 0.52 mL of DIEA (3mmol) in DCM (4 mL), purified using normal-phase (0→15% MeOH/DCM) and concentrated to obtain a pale-yellow solid as desired product, 14b. (160 mg, 65%), HPLC purity 95%. LCMS m/z 406 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.91 (s, 1H), 8.06 (d, J = 10.0 Hz, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.44 – 7.53 (m, 2H), 7.38 (d, J = 10.3 Hz, 1H), 3.62 (s, 8H), 2.57 (s, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₇Cl₂N₇O 406.0950; found 406.0941.

1-(4-(3-chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (14c).

Following General Method E using 37i (202 mg, 0.581 mmol) and 2-(3,4-dichlorophenyl)acetic acid (143 mg, 0.698 mmol), HOBt (78.5 mg, 0.581 mmol) and EDC-HCl (133 mg, 0.698 mmol) was treated with TEA (162 μL, 1.16 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (52.0 mg, 17%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.14 (d, J = 10.0 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 1.7 Hz, 1H), 7.47 (d, J = 10.0 Hz, 1H), 7.24(dd, J = 1.8, 8.2 Hz, 1H), 3.82 (s, 2H), 3.53 – 3.74 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₅Cl₃N₆O 425.0451; found 425.0437.

4-(3-chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-(3,4-dichlorophenyl)piperazine-1-carboxamide (14d).

General method D was followed, using N-(3,4-dichlorophenyl)piperazine-1-carboxamide hydrochloride 40 (100 mg, 0.322 mmol) and 3,6-dichloro[1,2,4]triazolo[4,3-b]pyridazine (91.0 mg, 0.482 mmol) dissolved in EtOH (2 mL) and treated with DIEA (160 μL, 0.966 mmol) with reaction time of 3h in the microwave. The crude mixture was concentrated, purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) and concentrated to obtain a white solid as the desired compound. (34.3 mg, 25%), HPLC purity 96%, LCMS m/z 426 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.91 (s, 1H), 8.16 (d, J = 10.0 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.45 – 7.54 (m, 2H), 3.45 – 3.74 (m, 8H) HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₄Cl₃N₇O 426.0404; found 426.0392.

2-(3,4-dichlorophenyl)-1-{4-[3-(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl]pipera zin-1-yl}ethan-1-one (14e).

General method D was followed using intermediate 35b (100 mg, 0.50 mmol) and 4 (147 mg, 0.540 mmol) was dissolved in ethanol and placed in a microwave vail, to which DIEA (0.13 mL, 0.740 mmol) was added. The vail was sealed and heated to 100 °C for 4 hours. Solvent was removed in vacuo, and the crude product was extracted in ethyl acetate, rinsing twice with water and once with brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The crude product was purified via normal phase flash chromatography (0–20% MeOH/EtOAc) to give the title compound (64.0 mg, 28%). LC-MS: m/z 459 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.01 (d, J=10.3 Hz, 1 H), 7.43 (d, J=8.3 Hz, 1 H), 7.39 (d, J=2.0 Hz, 1H), 7.12 (dd, J=8.2.02 Hz, 1 H), 7.07 (d, J=10.1 Hz, 1 H), 3.83 (d, J=5.3 Hz, 2 H), 3.74 (s, 2 H), 3.59 – 3.69 (m, 6 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₅Cl₂F₃N₆O 459.0715; found 459.0703.

N-(3,4-dichlorophenyl)-4-(3-(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxamide (14f).

General method D was Followed using intermediate 40 (53.7 mg, 0.241mmol) and 35b (50.0 mg, 0.161mmol) dissolved in EtOH (2 mL) and treated with DIEA (80 μL, 0.461 mmol) for 3 h. The mixture concentrated and purified by both normal phase (0→15% MeOH/DCM) and reverse phase to obtain a white solid product, 14f. (13.2 mg, 12%.), LCMS m/z 460(MH)⁺. HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.90 (s, 1 H), 8.31 (d, J=10.3 Hz, 1 H), 7.84 – 7.89 (m, 1 H), 7.64 (d, J=10.3 Hz, 1 H), 7.44 – 7.52 (m, 2 H), 3.58 – 3.73 (m, 8 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₄Cl₂F₃N₇O 460.0667; found 460.0658.

1-(4-(3-amino-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (14g).

Following General Method E using 37d (78.0 mg, 0.305 mmol) and 2-(3,4-dichlorophenyl)acetic acid (86.0 mg, 0.420 mmol), EDC-HCl (80.3 mg, 0.420 mmol), HOBt (57.0 mg, 0.422 mmol), TEA (97 μL, 0.700 mmol), and purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (15.0 mg, 11%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.80 (d, J = 10.0 Hz, 1H), 7.46 – 7.63 (m, 2H), 7.23 (dd, J = 2.0, 8.31Hz, 1H), 7.09 (d, J = 10.0 Hz, 1H), 6.12(s, 2H), 3.83 (s, 2H), 3.48 – 3.70 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₇Cl₂N₇O 406.0950; found 406.0938.

2-(3,4-dichlorophenyl)-1-(4-(3-morpholino-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)ethanone (14h).

Following General Method E using 37j (100 mg, 0.307 mmol) and 2-(3,4-dichlorophenyl)acetic acid (75.5 mg, 0.368 mmol), HOBt (41.4 mg, 0.307 mmol) and EDC-HCl (70.3 mg, 0.368 mmol) was treated with TEA (85.7 μL, 0.614 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (30.0 mg, 21%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (d, J = 10.3 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 1.7 Hz, 1H), 7.16 – 7.29 (m, 2H), 3.82 (s, 2H), 3.76 – 3.80 (m, 4H), 3.56 – 3.73 (m, 4H), 3.44 – 3.53 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₃Cl₂N₇O₂ 476.1368; found 476.1353.

2-(3,4-dichlorophenyl)-1-(4-{3-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl}piperazin-1-yl)et han-1-one (14i).

