Pharmacophore Establishment and Optimization of Saturated 1,6-Naphthyridine-Fused Quinazolinones that Inhibit Meningoencephalitis-Causing Naegleria fowleri

Abstract

Primary amoebic meningoencephalitis (PAM) is a human brain infection caused by Naegleria fowleri with a 97% mortality rate. Quinazolinones resulting from a Mannich-coupled domino rearrangement were recently identified as inhibitors of the amoeba. Herein, we resolved the effective concentrations for 25 pilot compounds and then, using the Mannich protocol and a key late-stage, N-demethylation/functionalization, we synthesized 53 additional analogs to improve potency, solubility and microsomal stability. We established an antiamoebic quinazolinone pharmacophore, culminating in (±)-trans-57b which featured the best combination of potency, selectivity index, solubility, and microsomal stability. Enantiomeric separation afforded (4aS,13bR)-57b (BDGR-20237) with a 41-fold potency advantage over its enantiomer. ADME and mouse pharmacokinetic profiling for BDGR-20237 revealed high brain penetrance but a limited half-life which did not statistically enhance the mouse survival in a pilot efficacy study. The pharmacophoric model, supported by 88 quinazolinones, several of which exhibit subnanomolar potency, will guide further scaffold optimization.

Graphical Abstract:

INTRODUCTION

Primary amoebic meningoencephalitis (PAM) is a rare, yet highly lethal, necrotic brain infection that is caused by the free-living amoeba Naegleria fowleri. ^1,2 Warm, freshwater ecosystems and soil are hospitable environments in which N. fowleri thrives, sustaining itself on microbiota.³ Potential for human infection occurs when amoeba-contaminated water enters the nasal cavity, typically encountered by children during recreational water activities in lakes or inadequately treated swimming pools and water parks.⁴ Alternatively, using infected tap water for nasal irrigation³ can introduce the amoeba unwittingly into the nasal mucosa where the amoebic trophozoite form migrates sequentially through the cribriform plate and olfactory bulb before infiltrating the frontal lobes of the brain.^4,5 Post-mortem analysis of the brains of PAM patients has shown severe tissue necrosis and hemorrhaging resulting from action of amoeba predation and the host immune response.^6–9

Symptoms of PAM, which include headache, fever, nausea, fatigue, vomiting, confusion, seizures, and coma, mimic what is observed in cases of bacterial or viral meningoencephalitis, thereby often delaying diagnosis and treatment.⁴ Infection is associated with a > 97% case fatality rate, and death typically results in 3–7 days after symptom onset.^8,10 Among the 157 reported cases in the United States between 1962 and 2022, there have been only four survivors,¹¹ and the infection has been documented in at least 39 nations worldwide.^4,12 Successful outcomes rely critically on early diagnosis and therapeutic intervention with repurposed drug cocktails (Figure 1).^6,13–15

Figure 1.

Drugs used alone or in combination for PAM.

The efficacy of these treatment regimens is largely anecdotal and limited in statistical significance.¹⁵ Nonetheless, in the absence of alternatives, amphotericin B is commonly used alone or in combination with fluconazole, rifampicin, or azithromycin.¹⁶ Miltefosine has also been used, but due to mixed results, additional studies are needed to determine its efficacy.¹⁷ Given the limited success with repurposed drugs, along with the ambiguity surrounding their efficacy and safety, there is a grave need for the development of brain penetrant, nontoxic and efficacious drugs that improve patient survival.

Following the study of a benzylamine series¹⁸ that inhibited N. fowleri amoeba, we examined other scaffolds for a parallel development opportunity. Recently, we disclosed a chemical methodology¹⁹ that paired a quinazolinone-based Mannich-like reaction with our dual rearrangement protocol termed as a Sequential Quinazolinone-Amidine Rearrangement Strategy (SQuAReS, B → D, Scheme 1).²⁰ An initial library of quinazolinones D bearing a terminal oblique ring fusion was prepared and represented a novel framework. We were delighted to discover that several members of the collection showed >90% inhibition of N. fowleri at 10 μM, thus warranting further studies.¹⁹ Here, we shifted from a synthetic methodology driven program to a medicinal chemistry campaign. First, we resolved 50% effective concentrations (EC₅₀ values) against N. fowleri Nf69 for the pilot compounds, and then expanded structure–activity and structure–property relationships (SAR and SPR, respectively) to establish a pharmacophore.

Scheme 1. Mannich-Coupled Sequential Quinazolinone-Amidine Rearrangement Strategy (SQuAReS).

Further, we sought to understand diastereomeric and enantiomeric influences on activity while refining potency, physiochemical properties, and other parameters to enable in vivo evaluation. Herein, we describe the synthesis and antiamoebic activity of 88 analogs that exposed pharmacophoric opportunities that were leveraged to afford exceptionally potent inhibitors of N. fowleri. Further, a prototype was profiled for potency against two amoeba strains, ADME, mouse pharmacokinetics, and preliminary in vivo efficacy to establish baseline parameters and guide further optimization.

RESULTS AND DISCUSSION

Chemistry.

Methodology derived compounds predominately featured a N-methyl substituent on the tertiary amine (R² of D, Scheme 1), so the first analogs were designed with this region preserved. Assembly of the quinazolinone core was done using N-Boc protected amino acids that were prepared from the hydrolysis of lactams 1a–b or protection of amine 3 to afford intermediates 2a–c (Scheme 2). A two step, one-pot dehydrative cyclization, promoted by triphenylphosphite in the presence of N-Boc-amino acids 2a–c, anthranilic acids 4, and neopentylamine delivered quinazolinone salts 5 after N-Boc deprotection. The salts 5 were used in a AgOTf-promoted Mannich-type reaction with N-Cbz aminopropanal to give cyclized products 6. The cis- and trans-isomers were isolated and taken forward as a mixture into the N-Cbz deprotection and dual rearrangement (quinazolinone to amidine formation, followed by a spontaneous intramolecular cyclization, SQuAReS, Scheme 1) to generate (±)-cis-8a-22a, 24a, 26a-31a ring-fused analogs and (±)-trans-8b-22b, 24b, 26b-30b analogs which were separated using normal phase chromatography. Alternatively, brominated intermediate 6 underwent Suzuki coupling to install a phenyl ring of 7 which was subsequently deprotected and basified to initiate the SQuAReS and afford (±)-cis-23a and (±)-trans-23b after chromatographic separation. A late-stage palladium catalyzed cyanation²¹ of (±)-trans-24b afforded (±)-trans-25b.

Scheme 2. Synthesis of N-Methyl Quinazolinone Analogs^a.

^aReagents and conditions: (a) KOH, EtOH, 100 °C, 24 h, then (b) Boc₂O, NEt₃, DMAP, 0 °C to rt, 24 h, 70–90%; (c) P(OPh)₃, pyr., 130 °C, 1 h; then (d) neopentlyamine, 145 °C 8 h, 20–80%; (e) HCl in dioxane or MeOH, 3 h (75–95%); (f) CbzNH(CH₂)₂CHO, AgOTf, 4 Å MS, MeCN, μW 150 °C 1 h, 50–90%; (g) PdCl₂(dppf), ArB(OH)₂, NaOAc, dioxane, H₂O, 95 °C, 82% (h) MsOH, HFIP, 50 °C, 3 h, then (i) NEt₃, CH₃CN, reflux to 150 °C, 4–18 h; 60–90%; (j) Pd₂(dba)₃, S-Phos, Zn(CN)₂, DMF, H₂O, μW 120 °C 1 h, 44%.

The most expedient way to integrate nonmethyl substituents on the tertiary amine was to demethylate the parent compounds, followed by NH-functionalization. Hence, (±)-trans-21b and (±)-trans-24b were N-demethylated^22–24 using 1-chloroethyl chloroformate to afford the corresponding NH products, (±)-trans-32b and (±)-trans-33b (Scheme 3). Notably, only the trans-six-membered, ring-fused isomers were successfully demethylated in this protocol. The cis-six-membered, ring-fused isomers were substantially less reactive under these conditions, showing <25% conversion, while also generating multiple side products. Additional reaction time, temperature and/or equivalents of ACE-Cl did not improve the outcome. The trans-NH compounds were acylated or alkylated to afford (±)-trans-34b-50b, 53b-57b, 59b-62b, 65b. For (±)-trans-51b-52b, 63b-64b, the N-ethanol intermediate was prepared, followed by O-mesylation and amine displacement. Lastly, 58b was prepared through cyanation²¹ of the chloride, 57b. Selected analogs were separated by chiral HPLC or supercritical fluid chromatography (SFC) to afford individual enantiomers.