General method D was followed using intermediate 35c (65 mg, 0.28 mmol) and 4 (85 mg, 0.31 mmol) were dissolved in ethanol and placed in a microwave vail, to which DIEA (0.08 mL, 0.42 mmol) was added. The vial was sealed and heated to 100 °C for 4 hours. Solvent was removed in vacuo, and the crude product was extracted in ethyl acetate, rinsing twice with water and once with brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The crude product was purified via normal phase flash chromatography (0–20% MeOH/EtOAc) to give the title compound (45.0 mg, 31%). LC-MS: m/z 467 (MH)⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.44 (d, J=7.3 Hz, 2H), 8.00 (d, J=10.0 Hz, 1H), 7.47–7.59 (m, 3H), 7.43 (d, J=8.3 Hz, 1H), 7.40 (s, 1H), 7.14 (d, J=8.3 Hz, 1H), 6.95(d, J=10.0 Hz, 1H), 3.87 (br.s., 2H), 3.76 (s, 2H), 3.68 (br.s., 2H), 3.59 (dd, J=11.2, 4.8 Hz, 4H). HRMS (ESI) m/z: [M + H]⁺. Calcd for C₂₃H₂₀Cl₂N₆O 467.1154; found 467.1144.

1-(4-(3-benzyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (14j).

General Method E was followed using intermediate 37a (228 mg, 0.580 mmol) with 2-(3,4-dichlorophenyl)acetic acid (143 mg, 0.700 mmol) to give the title compound (80.0 mg, 29%). LC-MS: m/z 481 (MH)⁺. ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.06 (d, J=10.0 Hz, 1 H), 7.57 (d, J=8.2 Hz, 1 H), 7.52 (d, J=1.8Hz, 1H), 7.18–7.38 (m, 7H), 4.36 (s, 2H), 3.82 (s, 2H), 3.57 (d, J=9.4 Hz, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₄H₂₂Cl₂N₆O 481.1310; found 481.1299.

2-(3,4-dichlorophenyl)-1-(4-(3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)ethanone (14k).

Following General Method E using 37b (202 mg, 0.636 mmol) and 2-(3,4-dichlorophenyl)acetic acid (156 mg, 0.761 mmol), HOBt (85.8 mg, 0.636 mmol) and EDC-HCl (146 mg, 0.636 mmol) was treated with TEA (177 μL, 1.27 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (88.0 mg, 30%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.79 (d, J = 5.9 Hz, 2H), 8.37 (d, J = 5.9 Hz, 2H), 8.25 (d, J = 10.23Hz, 1H), 7.47 – 7.71 (m, 3H), 7.25 (dd, J =1.7, 8.3 Hz, 1H), 3.85 (s, 2H), 3.58 – 3.78 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₁₉Cl₂N₇O 468.1106; found 468.1094.

2-(3,4-dichlorophenyl)-1-(4-(3-(2-(trifluoromethyl)pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)ethanone (14l).

Following General Method E using 37c (71.0 mg, 0.184 mmol) and 2-(3,4-dichlorophenyl)acetic acid (45.0 mg, 0.220 mmol), HOBt (24.8 mg, 0.184 mmol) and EDC-HCl (42.2 mg, 0.220 mmol) was treated with TEA (51.4 μL, 0.368 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O), further purified by silica gel chromatography with 0→30% MeOH/EtOAc to obtain white solid as the desired product. (29.0 mg, 29%), HPLC purity 97%. LC-MS: m/z 536 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.97 – 9.03 (m, 1 H) 8.87 (s, 1 H) 8.66 – 8.71 (m, 1 H) 8.30 (s, 1 H) 7.52 – 7.61 (m, 3 H) 7.22 – 7.29 (m, 1 H) 3.86 (s, 2 H) 3.62 – 3.77 (m, 8 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₃H₁₈Cl₂F₃N₇O, 536.0980; found 536.0969.

N-(3,4-dichlorophenyl)-4-(7-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxamide (15b).

Following General method D, using 35j (69.8 mg, 0.225 mmol) and 40 (56.8 mg, 0.337 mmol) dissolved EtOH (1 mL) and treated with DIEA (115 μL, 0.674 mmol) and heated in microwave at 100 °C for 3h. The crude mixture was then concentrated and purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain a white solid as desired product. (8.40 mg, 6%), HPLC purity 96%, LCMS m/z 381(MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.35 (d, J = 0.7 Hz, 1H), 8.82 – 9.00 (m, 1H), 8.02 – 8.19 (m, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.40 – 7.59 (m, 2H), 3.61 – 3.67 (m, 4H), 3.18 – 3.26 (m, 4H), 2.40 (d, J = 1.2 Hz, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₇Cl₂N₇O 406.0950; found 406.0941.

2-(3,4-dichlorophenyl)-1-(4-(7-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)ethanone (15a) and 2-(3,4-dichlorophenyl)-1-(4-(8-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazin-1-yl)ethanone (16a).

Following General Method E using 37f and 37g (50.0 mg, 0.196 mmol) and 2-(3,4-dichlorophenyl)acetic acid (48.3 mg, 0.236 mmol), HOBt (26.5 mg, 0.196 mmol) and EDC-HCl (45.0 mg, 0.236 mmol) was treated with TEA (54.0 μL, 0.393 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain the title compounds. 15a: white solid (13.0 mg, 16%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.07 (s, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 1.7 Hz, 1H), 7.24 (dd, J = 1.7, 8.3 Hz, 1H), 3.82 (s, 2H), 3.60 – 3.75 (m, 4H), 3.10 – 3.21 (m, 4H), 2.38 (s, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₈Cl₂N₆O 405.0997; found 405.0983. 16a: white solid (10.0 mg, 13%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 7.57 (d, J = 8.31 Hz, 1H), 7.52 (s, 1H), 7.16 – 7.30 (m, 2H), 3.82 (s, 2H), 3.45 – 3.70 (m, 8H), 2.53 (s, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₈Cl₂N₆O 405.0997; found 405.0985.

N-(3,4-dichlorophenyl)-4-(8-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxamide (16b).