Scheme 3. N-Demethylation/Alkylation Synthetic Sequence^a.

^aReagents and conditions: (a) ACE-Cl, DCE, reflux, 14–16 h, then (b) MeOH, reflux, 21 h, 50—80%; (c) RCOCl, NEt₃, DCM, 0 to 50 °C, 70–90%; (d) R²X, Et₃N, DMF 50–80 °C, 50–80%; (e) MsCl, NEt₃, DCE, 0 °C to rt, then (f) NHR³R⁴, rt to reflux, 17–49%; (g) Pd₂(dba)₃, S-Phos, Zn(CN)₂, DMF, H₂O, μW 120 °C 1 h, 50%.

Despite the focused methodological structural scope and lack of dose response data in our past chemistry study, the amoebic percent inhibition data did reveal some early insights. For instance, five-membered, ring-fused analogs and analogs with six-membered morpholino or piperazino ring fusions did not demonstrate appreciable inhibition at 10 μM. However, several analogs of interest had A-ring substituents, so we explored various core phenyl ring substitutions first (Table 1). Briefly, compounds were assessed in triplicate at multiple concentrations up to 10 μM against N. fowleri Nf69 to generate EC₅₀ values. Miltefosine, a positive control, inhibited the amoeba with an EC₅₀ = 35.7 ± 9.10 μM, a value similar to previously reported data.²⁵ Compounds were also tested at 8 concentrations up to 20 μM using human neuronal SH-SY5Y cells to evaluate toxicity.

Table 1.

A-Ring Substitution and Effect of Cis- or Trans-4a,13b Ring Fusion on Anti-Amoeba Activity

entry	cmpd	ring fusion	R1	R2	R3	R4	EC50 μMa	CC50 μMb
1	±−8a	cis	H	H	H	H	>10	>20
2	±−8b	trans					>10	>20
3	±−9a	cis	CH3	H	H	H	>10	>20
4	±−9b	trans					>10	>20
5	±−10a	cis	Cl	H	H	H	>10	>20
6	±−10b	trans					>10	>20
7	±−11a	cis	H	CH3	H	H	2.8 ± 0.3	>20
8	±−11b	trans					3.1 ± 0.5	>20
9	±−12a	cis	H	OCH3	H	H	>10	>20
10	±−12b	trans					7.3 ± 1.2	>20
11	±−13a	cis	H	CN	H	H	2.1 ± 0.3	>20
12	±−13b	trans					0.67 ± 0.05	>20
13	±−14a	cis	H	F	H	H	>10	>20
14	±−14b	trans					>10	>20
15	±−15a	cis	H	CF3	H	H	0.95 ± 0.08	>20
16	±−15b	trans					1.1 ± 0.3	>20
17	±−16a	cis	H	Cl	H	H	2.2 ± 0.2	>20
18	±−16b	trans					1.0 ± 0.3	>20
19	±−17a	cis	H	H	OCH3	H	1.8 ± 0.3	>20
20	±−17b	trans					1.5 ± 0.7	>20
21	±−18a	cis	H	H	F	H	>10	>20
22	±−18b	trans					>10	>20
23	±−19a	cis	H	H	CF3	H	6.9 ± 1.8	>20
24	±−19b	trans					0.99 ± 0.08	>20
25	±−20a	cis	H	H	Cl	H	>10	>20
26	±−20b	trans					6.0 ± 1.7	>20
27	±−21a	cis	H	Cl	Cl	H	0.26 ± 0.01	>20
28	±−21b	trans					0.20 ± 0.01	>20
29	±−22a	cis	H	CF3	Cl	H	0.27 ± 0.12	>20
30	±−22b	trans					0.34 ± 0.07	>20
31	±−23a	cis	H	Cl	Ph	H	3.4 ± 0.6	>20
32	±−23b	trans					2.1 ± 0.6	9.4
33	±−24a	cis	H	Cl	OCH3	H	0.38 ± 0.03	>20
34	±−24b	trans					0.43 ± 0.14	>20
35	±−25b	trans	H	CN	OCH3	H	3.8 ± 1.1	>20
36	±−26a	cis	H	Cl	H	OCH3	>10	>20
37	±−26b	trans					>10	>20
38	miltefosine						35.7 ± 9.10	NT

^aAveraged data from triplicate runs (n = 3) with ± SEM using N. fowleri (Nf69 strain, ATCC 30215) trophozoites.

^bHuman neuronal SH-SY5Y cells (ATCC CRL-2266); NT = not tested.

The absence of any core substituent other than hydrogen atoms resulted in poor inhibition (Table 1, entry 1, EC₅₀ > 10 μM), regardless of a cis- or trans-ring fusion. Methyl group or chlorine atom substitution at R¹ gave the same results. Substitution at R² alone (entries 7–11) revealed improved, single digit micromolar activity in several cases. A fluorine atom rendered the compound inactive (entries 13–14), and a methoxy group in this position afforded marginal activity for the trans-isomer while the cis isomer was inactive (entries 9–10). A chlorine atom or trifluoromethyl substituent reached similar potency for either diastereomer, in the range of EC₅₀ ~ 1–2 μM (entries 15–18). A nitrile group in this position showed a 3-fold preference over its trans-isomer (entries 11–12). Notable outcomes for R³ included a 7-fold preference for a trifluoromethyl substituent on the trans-ring fused scaffold versus its corresponding cis-isomer and a loss of activity for fluorination at this position.

Chlorine substitution at R² and R³ afforded a significant potency improvement (EC₅₀ ~ 200 nM range, entries 27–28, Table 1). Similar results were also observed for chlorine combined with trifluoromethyl or methoxy groups. Generally, substitution of the R² and/or R³ positions with electron withdrawing groups other than fluorine gave better potency with some trending preference for the trans-isomers; however, for the most potent compounds, the diastereomeric preference was negligible. Few examples featuring R⁴ substituents were prepared due to limited success of the Mannich reaction when R⁴ was a chlorine or bromine atom. Nonetheless, methoxy substituent at R⁴ led to poor amoeba inhibition, and in combination with an R² chloride atom, the pairing was unfavorable (entries 36–37). Except for phenyl substituted derivative, (±)-trans-23b, none of the compounds showed cytotoxicity in neuronal cells up to the highest tested concentrations of 20 μM.

Other skeletal modifications were surveyed (Figure 2). Pyridyl derivatives (±)-cis-27a and (±)-trans 27b were inactive (EC₅₀ > 10 μM). Analogs integrating the favored chlorinated or trifluoromethylated core with other changes included the ring-fused morpholino analogs (±)-cis-28a–30a and (±)-trans-28b–30b. The 5-membered, ring-fused analog 31a, isolated exclusively as a cis-isomer, was also prepared; however, none of the structural changes in this analog set showed antiamoebic activity or mammalian cytotoxicity when tested up to the highest concentration in each assay (EC₅₀ > 10 μM, CC₅₀ > 20 μM).

Figure 2.

Summary of analogs with heteroatom insertions or ring contractions made to the quinazolinone framework.

Early in the SAR development, we zeroed in on the dichloro-substitution of quinazolinones 21a–b due to the potency gained. In this case, the cis and trans isomers demonstrated virtually the same potency, but for other 6-membered, ring-fused compound pairs, there was an emerging trend that the trans isomer was preferred. Synthetically, the trans-isomer was generally produced in a greater yield than the corresponding cis-product, leading to a deficit of cis-isomer to work with, and when six-membered ring-fused analogs were subjected to the N-demethylation protocol (Scheme 3, conditions a,b), only the trans-isomers reacted productively to afford NH intermediates that could be further derivatized. Taken together, we used exclusively the trans-N-methyl products as starting materials to investigate the effects of other tertiary amine substituents. Dichloroquinazolinone (±)-trans-21b was selected as the initial framework for this survey (Table 2).