Following General method F, using 37f (428 mg, 0.460 mmol), 1,2-dichloro-4-isocyanatobenzene (104 mg, 0.550 mmol) in DCM (2 mL) and treated with DIEA (0.240 mL, 1.37 mmol). The crude mixture was concentrated and purified by normal phase (0→15% MeOH/DCM) to obtain a white solid as desired product. (63.8 mg, 34%), HPLC purity 95%, LCMS m/z 406 (MH)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.21 (s, 1H), 8.92 (s, 1H), 7.86 (dd, J = 0.7, 2.0 Hz, 1H), 7.42 – 7.59 (m, 2H), 7.29 (d, J = 1.2 Hz, 1H), 3.60 (s, 8H), 2.54 (d, J = 1.0 Hz, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₇Cl₂N₇O 406.0950; found 406.0941.

N-(3,4-dichlorophenyl)-4-(7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperazine-1-carboxamide (17b).

6-(piperazin-1-yl)-7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazine hydrochloride, 37h (200 mg, 0.824 mmol), 1,2-dichloro-4-isocyanatobenzene (252 mg, 1.34 mmol) dissolved in DMF (6 mL), and treated with DIEA (140 μL, 0.807 mmol). The mixture was stirred at room temperature for 16h, extracted using ethyl acetate (3×30 mL) and water (100 mL) three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo. The crude compound was purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) and further silica gel chromatography with 0→20% MeOH/EtOAc to obtain white solid as the desired product. (50.0 mg, 15%). HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (s, 1H), 8.52 (s, 1H), 7.84 (d, J = 1.7 Hz, 1H), 7.39 – 7.59 (m, 2H), 3.53 (d, J = 5.1 Hz, 8H), 2.95 – 3.10 (m, 2H), 2.75 – 2.91 (m, 2H), 2.54 (s, 1H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₇Cl₂N₇O 394.0950; found 394.0936.

2-(3,4-dichlorophenyl)-1-(4-(imidazo[1,2-b]pyridazin-6-yl)piperazin-1-yl)ethanone (18a).

Following General Method E using 37ap (102 mg, 0. 425 mmol) and 2-(3,4-dichlorophenyl)acetic acid (105 mg, 0.512 mmol), HOBt (57.4 mg, 0. 425 mmol) and EDC-HCl (97.5 mg, 0.512 mmol) was treated with TEA (119 μL, 0.851 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O), further purified by silica gel chromatography with 0→30% MeOH/EtOAc to obtain white solid as the desired product. (111 mg, 65%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (d, J = 0.73 Hz, 1H), 7.88 (dd, J = 0.5, 10.0 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.49 – 7.54 (m, 2H), 7.24 (dd, J = 2.1, 8.2 Hz, 1H), 7.20 (d, J = 10.0 Hz, 1H), 3.83 (s, 2H), 3.58 – 3.71 (m, 4H), 3.42 – 3.54 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₇Cl₂N₅O 390.0888; found 390.0876.

N-(3,4-dichlorophenyl)-4-(imidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxamide (18b).

Following General Method F, using 3,4-dichlorophenylisocyanate (30.0 mg, 0.144 mmol), 37ap (55.0 mg, 0.0293 mmol), DIEA (70 μL, 0.431 mmol), in ~1 mL DCM, and purified by normal-phase (0→20% MeOH/EtOAc) and concentrated to obtain a yellow solid as desired product, 18b. (15.5 mg, 28%). HPLC purity 96%. LCMS m/z 391 (MH)⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.76 – 7.82 (m, 1 H), 7.71 (s, 1 H), 7.61 – 7.64 (m, 1 H), 7.59 (s, 1 H), 7.35 (s, 1 H), 7.20 – 7.25 (m, 1 H), 6.79 – 6.85 (m, 1 H), 6.35 – 6.42 (m, 1 H), 3.59 – 3.72 (m, 8 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₆Cl₂N₆O 391.0841; found 391.0831.

2-(3,4-dichlorophenyl)-1-(4-(pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)ethanone (19).

Following General Method E using 37am (200 mg, 0. 834 mmol) and 2-(3,4-dichlorophenyl)acetic acid (205 mg, 1.00 mmol), HOBt (113 mg, 0. 834 mmol) and EDC-HCl (191 mg, 1.00 mmol) was treated with TEA (233 μL, 1.67 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O), further purified by silica gel chromatography with 0→20% MeOH/EtOAc to obtain white solid as the desired product. (315 mg, 97%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (d, J = 7.8 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.48 – 7.59 (m, 2H), 7.23 (dd, J = 1.8, 8.2 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.05 (d, J = 1.5 Hz, 1H), 3.82 (s, 2H), 3.54 – 3.72 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₇Cl₂N₅O 390.0888; found 390.0876.

N-(3,4-dichlorophenyl)-4-(imidazo[1,2-a]pyrazin-6-yl)piperazine-1-carboxamide (20).

Following General Method F, using 37v (15.2 mg, .0634 mmol), 3,4-dichlorophenylisocyanate (10.9 mg, 0.0580 mmol), DIEA (30 μL, 0.174 mmol) in DCM (1 mL), purified by normal-phase (0→20% MeOH/EtOAc) and concentrated to obtain white as the desired product. (5.10 mg, 22%), HPLC purity 96%. 1H NMR (400 MHz, CHLOROFORM-d) δ 7.64 (d, J = 2.69 Hz, 1H), 7.54 – 7.60 (m, 2H), 7.53 (d, J = 1.0 Hz, 1H), 7.36 – 7.41 (m, 1H), 7.34 (s, 1H), 7.24 (dd, J = 2.6, 8.7 Hz, 1H), 6.39 (s, 1H), 4.34 – 4.41 (m, 4H), 3.66 – 3.72 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₆Cl₂N₆O 391.0841; found 391.0832.

1-(4-(1H-indazol-6-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (21).