Table 2.

Effect of Changing the 3° Amine Substituent on (±)-Trans-Dichloroquinazolinones

entry	cmpd	R1	EC50 (μM)a	cc50 μMb (SI)c	MLM T1/2, mind	entry	cmpd	R1	EC50(μM)a	CC50 μMb (SI)c	MLM T1/2, mind
1	±-trans-21b	CH3	0.20 ± 0.01	>20	ND	13	±-trans-44b		>10	>20	ND
2	±-trans-32b	H	1.60 ± 0.18	>20	ND	14	±-trans-45b		>10	>20	ND
3	±-trans-34b		0.040 ± 0.003	>20	41.1	15	±-trans-46b		0.72 ± 0.08	>20	ND
4	±-trans-35b		0.007 ± 0.002	>20	19.7	16	±-trans-47b		0.30 ± 0.02	>20	ND
5	±-trans-36b		0.002 ± 0.001	4.8 (2400)	18.3	17	±-trans-48b		0.020 ± 0.02	>20	83.1
6	±-trans-37b		0.72 ± 0.05	>20	ND	18	±-trans-49b		0.023 ± 0.009	10.0 (435)	17.0
7	±-trans-38b		2.53 ± 0.58	>20	ND	19	±-trans-50b		0.0004 ± 0.0001	>20	9.8
8	±-trans-39b		0.0002 ± 0.0001	>20	21.9	20	±-trans-51b		0.18 ± 0.04	8.0 (44)	ND
9	±-trans-40b		0.48 ± 0.13	>20	ND	21	±-trans-52b		0.13 ± 0.02	6.4 (49)	ND
10	±-trans-41b		0.05 ± 0.01	>20	ND	22	±-trans-53b		0.006 ± 0.001	>20	109.0
11	±-trans-42b		>10	>20	ND	23	±-trans-54b		0.007 ± 0.002	>20	67.2
12	±-trans-43b		>10	>20	ND	24	±-trans-55b		0.011 ± 0.001	>20	75.2

^aAveraged data from triplicate runs (n = 3) with ± SEM using N. fowleri (NF69 strain, ATCC 30215) trophozoites; miltefosine (positive control) N. fowleri EC₅₀ = 35.7 ± 9.1 0 μM.

^bHuman neuronal SH-SY5Y cells (ATCC CRL-2266).

^cSI = selectivity index, CC₅₀/EC₅₀.

^dMetabolic stability in CD-1 strain mouse liver microsomes. ND – not determined.

The NH intermediate (±)-trans-32b showed an 8-fold loss in antiamoebic potency compared to the parent N-methyl analog, (±)-trans-21b, from which it was derived. Fortunately, several other appendages attached to this nitrogen atom resulted in spectacular potency (Table 2). For instance, installing an N-isobutyl group or a methylene-bridged cyclopropyl group registered EC₅₀ values of 7 and 2 nM, respectively (entries 4–5, Table 2), though some cytotoxicity was noted for the latter compound (selectivity index (SI) = 2400). A benzyl group was also well tolerated, but a scan of methylene-linked pyridyl groups revealed a trend in preference of 3 pyridyl ≫ 4-pyridyl ≫ 2-pyridyl in which the 3-pyridyl analog demonstrated an astonishing EC₅₀ of 0.2 nM (entry 8). The N-acylated derivatives bearing a bulky α substituent to the carbonyl group lost activity (11–14); however, submicromolar activity was restored with the less bulky dimethylaminoalkylamide (±)-trans-46b. The alkyl tethered alcohol and ethers (entries 17–19 and 22–24) were noteworthy performers with several landing in the single digit nanomolar potency range. Select analogs from this group were assessed for microsomal stability in mice before proceeding to additional structural refinements. Several of the most potent compounds showed suboptimal microsomal stability with half-lives of <60 min (entries 3–5, 8, 18–19). While (±)-trans-48b passed this benchmark with a half-life of 83 min, its kinetic solubility was limited (<10 μM). The morpholino derivative, (±)-trans-55b, was more soluble (206 μM), but still exhibited marginal microsomal stability (entry 24). Oxetane (±)-trans-53b was potent and showed the best stability of the tested analogs with a half-life of 109 min, though solubility was determined to be 40 μM (entry 22). Nonetheless, these were attractive structural starting points from which enhanced solubility and microsomal stability was expected to emerge with further optimization. As such, we sought to exchange one of the two aryl chlorine atoms with substituents that were anticipated to improve the profile.

We used oxetane (±)-trans-53b as a starting point for refinement (entry 10, Table 3). Exchange of the C11 chlorine atom with a methoxy group afforded (±)-trans-57b with an EC₅₀ of 29 nM, solubility of 198 μM, and microsomal stability half-life of >145 min. A combination of nitrile and methoxy groups (entry 14) was investigated due to the noted benefit on microsomal stability and lipophilic efficiency when replacing an aromatic chloride with an aromatic nitrile.²⁶ These changes, when paired with the oxetane, showed promising attributes, though they did not outperform the chloro/methoxy modification. Some of the best performing tertiary amine substituents assessed in earlier analogs, along with several others that reduced aliphatic carbon count or introduced solubilizing groups, were also surveyed. However, the combination of potency, solubility, and microsomal stability favored (±)-trans-57b.

Table 3.

Effects of A-Ring Substitution and 3° Amine Substituent on Potency, Solubility and Microsomal Stability and Resolution of Selected Racemic Analogs to Compare Enantiomeric Data

entry	cmpd	R1	R2	R3	EC50(μM)a	CC50 μMb	SIc	solubility μM	MLM, T1/2, mind
1	±-trans-15b				1.1 ± 0.3	>20	>18.2	159	21.2
2	trans-15b-ent-1	CF3	H	CH3	0.25 ± 0.02	>20	>80.0	109	10.9
3	trans-15b-ent-2				4.3 ± 2.3	>20	>4.7	ND	ND
4	±-trans-16b				1.0 ± 0.3	>20	>20.0	187	18.5
5	trans-16b-ent-1	Cl	H	CH3	0.28 ± 0.01	>20	>71.4	129	11.5
6	trans-16b-ent-2				4.0 ± 2.6	>20	>5.0	ND	ND
7	±-trans-21b				0.20 ± 0.01	>20	>100	138	ND
8	trans-21b-ent-1	Cl	Cl	CH3	0.086 ± 0.005	7.8	90.7	98.2	6.1
9	trans-21b-ent-2				2.6 ± 0.2	>20	>7.7	ND	ND
10	±-trans-53b	Cl	Cl		0.006 ± 0.001	>20	>33000	40.0	109.0
11	±-trans-57b				0.029 ± 0.003	5.0	172	198	>145
12	trans-57b-ent-1 BDGR-20236	Cl	OCH3		0.49 ± 0.081	>20	>40.8	29.1	75.6
13	trans-57b-ent-2 BDGR-20237				0.012 ± 0.001	2.3	191.7	23.9	48.2
14	±-trans-58b	CN	OCH3		0.12 ± 0.02	>20	>166.7	136	104.6
15	±-trans-59b	Cl	OCH3		0.015 ± 0.001	0.64	42.7	4.6	3.8
16	±-trans-60b	Cl	OCH3		0.023 ± 0.003	1.0	43.5	76.1	12.6
17	±-trans-61b	Cl	OCH3		0.11 ± 0.01	2.5	22.7	65.7	3.1
18	±-trans-62b				0.17 ± 0.04	1.8	10.6	ND	ND
19	trans-62b-ent-1	Cl	OCH3		0.62 ± 0.30	>20	>32.3	ND	ND
20	trans-62b-ent-2				0.027 ± 0.003	1.0	38.0	2.6	ND
21	±-trans-63b	Cl	OCH3		0.52 ± 0.06	>20	>38.5	43.1	29.8
22	±-trans-64b	Cl	OCH3		0.74 ± 0.25	>20	>27.0	172	>145
23	±-trans-65b	Cl	OCH3		0.12 ± 0.01	>20	>166.7	3.8	ND
24		miltefosine			35.7 ± 9.10	ND	ND	ND	ND

^aAveraged data from triplicate runs (n = 3) with ± SEM using N. fowleri (Nf69 strain, ATCC 30215) trophozoites.

^bHuman neuronal SH-SY5Y cells (ATCC CRL-2266).

^cSI = selectivity index, CC₅₀/EC₅₀.

^dMetabolic stability in CD-1 strain mouse liver microsomes. ND = not determined.

We had mainly been working with racemic analogs, as chiral separation proved challenging; however, we were able to spot check the behavior of selected, enantioenriched (>96–99%) compounds that were separated (Table 3). Chiral prep HPLC conditions were identified for several early analogs featuring an N-methyl substituent, including (±)-trans-15b, (±)-trans-16b, and (±)-trans-21b. In all cases in which the enantiomers were resolved for a given compound, the isomer with the shortest retention time was given a suffix of “ent-1” while the isomer with the longer retention time was labeled ‘ent-2’. These three compounds, separated on the same chiral column matrix, showed that the enantiomer with the shortest retention time was substantially more active than the later eluting isomer (entries 2/3, 5/6 and 8/9, Table 3). For (±)-trans-21b, a >30-fold difference was noted for the more active enantiomer. Evaluation of the most potent enantiomers for these three early analogs showed impaired MLM stability which was a focus of further optimization. For (±)-trans-62b, a different chiral column was employed successfully, revealing that the late-eluting enantiomer was the more potent of the two; however, suboptimal microsomal stability was observed for this analog, too. Despite several attempts to use standard chiral HPLC to separate the enantiomers of one of the best performing analogs, (±)-trans-57b, complete resolution could not be achieved with our techniques and equipment. Ultimately, the separation was successfully performed using chiral SFC.

With separated isomers in hand, trans-57-ent-2 was shown to have a 41-fold advantage in potency over the other isomer (EC₅₀ = 12 nM, entry 13, Table 3). Solubility and microsomal stability were reduced compared to the racemate; however, the values were reasonable to work with in terms of benchmarking spectrum of activity across another N. fowleri strain and establishing physiochemical and initial ADME parameters. In parallel, each enantiomer was individually crystallized and analyzed by single-crystal X-ray crystallography to unequivocally establish the respective absolute configuration (Figure 3). The less active isomer, trans-57b-ent-1, designated BDGR-20236 internally, was determined to possess the 4aR,13bS-configuration at the ring fusion while the more potent isomer, trans-57b-ent-2 (BDGR-20237) had the 4aS, 13bR-configuration.

Figure 3.

X-ray derived molecular drawings of resolved enantiomers (4aR,13bS)-57b-ent-1 (BDGR-20236) and (4aS,13bR)-57b-ent-2 (BDGR-20237), each bottom panel shown with 50% probability ellipsoids.

Both enantiomers of trans-57b were evaluated against N. fowleri TY strain, isolated from a patient in Virginia, to assess potential differences in susceptibility compared to Nf69 (origin: Australia) which may be genetically distinct (Table 4). Exquisite congruency was observed for both enantiomers across the two strains. Physiochemical and ADME parameters were then benchmarked. The obtained log D value of 2.3 was within an ideal range for drug-likeness. Kinetic aqueous solubility was modest at 24–29 μM.

Table 4.

Strain Potency, Physiochemical, and ADME Data

	BDGR-20236 (4aR,13bS)-57b-ent-1	BDGR-20237 (4aS,13bR)-57b-ent-2
N. fowleri EC50, μM	Nf69 straina	0.49 ± 0.081	0.012 ± 0.001
	TY strainb	0.47 ± 0.096	0.014 ± 0.002
log D7.4c		2.3	2.3
solubilityd		29 μM	24 μM
BBB PAMPA, Peavg (nm/s)e		182, CNS+	172, CNS+
MLM, T1/2f		75.6 min	48.2 min
HLM, T1/2		80.3 min	92.7 min
plasma stability, T1/2f,g		>289 min	>289 min
plasma protein bindingf264		74% bound	74% bound

^aN. fowleri (Nf69 strain, ATCC 30215) trophozoites.

^bN. fowleri (TY strain, ATCC 30107) trophozoites; Averaged data from triplicate runs (n = 3) with ± SEM.

^cIn octanol/phosphate buffer, pH 7.4.

^dKinetic solubility, 50 mM phosphate buffer.

^eAveraged data from duplicate runs, 4 h; using positive and negative controls, P_e > 15 nm/s = CNS+; P_e ≤ 15 = CNS-.

^fCD-1 mouse.

^gMicrosomal stability expressed in half-life, 60 min exposure, +NADPH.

We assessed the likelihood of brain penetration using an in vitro blood–brain barrier (BBB) parallel artificial membrane permeability assay (PAMPA),²⁷ results of which predicted excellent BBB permeability compared to controls. The MLM stability was better for the less active isomer, BDGR-20236; however marginally, by 27 min. Mouse plasma stability was superb, and a significant free fraction (26%) was observed in mice. Taken together, BDGR-20237, while not ideal in terms of solubility and metabolic stability, was deemed suitable for a mouse pharmacokinetic (PK) study to assess exposure in plasma and brain tissue. Mice (n = 3/group) were dosed independently with a single bolus of BDGR-20237 (11 mg/kg) by either intravenous (IV) or oral (PO) delivery. Plasma concentrations were assessed at six time points over 12 h, and brain concentrations were determined at the experimental end point. Collected PK parameters (Table 5) revealed a half-life of ~1 h by either route and a linear decrease in plasma exposure over 12 h in both the IV and PO dosed cohorts with bioavailability determined to be 38%.

Table 5.

PK Parameters for BDGR-20237 in CD-1 Mice^a

parameter		dose mg/kgb	route	resultc
half-life, T1/2 (h)d		11	IV	1.0 ± 0.2
			PO	1.1 ± 0.4
Vd (L/kg)d		11	IV	3.7 ± 0.2
Cl (mL/min/kg)d		11	IV	40.8 ± 7.6
plasma AUCinf (ng/h/mL)d		11	IV	5011 ± 857
			PO	1920 ± 194
plasma conc. at single time points (ng/mL)	0.5 hd	11	IV	2113 ± 238
			PO	469 ± 81
	2 hd	11	IV	882 ± 189
			PO	459 ± 109
	4 hd	11	IV	281 ± 110
			PO	254 ± 18
	8 hd	11	IV	18.1 ± 15.0
			PO	21.1 ± 5.5
	12 hd	11	IV	2.6 ± ND
			PO	2.0 ± ND
	2h	44	IP	3849 ± 390
	4h	44	IP	1917 ± 409
brain conc. (ng/g)e	12 hd	11	IV or PO	low to BLDg
	4h	44	IP	9089 ± 2392
B/P ratiof	4h	44	IP	4.7
bioavailability, %Fd		11	PO	38%

^aFemale CD-1 mice, n = 3/group.

^bActual dose reported rather than intended 10 or 50 mg/kg doses.

^cAveraged data from triplicate runs with standard deviation reported.

^dData from 12 h experiment.

^eBrain homogenate.

^fBrain-to-plasma ratio obtained from the ratio of brain concentration divided by plasma concentration, each respective data point at 4 h.

^gBLD = below levels of detection for two of three mice in each group; brain concentrations for: one mouse in IV group: 13.3 ng/mL; one mouse in PO group: 10.4 ng/mL.

Using a 11 mg/kg dose, the concentration of BDGR-20237 in brain tissue at 12 h was shown to be near or below the limits of detection (BLD). To verify brain penetration, a follow up PK experiment in mice (n = 3/group) was done with a single intraperitoneal (IP) bolus of BDGR-20237 at a dose of 44 mg/kg with plasma concentrations determined at 2 and 4 h. Brain exposure was determined at 4 h (Table 5). We observed that increasing the dose by 5-fold (IP dosing) compared to the first experiment boosted the plasma exposure such that at the 4 h time point, exposure was ~8-fold higher than that observed with IV or PO dosing at the lower dose. Furthermore, at 4 h we observed high brain exposure with a brain-to-plasma ratio (B/P) of 4.7.