Following General Method E using 37ae (131 mg, 0.549 mmol) and 2-(3,4-dichlorophenyl)acetic acid (135 mg, 0.658 mmol), HOBt (74.1 mg, 0.549 mmol) and EDC-HCl (126 mg, 0.658 mmol) was treated with TEA (153 μL, 1.10 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (47.0 mg, 22%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (s, 1 H) 7.58 (dd, J=8.7, 2.6 Hz, 2 H) 7.53 (d, J=2.0 Hz, 1 H) 7.24 (dd, J=8.3, 2.2 Hz, 1 H) 6.95 (dd, J=9.0, 2.0 Hz, 1 H) 6.80 (s, 1 H) 3.83 (s, 2 H) 3.59 – 3.75 (m, 4 H) 3.08 – 3.24 (m, 4 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₈Cl₂N₄O 389.0936; found 389.0922.

1-(4-(1H-pyrrolo[2,3-b]pyridin-6-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (22).

Following General Method E using 37an (84.0 mg, 0.352 mmol) and 2-(3,4-dichlorophenyl)acetic acid (86.5 mg, 0.422 mmol), HOBt (47.5 mg, 0.352 mmol) and EDC-HCl (80.6 mg, 0.422 mmol) was treated with TEA (98.2 μL, 0.704 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain pink solid as the desired product. (29.0 mg, 21%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.09 (br. s., 1H), 7.74 (d, J = 8.6 Hz, 1H), 7.47 – 7.60 (m, 2H), 7.24 (dd, J = 2.1, 8.2 Hz, 1H), 7.07 (dd, J = 2.4, 3.4 Hz, 1H), 6.66 (d, J = 8.6 Hz, 1H), 6.24 (dd, J = 2.0, 3.4 Hz, 1H), 3.81 (s, 2H), 3.55 – 3.69 (m, 4H), 3.39 – 3.52 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₈Cl₂N₄O 389.0936; found 389.0924.

1-(4-(1H-benzo[d]imidazol-6-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (23).

Following General Method E using 37ag (100 mg, 0.419 mmol) and 2-(3,4-dichlorophenyl)acetic acid (103 mg, 0.502 mmol), HOBt (56.5 mg, 0.419 mmol) and EDC-HCl (96.0 mg, 0.503 mmol) was treated with TEA (117 μL, 0.838 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) and silica gel chromatography with 0→20% MeOH/EtOAc to obtain white solid as the desired product. (18.0 mg, 11%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.06 (s, 2 H) 7.57 (d, J=8.3 Hz, 1 H) 7.52 (d, J=1.7 Hz, 1 H) 7.45 (d, J=9.0 Hz, 1 H) 7.24 (dd, J=8.2, 1.83 Hz, 1 H) 7.02 (br. s., 1 H) 6.93 – 6.99 (m, 1 H) 3.82 (s, 2 H) 3.66 (dt, J=10.2, 5.0 Hz, 4 H) 2.93 – 3.17 (m, 4 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₈Cl₂N₄O 389.0936; found 389.0922.

1-(4-(benzo[c][1,2,5]oxadiazol-5-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (24).

Following General Method E using 37ah (37.7 mg, 0.157 mmol), and 2-(3,4-dichlorophenyl)acetic acid (38.5 mg, 0.188 mmol), HOBt (113 mg, 0.157 mmol) and EDC-HCl (35.9 mg, 0.188 mmol) was treated with TEA (43.7 μL, 0.313 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain dark brown solid as the desired product. (20.0 mg, 33%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.88 (d, J = 9.8 Hz, 1H), 7.68 (dd, J = 1.7, 9.8 Hz, 1H), 7.48 – 7.61 (m, 2H), 7.24 (dd, J = 1.7, 8.3 Hz, 1H), 6.83 (s, 1H), 3.83 (s, 2H), 3.60 – 3.74 (m, 4H), 3.34 – 3.46 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₆Cl₂N₄O₂ 391.0729; found 391.0720.

2-(3,4-dichlorophenyl)-1-(4-(quinolin-2-yl)piperazin-1-yl)ethanone (25).

Following General Method E using 37au (100 mg, 0.400 mmol) and 2-(3,4-dichlorophenyl)acetic acid (98.6 mg, 0.481 mmol), HOBt (54.1 mg, 0.400 mmol) and EDC-HCl (91.9 mg, 0.481 mmol) was treated with TEA (112 μL, 0.80 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain brown solid as the desired product. (15.0 mg, 9%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.07 (d, J=9.3 Hz, 1 H) 7.72 (d, J=8.1 Hz, 1 H) 7.56 (t, J=9.2 Hz, 4 H) 7.20 – 7.32 (m, 3 H) 3.83 (s, 2 H) 3.57 – 3.77 (m, 8 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₁₉Cl₂N₃O 400.0983; found 400.0971.

1-(4-(1,5-naphthyridin-2-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (26).

Following General Method E using 37at (100 mg, 0.400 mmol) and 2-(3,4-dichlorophenyl)acetic acid (98.5 mg, 0.481 mmol), HOBt (54.0 mg, 0.400 mmol) and EDC-HCl (91.7 mg, 0.481 mmol) was treated with TEA (112 μL, 0.800 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (55.0 mg, 34%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.52 – 8.65 (m, 1H), 8.09 (d, J = 9.3 Hz, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.46 – 7.61 (m, 4H), 7.25 (dd, J = 1.7, 8.3 Hz, 1H), 3.83 (s, 2H), 3.75 (dd, J = 5.6, 11.7 Hz, 4H), 3.59 – 3.71 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₁₈Cl₂N₄O 401.0936; found 401.0922.

1-(4-(2,7-naphthyridin-3-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (27).

Following General Method E using 37as (70.0 mg, 0.280 mmol) and 2-(3,4-dichlorophenyl)acetic acid (68.9 mg, 0.336 mmol), HOBt (37.8 mg, 0.280 mmol) and EDC-HCl (64.2 mg, 0.336 mmol) was treated with TEA (78.0 μL, 0.559 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (12.0 mg, 11%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d6) δ 9.23 (d, J=0.7 Hz, 1 H), 8.10 (d, J=10.0 Hz, 1 H), 7.38 (d, J=10.0 Hz, 1 H), 6.77 – 6.89 (m, 2 H), 6.71 (d, J=8.1 Hz, 1 H), 5.98 (s, 2 H), 3.68 (s, 2 H), 3.57 – 3.66 (m, 4 H), 3.51 (d, J=4.9 Hz, 4 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₁₈Cl₂N₄O 401.0936; found 401.0924.