Considering these outcomes, the efficacy of BDGR-20237 was assessed using female CD-1 mice which were intranasally infected with N. fowleri Nf69 trophozoites. The study design included three groups of mice (n = 6/group) that were infected on day 0 (Figure 4, panel A). After a 48 h induction period to permit trophozoites to establish brain infection (days 0–2), separate cohorts of mice were treated for 5 days via IP administration using amphotericin B (5 mg/kg, once a day, QD) or BDGR-20237 (11 mg/kg, twice daily, BID). A control group of infected mice received only the vehicle during the treatment window. The 14-day study concluded with the brain tissue of surviving mice being cultured for viable amoeba.

Figure 4.

(A) Study design of mouse efficacy study using N. fowleri Nf69. (B) Kaplan–Meier survival data for infected mice treated with amphotericin (B, C) Kaplan–Meier survival data for infected mice treated with BDGR-20237.

The onset of neurological symptoms in the infected mice occurred more rapidly than typically observed, as the pathogenicity of the cultured amoeba can be challenging to consistently calibrate at the inception of the study. The amoeba was lethal, resulting in the expiration of all infected mice lacking treatment by day 6 of the experiment. Two of the six amphotericin B treated mice survived and exhibited normal behavior. Upon assessment of brain tissue from the two survivors, amoeba was detected in one. Resultantly, survivability of the amphoB treated group was 17% (Figure 4, panel B). Infected mice treated with BDGR-20237 succumbed to infection by day 7 (Figure 4, panel C). Neither of the two treatment groups (i.e., amphoB or BDGR treated animals), despite the survivors from the amphotericin treated cohort, provided statistically significant extension of life over vehicle controls, though both treatment groups were “trending” toward significance (p value >0.1). Complications from the rapid disease onset may have obscured this value. Nonetheless, considering the ADME and PK profiles for BDGR-20237, along with the pilot mouse efficacy assessment, it is likely that the 11 mg/kg BID dosing over 5 days was insufficient to maintain adequate brain concentrations to provide protection to infected mice. While the 50 mg/kg PK study comparatively revealed much greater plasma and brain exposure and mice did not show any clinical signs of adverse events after administration of a single bolus, the brain concentration of the compound was high at this dose (est. ~20 μM), and there was concern that repeat dosing at this concentration may lead to toxicity given the cytotoxicity that was observed with neuronal cells. Consequently, an assessment of the maximum tolerated dose (MTD) will be performed to guide optimal dosing in future efficacy studies.

CONCLUSIONS

Although rare, the human brain infection caused by N. fowleri is highly lethal. Repurposed drugs, though limited in successful intervention, are frequently used to combat the infection due to lack of investment in the discovery and development of safe and effective CNS-penetrant drugs specifically designed to address PAM. Out of a recent synthetic methodology aimed at constructing novel, pharmacologically biased heterocyclic frameworks, we fortuitously discovered inhibitory activity against the N. fowleri amoeba. These quinazolinones contain an oblique ring fusion with an optimally positioned nitrogen atom, signifying a new asset in the bleak arsenal of scaffolds that show promise against the pathogen. Implementation of the Mannich coupled domino rearrangement and execution of a key late stage N-demethylation/functionalization procedure enabled efficient analog diversification, resulting in the design and synthesis of an 88-member compound collection. These quinazolinones defined a pharmacophoric model from which to optimize several tunable regions. Exceptionally potent analogs with subnanomolar activity were identified, and as is most often the case, compound advancement depended critically on a comprehensive balance of potency, cytotoxicity, solubility, and microsomal stability. From this effort, a single enantiomer, BDGR-20237, exhibited equivalent double-digit nanomolar potency for two wild type strains of N. fowleri, and though some cytotoxicity was noted in a human neuronal cell line, the selectivity index of 192 provided a reasonable window to benchmark mouse PK parameters at two doses and confirm brain exposure. Despite some limitations in ADME and PK, a pilot study of BDGR-20237 was performed in a mouse infection model, revealing that further refinements are necessary, and a strategy for dosing frequency and duration will be required. As these compounds emerged from a phenotypic screen, studies are underway to elucidate a mechanism of action for this chemical series. Nonetheless, this new quinazolinone prototype represents a promising development scaffold that will enhance our understanding of N. fowleri vulnerability, thereby generating opportunity for advancement of new therapeutics.

EXPERIMENTAL SECTION

General Chemistry Experimental Section.

The purity of all final compounds was confirmed to be ≥95% by HPLC/MS. Commercial reagents and solvents were purchased from Alfa Aesar, Ambeed, Combi-Blocks, Fischer Scientific, VWR International, Chem-Impex Int’l, Inc., Acros Organics, etc. Chromatographic purifications were accomplished using a Teledyne Isco CombiFlash Rf 300 purification system with either Redi-sep silica gel columns (normal-phase) or Redi-sep gold C18 columns (reverse phase). LCMS/HPLC analysis was used to monitor reactions and determine purity and was performed on an either an Agilent 1290 Infinity II HPLC system with a 1290 Infinity II Diode Array Detector and an Agilent 6120 Quadrupole LC-MS system with the following parameters: Poroshell 120 EC-C18, 1.9 μm column, UV detection wavelength = 254 nm, flow rate = 1.0 mL/min, gradient = 20–100% LC-MS grade methanol over 4 min; the organic mobile phase and aqueous mobile phase contained 0.1% LC-MS grade formic acid or an Shimadzu LCMS2020 system with a SPD-M40 photodiode array detector with the following parameters: Shim-Pack Velox SP-C18 2.7 μm column, UV detection wavelength = 254 nm, flow rate = 1.0 mL/min, gradient = 20–100% LC-MS grade methanol over 4 min; the organic mobile phase and aqueous mobile phase contained 0.1% LC-MS grade formic acid. Preparative HPLC chiral separations were performed on an ACCQPrep HP150 using either a Regis Reflect I-Cellulose B (5 μm (3,5-diphenylcarbamate), 25 cm × 21.1 mm) column or a Regis (R,R)-Welk-O 1 (5 μm Kromasil, 25 cm × 21.1 mm) column. Analysis of enantiopurity was done using a chiral phase HPLC (Agilent 1200- Series HPLC) wither either a Reflect ICellulose B5 μm (3,5-diphenylcarbamate), 25 cm × 21.1 mm column or a (R,R)-Welk-O 1 5 μm Kromasil, 25 cm × 21.1 mm column. Microwave irradiated (MWI) reactions were carried out using a Biotage Initiator+ Fourth Generation microwave synthesizer. High resolution mass spectra (HRMS) were obtained by the Analytical Instrument Center at the School of Pharmacy on an Electron Spray Injection (ESI) mass spectrometer. ¹H, ¹⁹F, and ¹³C nuclear magnetic resonance (NMR) spectra were obtained on either a Varian 500 MHz or a Bruker Ascend 400 MHz spectrometer in chloroform-d. DMSO-d₆, D₂O, or acetic-acid-d₆. The chemical shifts (δ) reported are given in parts per million (ppm). The following abbreviations were used to describe peak splitting patterns: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, p = pentuplet, dd = doublet of doublet, dt = doublet of triplet, dq = doublet of quartet, td = triplet of doublet, tt = triplet of triplet, qd = quartet of doublet, ddd = doublet of doublet of doublet, tdd = triplet of doublet of doublet, dddd = doublet of doublet of doublet of doublet and m = multiplet. Coupling constants, J, which were reported in Hertz (Hz). The following compounds were previously reported, and the analytical data obtained matched what was previously reported:¹⁹ intermediates 2a–c, and final compounds: (±)-cis-8–9a, (±)-cis-11–14a, (±)-cis-16–20a, (±)-cis-27–28a, (±)-trans-8–9b, (±)-trans-11–14b, (±)-trans-16–20b, (±)-trans-27–28b.

General Procedure 1: Synthesis of N-Boc-Protected Quinazolinones.

Substituted 2-aminobenzoic acid 4 (1 equiv), N-Boc-protected amino-acid 2 (1.2 equiv), and P(OPh)₃ (2.2 equiv) were combined in anhydrous pyridine (0.12 M). After heating thermally at 130 °C in a pressure relief vial for 1 h, the reaction was cooled to rt, and neopentyl amine (2 equiv) was added. The reaction mixture was heated thermally to 145 °C for 8 h. After cooling, solvent was removed under reduced pressure. The crude oil was dissolved in EtOAc (80 mL), and sequentially washed with 10% citric acid, sat. aq NaHCO₃, and brine. The separated organic extract was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford S1–S10.

General Procedure 2: Synthesis of Hydrochloride Salts 5a–5j from N-Boc-Protected Quinazolinones.

In a round-bottom flask, the N-Boc-protected quinazolinone S1–S10 (1 equiv) was suspended in either 4 N HCl in dioxane (0.16 M) or 3 N HCl in MeOH (0.16 M) for 2–3 h or until TLC or LCMS showed starting material consumption. The reaction mixture was poured into Et₂O and stirred for 10 min. The product HCl salt was then collected by vacuum filtration and dried in a vacuum oven at 60 °C to afford 5a–5j.

General Procedure 3: Synthesis of Mannich-Cyclized Products 6a–6j.

HCl salts 5 (1 equiv), benzyl (3-oxopropyl)carbamate (4 equiv), AgOTf (1 equiv) and activated/Ar-cooled 4 Å MS were placed in an oven-dried 2–5 or 10–20 mL μW vial. The vessel was sealed, evacuated, and backfilled twice with Ar (g), followed by the addition of degassed anhydrous CH₃CN (0.15 M). The reaction mixture was then heated by μW irradiation at 150 °C for 1 h. The reaction was filtered over Celite, and the collected organic filtrate was concentrated, then diluted with 20% IPA/CHCl₃ (65 mL) and washed with sat. aq NaHCO₃ (50 mL). The separated organic extract was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was suspended in MeOH (10 mL) and absorbed to a 10 g HF-Mega BE-SCX column (Agilent). After rinsing with 3 × 60 mL of MeOH, the absorbed crude material was obtained by washing with 7N NH₃ in MeOH (60 mL). After concentration, the residue was purified by flash chromatography to afford 6a–6j.

General Procedure 4: Suzuki Cross-Coupling of Mannich-Cyclized Intermediate to Afford 7.

Mannich product 6e was placed in a 20–40 mL oven-dried pressure relief vial with Pd(dppf)₂Cl₂ (0.1 equiv), NaOAc (4.5 equiv) and PhB(OH)₂ (1.5 equiv). The vial was sealed, evacuated, and backfilled with Ar (g). Degassed anhydrous dioxane/water (3:1) was added, and the vial was heated to 95 °C for 24 h. The reaction mixture was diluted with DCM, washed with H₂O, followed by brine. The separated organic extract was dried over Na₂SO₄, filtered, and concentrated, affording a residue that was purified by normal phase chromatography to afford Suzuki product 7.

General Procedure 5: N-Cbz Deprotection and Quinazolinone/Amidine Rearrangement (SQuAReS).

In a round-bottom flask, compound 6 or 7 (1 equiv) was suspended in HFIP²⁸ (0.2 M). MsOH (5 equiv) was added, and the reaction was heated for 3 h at 50 °C. After 3 h, the reaction was cooled to rt and quenched with MeOH (0.2 M). Solvent was removed under reduced pressure by azeotroping with chloroform (3×). The residue was transferred to a new sealed tube, suspended in CH₃CN (0.08 M), and NEt₃ (15 equiv) was added. The tube was sealed and heated to 80 °C, generally for 8 h, or until the free amine was fully converted to the cyclized product, as monitored by LCMS. Upon completion, solvent was removed under reduced pressure. The residue was then suspended in 20% IPA/CHCl₃ (120 mL) and washed with brine (80 mL). The separated organic extract was dried over Na₂SO₄, filtered, and concentrated. The residue was then purified by normal phase chromatography to afford (±)-cis-8–24a, (±)-cis-26-31a, (±)-trans-8–24b, or (±)-trans-26–30b.

General Procedure 6: N-Demethylation of Quinazolinones.

To a sealed, oven-dried pressure relief vial containing either (±)-trans-21b or (±)-trans-25b that was evacuated and backfilled with Ar (g) was added DCM (0.2 M). After the dropwise addition of ACE-Cl (4 equiv), the reaction was allowed to stir at rt for 10 min before heating to reflux for 14–16 h. After solvent and excess ACE-Cl was removed under reduced pressure, the crude reaction mixture was suspended in anhydrous methanol (0.02 M) and heated to reflux for 21 h. Upon completion, solvent was removed under reduced pressure. The crude reaction mixture was then suspended in 20% IPA/CHCl₃ (250 mL) and washed with sat. aq NaHCO₃ (150 mL). After drying the separated organic phase over Na₂SO₄ and filtering, the residue resulting from concentration was purified by normal phase chromatography to afford (±)-trans-32b or (±)-trans-33b.

General Procedure 7: N-Alkylation of NH-Quinazolinones.

In a 2 dram pressure relief vial flushed with Ar, (±)-trans-32b or 33b (1 equiv), alkyl-halide (4 equiv), and Et₃N (6 equiv) were suspended in anhydrous DMF (0.25 M) and heated at 50–80 °C until starting material was consumed, as indicated by LCMS (~20 h). The reaction was diluted with 20% IPA/CHCl₃ (60 mL), washed sequentially with sat. aq NaHCO₃ (1 × 40 mL), H₂O (2 × 40 mL) and brine (1 × 40 mL). The separated organic extract was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by normal phase chromatography to afford (±)-trans-34–41b, (±)-trans-47–50b, (±)-trans-53–57b, (±)-trans-59–62b, and (±)-trans-65b.

General Procedure 8: N-Acylation of NH-Quinazolinones.

In a 2 dram pressure relief vial flushed with Ar, (±)-trans-32b or 33b (1 equiv) and Et₃N (2 equiv) were suspended in anhydrous DCM (0.25 M) and cooled to 0 °C before an appropriately substituted acid chloride (1.2 equiv) was added dropwise. After stirring at 0 °C for 10 min, the reaction was allowed to warm to rt and then stirred until LCMS showed minimal or no starting material (~18–24 h). The reaction was diluted with 20% IPA/CHCl₃ and washed with sat. aq NaHCO₃. The separated organic extract was dried over Na₂SO₄, filtered, and concentrated to afford a residue that was purified by normal phase chromatography to afford (±)-trans-42–46b.

General Procedure 9: N-Alkylation of N-Ethanolamine.

In a 2 dram pressure relief vial, (±)-trans-48b or 56b (1 equiv) and Et₃N (2 equiv) were suspended in anhydrous DCE (0.25 M) and then cooled to 0 °C. At 0 °C, MsCl (1.2 equiv) was added dropwise, and then the reaction was allowed to warm to rt and stirred for 2 h. Amine (6 equiv) was added dropwise, and the reaction stirred at rt for 22 h. The reaction was diluted with 20% IPA/CHCl₃ (60 mL), washed sequentially with sat. aq NaHCO₃ (40 mL), water (40 mL), and brine (40 mL). The separated organic extract was dried over Na₂SO₄, filtered, and concentrated to afford a residue that was purified by normal phase chromatography to give (±)-trans-51–52b or (±)-trans-63–64b.

(±)-(4aR,13bR)-9-Chloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-cis-10a and (±)-(4aR,13bS)-9-Chloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-trans-10b.

Step 1: Synthesis of tert-Butyl (4-(5-Chloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)butyl)(methyl)carbamate (S1).

Compound S1 was synthesized according to general procedure 1 from 2-amino-6-chloro-benzoic acid and 2a. Purification by flash chromatography (5–25% X/hexanes, X = 4:1 EtOAc/DCM) delivered S1 as a yellow oil (1.25 g, 70%). ¹H NMR (400 MHz, CDCl₃) δ 7.56–7.45 (m, 2H), 7.39 (dd, J = 7.5, 1.5 Hz, 1H), 4.61–3.62 (m, 2H), 3.25 (t, J = 7.1 Hz, 2H), 2.84 (d, J = 6.1 Hz, 5H), 1.76 (t, J = 7.8 Hz, 2H), 1.68–1.57 (m, 2H), 1.44 (s, 9H), 0.99 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 161.0, 155.9, 149.4, 134.1, 133.5, 129.0, 126.1, 117.8, 79.4, 52.2, 48.7, 47.9, 35.6, 34.5, 34.2, 28.8, 28.5, 27.3, 24.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₃H₃₅ClN₃O₃, 436.2361. Found 436.2353.