4-(1H-benzo[d]imidazol-2-yl)-N-(3,4-dichlorophenyl)piperazine-1-carboxamide and N-(3,4-dichlorophenyl)-4-phenylpiperazine-1-carboxamide (28) was obtained from the work by Jumani et al.⁶

1-(4-(benzo[d]oxazol-2-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (29a).

Following General Method E using 37av (100 mg, 0.417 mmol) and 2-(3,4-dichlorophenyl)acetic acid (103 mg, 0.502 mmol), HOBt (56.3 mg, 0.417 mmol) and EDC-HCl (95.6 mg, 0.502 mmol) was treated with TEA (116 μL, 0.834 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O), further purified by silica gel chromatography with 0→20% MeOH/EtOAc to obtain white solid as the desired product. (45.0 mg, 28%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.57 (d, J=8.1 Hz, 1 H) 7.52 (d, J=1.7 Hz, 1 H) 7.42 (d, J=7.8 Hz, 1 H) 7.31 (d, J=8.1 Hz, 1 H) 7.24 (d, J=8.1 Hz, 1 H) 7.16 (t, J=7.7 Hz, 1 H) 7.02 – 7.07 (m, 1 H) 3.83 (s, 2 H) 3.55 – 3.72 (m, 8 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₇Cl₂N₅O₂ 390.0776; found 390.0763.

1-(4-(benzo[d]thiazol-2-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (29b).

Following General Method E using 37ai (176 mg, 0.688 mmol) and 2-(3,4-dichlorophenyl)acetic acid (169 mg, 0.824 mmol), HOBt (93.0 mg, 0.688 mmol) and EDC-HCl (158 mg, 0.824 mmol) was treated with TEA (192 μL, 1.38 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O), and further purified by silica gel chromatography with 0→20% MeOH/EtOAc to obtain white solid as the desired product. (210 mg, 75%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.79 (d, J=7.8 Hz, 1 H) 7.45 – 7.60 (m, 3 H) 7.26 – 7.33 (m, 1 H) 7.24 (dd, J=8.2, 1.6 Hz, 1 H) 7.10 (t, J=7.6 Hz, 1 H) 3.83 (s, 2 H) 3.50 – 3.74 (m, 8 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₇Cl₂N₃OS 406.0548; found 406.0536.

1-(4-(2-aminoimidazo[1,2-b]pyridazin-6-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (30a)⁵³.

Intermediate 50 (20.0 mg, 0.0414 mmol), and hydroxylamine hydrochloride (14.4 mg, 0.207 mmol) were weighed into a round bottom flask with a magnetic stirrer. Th mixture was dissolved with 10 mL of EtOH and 5 mL of water, and heated for 48 h at 120 °C. The reaction was then allowed to cool to room temperature, concentrated and purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (10.0 mg, 59%), HPLC purity 95%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.42 – 7.68 (m, 3H), 7.17 – 7.32 (m, 1H), 6.97 – 7.05 (m, 1H), 6.80 – 6.91 (m, 1H), 4.88 – 5.08 (m, 2H), 3.79 – 3.87 (m, 2H), 3.51 – 3.72 (m, 4H), 3.35 – 3.43 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₈Cl₂N₆O 405.0997; found 405.0978.

Ethyl 6-(4-(2-(3,4-dichlorophenyl)acetyl)piperazin-1-yl)imidazo[1,2-b]pyridazine-2-carboxylate (30b).

Following General Method E using 37k (100 mg, 0.321 mmol) and 2-(3,4-dichlorophenyl)acetic acid (78.9 mg, 0.385 mmol), HOBt (43.3 mg, 0.321 mmol) and EDC-HCl (73.5 mg, 0.385 mmol) was treated with TEA (89.5 μL, 0.641 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (55.0 mg, 37%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (s, 1H), 7.92 (d, J = 10.3 Hz, 1H), 7.49 – 7.60 (m, 2H), 7.35 (d, J = 10.0 Hz, 1H), 7.24(d, J = 7.6 Hz, 1H), 4.28 (q, J = 6.8 Hz, 2H), 3.82 (s, 2H), 3.44 – 3.74 (m, 8H), 1.30 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₁Cl₂N₅O₃ 462.1100; found 462.1087.

Butyl 6-(4-(2-(3,4-dichlorophenyl)acetyl)piperazin-1-yl)imidazo[1,2-b]pyridazine-2-carboxylate (30c).

A clean, dry 50 mL round bottom flask with stirring bar was charged with 37l (225 mg, 0.662), dissolved in DCM (6 mL) and treated with TEA (184 μL, 1.32 mmol). (2-(3,4-dichlorophenyl)acetyl chloride (222 mg, 0.993 mmol) prepared by treating with SOCl₂, was added in droplets while the flask is placed in an ice bath. The reaction mixture was stirred at ~0°C for 20 mins, then allowed to stir at room temperature for 16h. The reaction mixture was then concentrated and extracted with water and EtOAc, dried with Na₂SO₄. The dried organic was then concentrated and purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (218 mg, 67%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (s, 1H), 7.93 (d, J = 10.3 Hz, 1H), 7.45 – 7.62 (m, 2H), 7.34 (d, J = 10.0 Hz, 1H), 7.17 – 7.28 (m, 1H), 4.24 (t, J = 6.2 Hz, 2H), 3.43 – 3.91 (m, 10H), 1.56 – 1.80 (m, 2H), 1.31 – 1.53 (m, 2H), 0.93 (t, J = 7.1 Hz, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₃H₂₅Cl₂N₅O₃ 490.1413; found 490.1399.