Step 2: Synthesis of 5-Chloro-2-(4-(methylamino)butyl)-3-neopentylquinazolin-4(3H)-one bis-Hydrochloride Salt (5a).

Compound 5a was synthesized according to general procedure 2 from S1, which afforded 5a as a white solid (0.90 g, 84%).

¹H NMR (400 MHz, DMSO) δ 9.14 (d, J = 7.6 Hz, 2H), 7.83–7.71 (m, 2H), 7.58 (dd, J = 7.5, 1.5 Hz, 1H), 4.06 (s, 2H), 3.03 (t, J = 7.0 Hz, 2H), 2.90 (p, J = 6.7 Hz, 2H), 2.50–2.45 (m, 4H), 1.80 (dt, J = 10.3, 6.3 Hz, 4H), 0.93 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 160.6, 159.0, 145.4, 134.9, 132.9, 129.6, 123.2, 116.5, 51.9, 47.6, 34.0, 33.0, 32.3, 28.1, 24.7, 23.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₂₇ClN₃O, 336.1837. Found 336.1837.

Step 3: Synthesis of Benzyl (2-(3-(5-Chloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-methylpiperidin-2-yl)ethyl)-carbamate (6a).

Compound 6a was synthesized according to general procedure 3 from 5a. Purification by reverse phase flash chromatography (10–90% MeOH/H₂O) which afforded 6a as an off-white solid (0.44 g, 69%). 6a was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-9-Chloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-10a and (±)-(4aR,13bS)-9-Chloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-10b.

Both diastereomers of 10 were synthesized according to general procedure 5 starting from 6a. Purified by flash chromatography10–60% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH). (±)-cis-10a and trans-10b: 138 mg (76%).

(±)-cis-10a, isolated as a light brown solid (92 mg).

¹H NMR (400 MHz, CDCl₃) δ 7.55–7.45 (m, 2H), 7.37 (dd, J = 6.6, 2.4 Hz, 1H), 4.07 (ddd, J = 14.2, 5.8, 3.9 Hz, 1H), 3.95 (ddd, J = 14.2, 11.2, 5.0 Hz, 1H), 2.97 (q, J = 3.6 Hz, 1H), 2.84–2.72 (m, 2H), 2.53 (dt, J = 6.0, 3.1 Hz, 1H), 2.35 (dtd, J = 14.4, 5.1, 3.9 Hz, 1H), 2.27 (s, 3H), 2.17 (td, J = 10.8, 3.3 Hz, 1H), 1.92 (dddd, J = 14.2, 11.2, 5.9, 2.7 Hz, 1H), 1.69–1.47 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 156.9, 150.0, 133.7, 133.2, 128.7, 126.3, 117.4, 57.8, 55.8, 42.9, 42.0, 39.8, 26.8, 24.8, 22.6. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₉ClN₃O, 304.1211. Found 304.1202. HPLC Purity: ≥ 99%, RT = 1.90 min.

(±)-trans-10b, isolated as a light brown solid (46 mg).

¹H NMR (400 MHz, CDCl₃) δ 7.56–7.50 (m, 2H), 7.40 (dd, J = 6.4, 2.6 Hz, 1H), 4.16 (ddd, J = 14.5, 6.1, 4.8 Hz, 1H), 4.02 (ddd, J = 14.9, 9.6, 5.9 Hz, 1H), 2.96 (ddt, J = 11.7, 4.1, 1.9 Hz, 1H), 2.77–2.69 (m, 1H), 2.65 (td, J = 11.1, 3.7 Hz, 1H), 2.44 (ddt, J = 13.4, 5.9, 4.7 Hz, 1H), 2.36 (s, 3H), 2.10 (td, J = 12.0, 3.2 Hz, 1H), 1.96 (dt, J = 10.4, 5.2 Hz, 1H), 1.91–1.70 (m, 3H), 1.43 (tdd, J = 13.0, 11.5, 4.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.2, 156.4, 150.1, 133.9, 133.5, 129.0, 126.5, 117.5, 62.3, 56.9, 44.9, 42.7, 40.8, 27.2, 26.7, 25.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₉ClN₃O, 304.1211. Found 304.1200. HPLC Purity: ≥98%, RT = 1.99 min.

(±)-(4aR,13bR)-4-Methyl-10-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Cis-15a and (±)-(4aR,13bS)-4-Methyl-10-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-15b.

Step 1: Synthesis of tert-Butyl Methyl(4-(3-neopentyl-4-oxo-6-(trifluoromethyl)-3,4-dihydroquinazolin-2-yl)butyl)carbamate (S2).

Compound S2 was synthesized according to general procedure 1 from 2-amino-5-trifluoromethyl-benzoic acid and 2a.

Purification by flash chromatography (5–20% X/hexanes, X = 4:1 EtOAc/DCM) delivered S2 as a yellow oil (1.16 g, 56%). ¹H NMR (400 MHz, CDCl₃) δ 8.54–8.49 (m, 1H), 7.89 (dd, J = 8.6, 2.2 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 4.12 (s, 2H), 3.28 (t, J = 6.9 Hz, 2H), 2.88 (d, J = 29.4 Hz, 5H), 1.79 (d, J = 9.8 Hz, 2H), 1.65 (p, J = 7.6 Hz, 2H), 1.45 (s, 9H), 1.01 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 162.5, 156.0, 149.1, 130.4, 129.7, 127.8, 125.2 (q, J = 4.2 Hz), 122.6, 120.5, 115.5, 79.5, 52.6, 47.9, 35.8, 34.8, 34.3, 28.8, 28.6, 27.4, 24.6. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.31. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₄H₃₅F₃N₃O₃, 470.2625. Found 470.2620.

Step 2: Synthesis of 2-(4-(Methylamino)butyl)-3-neopentyl-6-(trifluoromethyl)quinazolin-4(3H)-one bis-hydrochloride Salt (5b).

Compound 5b was synthesized according to general procedure 2 from S2, which afforded 5b as a white solid (0.71 g, 80%). ¹H NMR (400 MHz, DMSO) δ 8.92 (d, J = 7.9 Hz, 2H), 8.16 (d, J = 2.2 Hz, 1H), 7.92 (dd, J = 8.7, 2.2 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 6.02 (s, 1H), 3.93 (s, 2H), 2.81 (t, J = 7.1 Hz, 2H), 2.73 (q, J = 6.8 Hz, 2H), 2.32 (m, 3H), 1.72–1.53 (m, 4H), 0.76 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 161.3, 160.9, 148.0, 130.5, 127.5, 126.5 (q, J = 32.7 Hz), 124.0 (q, J = 4.1 Hz), 122.4, 119.9, 51.9, 47.8, 34.2, 34.0, 32.2, 28.3, 24.8, 23.7. ¹⁹F NMR (376 MHz, DMSO) δ − 61.6. HRMS (ESI) m/z: [M + H]+ Calcd for C₁₉H₂₇F₃N₃O, 370.2101. Found 370.3709.

Step 3: Synthesis of Benzyl (2-(1-Methyl-3-(3-neopentyl-4-oxo-6-(trifluoromethyl)-3,4-dihydroquinazolin-2-yl)piperidin-2-yl)ethyl)-carbamate (6b). Compound 6b was synthesized according to general procedure 3 from 5b.

Purification by reverse phase flash chromatography (10–90% MeOH/H₂O) which afforded 6b as an off-white solid (319 mg, 76%). 6b was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-4-Methyl-10-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-15a and (±)-(4aR,13bS)-4-Methyl-10-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-15b.

Both diastereomers of 15 were synthesized according to general procedure 5 starting from 6b. Purified by flash chromatography: 5–50% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH). (±)-cis-15a and (±)-trans-15b: 128 mg (79%).