6-(4-(2-(3,4-dichlorophenyl)acetyl)piperazin-1-yl)imidazo[1,2-b]pyridazine-2-carboxylic acid (30d).

A 50 mL round bottom flask with magnetic stirrer was added to compound 30b (20.0 mg, 0.0433), and LiOH monohydrate (50.0 mg, 1.19 mmol). The solid mixture was stirred in THF (6 mL) for 16h and quenched with 1M HCl, after which the mixture was extracted using ethyl acetate and water three times. After which, the organic fraction was then dried over Na₂SO₄ and concentrated under vacuo. The crude compound was purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (11.0 mg, 63%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 7.91 (d, J = 10.3 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.52 (s, 1H), 7.32 (d, J = 10.0 Hz, 1H), 7.24(d, J = 8.6 Hz, 1H), 3.82 (s, 2H), 3.48 – 3.73 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₇N₇O₃ 434.0787; found 434.0772.

6-(4-(2-(3,4-dichlorophenyl)acetyl)piperazin-1-yl)-N,N-dimethylimidazo[1,2-b]pyridazine-2-carboxamide (30e).

Following General Method E using 37m (100 mg, 0.322 mmol), 2-(3,4-dichlorophenyl)acetic acid (79.1 mg, 0.386 mmol), EDC-HCl (73.7 mg, 0.386), HOBt (43.4 mg, 0.322 mmol), TEA (89.8 μL, 0.643 mmol), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (20.0 mg, 14%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.16 (s, 1H), 7.92 (d, J = 10.0 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 10.0 Hz, 1H), 7.24 (dd, J = 2.0, 8.31 Hz, 1H), 3.82 (s, 2H), 3.58 – 3.72 (m, 4H), 3.52 (br. s., 4H), 3.41 (br. s., 3H), 2.99 (br. s., 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₂Cl₂N₆O₂ 461.1259; found 461.1245.

2-(3,4-dichlorophenyl)-1-(4-(2-(morpholine-4-carbonyl)imidazo[1,2-b]pyridazin-6-yl)piperazin-1-yl)ethanone (30f).

Following General Method E using 37n (100 mg, 0.283 mmol), 2-(3,4-dichlorophenyl)acetic acid (69.7 mg, 0.340 mmol), EDC-HCl (65.0 mg, 0.340), HOBt (38.3 mg, 0.283 mmol), TEA (79.1 μL, 0.567 mmol), and purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (15.0 mg, 11%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (s, 1H), 7.93 (d, J = 10.3 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 10.0 Hz, 1H), 7.24 (dd, J = 2.1, 8.2 Hz, 1H), 4.22 (br. s., 2H), 3.83 (s, 2H), 3.48 – 3.71 (m, 14H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₃H₂₄Cl₂N₆O₃ 503.1365; found 503.1350.

2-(3,4-dichlorophenyl)-1-(4-(2-methylthiazolo[4,5-b]pyridin-5-yl)piperazin-1-yl)ethanone (31a).

Following General Method E using 37p (27.0 mg, 0.0998 mmol) and 2-(3,4-dichlorophenyl)acetic acid (24.5 mg, 0.120 mmol), HOBt (57.4 mg, 0. 0998 mmol) and EDC-HCl (22.9 mg, 0.120 mmol) was treated with TEA (28.0 μL, 0.199 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (10.0 mg, 24%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 9.0 Hz, 1H), 7.49 – 7.63 (m, 2H), 7.19 – 7.33 (m, 1H), 6.94 – 7.04 (m, 1H), 3.83 (s, 2H), 3.50 – 3.72 (m, 8H), 2.77 (s, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₈Cl₂N₄OS 421.0657; found 421.0643.

1-(4-(2-aminothiazolo[4,5-b]pyridin-5-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (31b).

Following General Method E using 37o (140 mg, 0.515 mmol) and 2-(3,4-dichlorophenyl)acetic acid (127 mg, 0.620 mmol), HOBt (69.5 mg, 0.515 mmol) and EDC-HCl (118 mg, 0.618 mmol) was treated with TEA (144 μL, 1.03 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O), further purified by silica gel chromatography with 0→20% MeOH/EtOAc to obtain white solid as the desired product. (30.0 mg, 23%), ¹H NMR (400 MHz, DMSO-d₆) δ 7.79 (d, J = 8.6 Hz, 1H), 7.65 (s, 2H), 7.56 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.24 (dd, J = 2.0, 8.31 Hz, 1H), 6.51 (d, J = 8.8 Hz, 1H), 3.81 (s, 2H), 3.61 (d, J = 19.8 Hz, 4H), 3.45 (d, J = 15.2 Hz, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₇Cl₂N₅OS 422.0609; found 422.0592.

N-(5-(4-(2-(3,4-dichlorophenyl)acetyl)piperazin-1-yl)thiazolo[4,5-b]pyridin-2-yl)benzamide (31c).

Following General Method E using 37r (91.0 mg, 0.242 mmol) and 2-(3,4-dichlorophenyl)acetic acid (60.0 mg, 0.293 mmol), HOBt (32.7 mg, 0.242 mmol) and EDC-HCl (55.5 mg, 0.293 mmol) was treated with TEA (67.6 μL, 0.484 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (46.0 mg, 36%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.81 – 13.05 (m, 1H), 8.18 (d, J = 8.8 Hz, 1H), 8.08 – 8.15 (m, 2H), 7.66 (d, J = 7.1 Hz, 1H), 7.51 – 7.62 (m, 4H), 7.24 (dd, J = 2.1, 8.2 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 3.83 (s, 2H), 3.52 – 3.72 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₅H₂₁Cl₂N₅O₂S 526.0871; found 526.0861.

N-(5-(4-(2-(3,4-dichlorophenyl)acetyl)piperazin-1-yl)thiazolo[4,5-b]pyridin-2-yl)isonicotinamide (31d).

A clean, dry 50 mL round bottom flask with stirring bar was charged with 37t (60.0 mg, 0.159 mmol), dissolved in DCM (6 mL) and treated with TEA (44.4 μL, 0.318 mmol). (2-(3,4-dichlorophenyl)acetyl chloride (53.3 mg, 0.240 mmol) prepared by treating with SOCl₂, which was added in droplets while the flask is placed in an ice bath. The reaction mixture was stirred at ~0°C for 20 mins, then allowed to stir at room temperature for 16h. The reaction mixture was allowed to cool to room temperature, concentrated and extracted with water and EtOAc, dried with Na₂SO₄. The dried organic was then concentrated and purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (6 mL), to obtain yellow solid as the desired product. (10.0 mg, 12%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.37 – 13.21 (m, 1H), 8.82 (d, J = 5.6 Hz, 1H), 8.17 (d, J = 8.8 Hz, 1H), 8.00 (d, J = 5.6 Hz, 1H), 7.49 – 7.63 (m, 3H), 7.18 – 7.32 (m, 2H), 6.92 (d, J = 8.8 Hz, 1H), 3.83 (s, 2H), 3.51 – 3.71 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₄H₂₀Cl₂N₆O₂S 527.0824; found 527.0809.

2-(3,4-dichlorophenyl)-1-(4-(2-methylbenzo[d]thiazol-5-yl)piperazin-1-yl)ethanone (32a).

Following General Method E using 37al (170 mg, 0.630 mmol) and 2-(3,4-dichlorophenyl)acetic acid (155 mg, 0.756 mmol), HOBt (85.0 mg, 0.630 mmol) and EDC-HCl (144 mg, 0.756 mmol) was treated with TEA (176 μL, 1.26 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O), further purified by silica gel chromatography with 0→20% MeOH/EtOAc to obtain brown solid as the desired product. (112 mg, 42%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.83 (d, J=8.8 Hz, 1 H) 7.50 – 7.59 (m, 2 H) 7.40 (d, J=2.4 Hz, 1 H) 7.24 (dd, J=8.3, 2.0 Hz, 1 H) 7.15 (dd, J=8.8, 2.4 Hz, 1 H) 3.83 (s, 2 H) 3.67 (dt, J=16.6, 4.9 Hz, 4 H) 3.11 – 3.23 (m, 4 H) 2.75 (s, 3 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₁₉Cl₂N₃OS 420.0704; found 420.0692.

1-(4-(2-aminobenzo[d]thiazol-5-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (32b).

Following General Method E using 37q (93.0 mg, 0.343 mmol) and 2-(3,4-dichlorophenyl)acetic acid (84.5 mg, 0.412 mmol), HOBt (46.4 mg, 0.343 mmol) and EDC-HCl (78.7 mg, 0.412 mmol) was treated with TEA (95.9 μL, 0.687 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (11.0 mg, 8%), HPLC purity 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.57 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.36 (s, 2H), 7.24 (d, J = 2.0 Hz, 1H), 6.91 (d, J = 2.2 Hz, 1H), 6.68 – 6.73 (m, 1H), 3.81 (s, 2H), 3.64 (d, J = 16.6 Hz, 4H), 3.00 – 3.14 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₈Cl₂N₄OS 421.0657; found 421.0644.

N-(5-(4-(2-(3,4-dichlorophenyl)acetyl)piperazin-1-yl)benzo[d]thiazol-2-yl)benzamide (32c).

Following General Method E using 37s (100 mg, 0.267 mmol) and 2-(3,4-dichlorophenyl)acetic acid (65.6 mg, 0.320 mmol), HOBt (36.0 mg, 0.267 mmol) and EDC-HCl (61.1 mg, 0.320 mmol) was treated with TEA (74.5 μL, 0.533 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain brown solid as the desired product. (20.0 mg, 14%), HPLC purity 96%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.69 – 12.86 (m, 1H), 8.09 – 8.18 (m, 2H), 7.81 – 7.87 (m, 1H), 7.63 – 7.70 (m, 1H), 7.58 (d, J = 8.3 Hz, 4H), 7.22 – 7.29 (m, 2H), 7.07 – 7.13 (m, 1H), 3.84 (s, 2H), 3.59 – 3.76 (m, 4H), 3.13 – 3.24 (m, 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₆H₂₂Cl₂N₄O₂S 525.0919; found 525.0908.

6-(4-(2-(3,4-dichlorophenyl)acetyl)piperazin-1-yl)benzo[d]thiazol-2(3H)-one (32d).

Following General Method E using 37aa (95.0 mg, 0.350 mmol) and 2-(3,4-dichlorophenyl)acetic acid (86.0 mg, 0.420 mmol), HOBt (47.2 mg, 0.350 mmol) and EDC-HCl (80.1 mg, 0.420 mmol) was treated with TEA (97.6 μL, 0.70 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (37.0 mg, 30%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆), δ 11.41 – 11.91 (m, 1 H), 7.45 – 7.66 (m, 2 H), 7.12 – 7.29 (m, 2 H), 6.87 – 7.05 (m, 2 H), 3.80 (s, 2 H), 3.63 (d, J=16.6 Hz, 4 H), 3.04 (br. s., 4 H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₇Cl₂N₃O₂S 349.0874; found 349.0865.

1-(4-(1H-pyrazolo[4,3-b]pyridin-5-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (33a).

Following General Method E using 37ak (100 mg, 0.417 mmol) and 2-(3,4-dichlorophenyl)acetic acid (103 mg, 0.502 mmol), HOBt (56.3 mg, 0.417 mmol) and EDC-HCl (95.6 mg, 0.502 mmol) was treated with TEA (116 μL, 0.834 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O), further purified by silica gel chromatography with 0→20% MeOH/EtOAc to obtain yellow solid as the desired product. (20.0 mg, 12%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.92 (br. s., 1H), 7.92 (s, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.49 – 7.61 (m, 2H), 7.24 (dd, J = 2.0, 8.3 Hz, 1H), 7.10 (d, J = 9.3 Hz, 1H), 3.82 (s, 2H), 3.63 (dd, J = 5.4, 17.85 Hz, 4H), 3.50 (br. s., 4H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₁₇Cl₂N₅O 390.0888; found 390.0876.

2-(3,4-dichlorophenyl)-1-(4-(1-methyl-1H-pyrazolo[4,3-b]pyridin-5-yl)piperazin-1-yl)ethanone (33b).

Following General Method E using 37z (50.0 mg, 0.197 mmol) and 2-(3,4-dichlorophenyl)acetic acid (48.5 mg, 0.237 mmol), HOBt (26.6 mg, 0.197 mmol) and EDC-HCl (45.2 mg, 0.237 mmol) was treated with TEA (55.0 μL, 0.394 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (11.0 mg, 14%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.95 – 8.01 (m, 1H), 7.83 – 7.89 (m, 1H), 7.55 – 7.59 (m, 1H), 7.50 – 7.54 (m, 1H), 7.21 – 7.27 (m, 1H), 7.11 – 7.19 (m, 1H), 3.99 (s, 3H), 3.80 – 3.86 (m, 2H), 3.57 – 3.69 (m, 4H), 3.47 – 3.55 (m, 3H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₁₉Cl₂N₅O 404.1045; found 404.1034.

1-(4-(1-benzyl-1H-pyrazolo[4,3-b]pyridin-5-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (33c).

Following General Method E using 37x (200 mg, 0.606 mmol) and 2-(3,4-dichlorophenyl)acetic acid (149 mg, 0.727 mmol), HOBt (81.8 mg, 0.606 mmol) and EDC-HCl (139 mg, 0.727 mmol) was treated with TEA (169 μL, 1.21 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain white solid as the desired product. (222 mg, 77%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 9.3 Hz, 1H), 7.93 (d, J = 0.7 Hz, 1H), 7.54 – 7.58 (m, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.16 – 7.34 (m, 6H), 7.13 (d, J = 9.5 Hz, 1H), 5.59 (s, 2H), 3.81 (s, 2H), 3.43 – 3.69 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₅H₂₃Cl₂N₅O 480.1358; found 480.1344.

1-(4-(2-benzyl-2H-pyrazolo[4,3-b]pyridin-5-yl)piperazin-1-yl)-2-(3,4-dichlorophenyl)ethanone (34).

Following General Method E using 37y (100 mg, 0.303 mmol) and 2-(3,4-dichlorophenyl)acetic acid (74.6 mg, 0.364 mmol), HOBt (40.9 mg, 0.303 mmol) and EDC-HCl (69.5 mg, 0.364 mmol) was treated with TEA (84.6 μL, 0.606 mmol) in DMF (6 mL), purified by reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain cream white solid as the desired product. (15.0 mg, 10%), HPLC purity 97%. ¹H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.21 – 7.37 (m, 6H),7.11 (d, J = 9.5 Hz, 1H), 5.53 (s, 2H), 3.81 (s, 2H), 3.43 – 3.70 (m, 8H). HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₅H₂₃Cl₂N₅O 480.1358; found 480.1345.

2-(3,4-dichlorophenyl)-1-(4-(2-(3,4-dimethyl-1H-pyrrol-1-yl)imidazo[1,2-b]pyridazin-6-yl)piperazin-1-yl)ethanone (51).

Following General Method E using 37u (100 mg, 0.300 mmol) and 2-(3,4-dichlorophenyl)acetic acid (73.9 mg, 0.360 mmol), HOBt (40.1 mg, 0.300 mmol) and EDC-HCl (68.9 mg, 0.360) mmol) was treated with TEA (83.9 μL, 0.601 mmol) in DMF (6 mL), purified by only reverse-phase HPLC (5→95% CH₃CN/H₂O) to obtain yellow solid as the desired product. (20.0 mg, 14%), LCMS m/z 483 (MH)⁺.

Antiparasitic activity.

Anticryptosporidial activity was measured using a previously described high-content microscopy assay, a human ileocecal adenocarcinoma (HCT-8) cell infection model and C. parvum Iowa isolate oocysts (Bunch Grass Farms).¹⁸ Oocysts were treated to induce excystation (10 mM HCL (10 min, 37° C) followed by 2 mM sodium taurocholate in PBS (10 min, 16 °C)) and used to infect nearly confluent HCT-8 cell monolayers grown in 384-well clear-bottomed plates. Compounds were added after 3 hours incubation, followed by incubation for an additional 45 hours (37°C, 5% CO₂). Wells were then washed 3 times with PBS containing 111 mM D-galactose, fixed with 4% formaldehyde, permeabilized with 0.25% Triton X-100, blocked with 4% bovine serum albumin (BSA), and stained for fluorescence microscopy using fluorescein-labeled Vicia villosa lectin (Vector Laboratories) to label the parasitophorous vacuoles and Hoechst 33258 (Anaspec) to label the cell nuclei. A Nikon Eclipse Ti2000 epifluorescence microscope with an automated stage was programmed to focus on the center of each well and take a 3×3 composite image using an EXi blue fluorescence microscopy camera (QImaging) with a 20X objective (NA = 0.45). Nuclei and parasite images were separately exported as .tif files, and parasites and host cells were counted using custom NIH ImageJ macros. Dose-response curves were plotted, and half maximal effective concentrations (EC₅₀) and 90% effective concentrations (EC₉₀) were calculated using GraphPad Prism software version 6.01.

Scheme 3.

Reagents and Conditions: (a) NH₂NH₂, EtOH, 85°C, 3–24h, 80–90%; (b) R¹CO₂H, 100°C, 1h, 80–90%; (c) R¹CO₂H, POCl₃, 130°C, 24h, 60%; (d) R¹CO₂H, EDC-HCl, HOBT, TEA, DMF, 87%; then POCl_3, 130°C, 24h, 60%.

Abbreviations.

ADME

absorption, distribution, metabolism and elimination

CC₅₀

50% cytotoxic concentration

Cp

Cryptosporidium parvum

EC₅₀

50% efficacious concentration

SI

Selectivity Index

NTZ

nitazoxanide

PK

pharmacokinetic