(±)-cis-15a was isolated as a tan solid (49 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.53 (d, J = 2.1 Hz, 1H), 7.86 (dd, J = 8.6, 2.2 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 4.14 (ddd, J = 14.2, 5.8, 3.9 Hz, 1H), 4.02 (ddd, J = 14.0, 11.2, 5.0 Hz, 1H), 3.01 (p, J = 4.8, 4.1 Hz, 1H), 2.91–2.73 (m, 2H), 2.56 (dt, J = 5.9, 3.1 Hz, 1H), 2.37 (dq, J = 14.3, 4.8 Hz, 1H), 2.28 (s, 3H), 2.19 (td, J = 10.7, 3.8 Hz, 1H), 1.96 (dddd, J = 14.3, 11.3, 5.8, 2.7 Hz, 1H), 1.59 (dddd, J = 21.9, 11.5, 8.4, 4.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.2, 157.8, 149.7, 130.1 (q, J = 3.3 Hz), 128.1, 125.1, 124.6 (q, J = 4.1 Hz), 122.4, 120.0, 62.2, 56.8, 44.9, 42.6, 40.5, 27.1, 26.4, 25.0. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.25. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₉F₃N₃O, 338.1375. Found 338.1464. HPLC purity: ≥99%, RT = 2.21 min.

(±)-trans-15b was isolated as a tan solid (79 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.54 (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.6, 2.2 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H), 4.25–4.10 (m, 2H), 3.00 (ddt, J = 11.7, 4.1, 1.8 Hz, 1H), 2.73 (qd, J = 12.1, 10.8, 4.0 Hz, 2H), 2.46 (dq, J = 13.4, 5.4 Hz, 1H), 2.39 (s, 3H), 2.14 (td, J = 12.0, 3.2 Hz, 1H), 2.01 (td, J = 10.2, 4.7 Hz, 1H), 1.94–1.74 (m, 3H), 1.56–1.43 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 161.2, 157.8, 149.7, 130.1 (q, J = 3.3 Hz), 128.1, 125.1, 124.6 (q, J = 4.1 Hz), 122.4, 120.0, 62.2, 56.8, 44.9, 42.6, 40.5, 27.1, 26.4, 25.0. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.25. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₉F₃N₃O, 338.1375. Found 338.1471. HPLC purity: ≥99%, RT = 2.51 min.

(4aR,13bS)-4-Methyl-10-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-15b-ent 1 and (4aS,13bR)-4-Methyl-10-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, Trans-15b-ent 2.

60 mg of (±)-trans-15b was separated under the following conditions: Reflect I-Cellulose B5 μm (3,5-diphenylcarbamate), 25 cm × 21.1 mm. 2–15% IPA/hexanes, 1.5 mL injection, 21 mL/min.

(4aR,13bS)-4-Methyl-10-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, trans-15b-ent 1.

Absolute stereochemistry arbitrarily assigned. 22.8 mg isolated from chiral separation. (96% ee, RT = 12.93 min).

¹H NMR (400 MHz, CDCl₃) δ 8.53 (d, J = 2.1 Hz, 1H), 7.88 (dd, J = 8.6, 2.2 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 4.23–4.10 (m, 2H), 2.98 (dq, J = 11.5, 2.2 Hz, 1H), 2.79–2.66 (m, 2H), 2.45 (dq, J = 13.4, 5.4 Hz, 1H), 2.37 (s, 3H), 2.12 (td, J = 12.0, 3.1 Hz, 1H), 2.00 (td, J = 10.1, 4.7 Hz, 1H), 1.91–1.74 (m, 3H), 1.48 (qd, J = 12.6, 3.7, 1.6 Hz, 1H).¹³C NMR (101 MHz, CDCl₃) δ 161.4, 158.0, 149.8, δ 130.28 (q, J = 3.2 Hz), 128.2, 125.3, 124.71 (q, J = 4.1 Hz), 122.6, 120.2, 62.3, 56.9, 45.0, 42.8, 40.7, 27.3, 26.6, 25.1. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.25. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₉F₃N₃O, 338.1375. Found 338.1470. HPLC purity: ≥98%, RT = 2.51 min.

(4aS,13bR)-4-Methyl-10-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-15b-ent 2.

Absolute stereochemistry arbitrarily assigned. 20.6 mg isolated from chiral separation. (97% ee, RT = 15.08 min).

¹H NMR (400 MHz, CDCl₃) δ 8.53 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.7, 2.2 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 4.26–4.09 (m, 2H), 2.98 (dt, J = 11.9, 3.3 Hz, 1H), 2.80–2.66 (m, 2H), 2.37 (s, 4H), 2.12 (td, J = 11.9, 3.1 Hz, 1H), 2.00 (td, J = 10.2, 4.8 Hz, 1H), 1.91−1.76 (m, 3H), 1.48 (qd, J = 13.0, 12.4, 4.1 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 161.4, 158.0, 149.8, δ 130.28 (q, J = 3.3 Hz), 128.2, 125.3, 124.71 (q, J = 4.1 Hz), 122.6, 120.2, 62.3, 56.9, 45.1, 42.8, 40.7, 27.3, 26.6, 25.1. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.25. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₉F₃N₃O, 338.1375. Found 338.1471. HPLC purity: ≥99%, RT = 2.51 min.

(4aR,13bS)-10-Chloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-16b-ent-1 and (4aS,13bR)-10-Chloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-16b-ent-2.

50 mg of (±)-trans-16b was separated under the following conditions: Reflect I-Cellulose B5 μm (3,5-diphenylcarbamate), 25 cm × 21.1 mm. 2–15% IPA/hexanes, 1.5 mL injection, 21 mL/min.

(4aR,13bS)-10-Chloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-16b-ent-1.

Absolute stereochemistry arbitrarily assigned. Fourteen mg isolated from chiral separation (>99% ee, RT = 9.17 min). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J = 2.4 Hz, 1H), 7.63 (dd, J = 8.8, 2.4 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 4.21–4.09 (m, 2H), 2.98 (ddt, J = 11.6, 4.0, 1.9 Hz, 1H), 2.75–2.65 (m, 2H), 2.47–2.40 (m, 1H), 2.37 (s, 3H), 2.11 (td, J = 12.0, 3.2 Hz, 1H), 1.99 (td, J = 10.2, 5.0 Hz, 1H), 1.89–1.73 (m, 3H), 1.53–1.41 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 161.1, 156.0, 146.2, 134.6, 132.1, 128.9, 126.0, 121.4, 62.4, 56.9, 44.9, 42.8, 40.6, 27.3, 26.6, 25.1. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₉ClN₃O, 304.1211. Found 304.1209. HPLC purity: ≥95%, RT = 2.02 min.

(4aS,13bR)-10-Chloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-16b-ent-2.

Absolute stereochemistry arbitrarily assigned. 14.1 mg isolated from the chiral separation (>99% ee, RT = 11.03 min).

¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 2.4 Hz, 1H), 7.63 (dd, J = 8.8, 2.4 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 4.21–4.09 (m, 2H), 2.98 (ddt, J = 11.6, 4.1, 1.9 Hz, 1H), 2.71 (ddd, J = 12.5, 7.4, 3.8 Hz, 2H), 2.43 (dq, J = 13.3, 5.5 Hz, 1H), 2.37 (s, 3H), 2.12 (td, J = 12.0, 3.2 Hz, 1H), 1.98 (td, J = 10.3, 5.0 Hz, 1H), 1.88–1.73 (m, 3H), 1.53–1.41 (m, 1H).¹³C NMR (101 MHz, CDCl₃) δ 161.1, 156.0, 146.2, 134.6, 132.1, 128.9, 126.0, 121.4, 62.4, 56.9, 44.8, 42.8, 40.6, 27.3, 26.6, 25.1. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₉ClN₃O, 304.1211. Found 304.1209. HPLC purity: ≥97%, RT = 2.02 min.

(±)-(4aR,13bR)-10,11-Dichloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Cis-21a and (±)-(4aR,13bS)-10,11-Dichloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-21b.

Step 1: Synthesis of tert-Butyl (4-(6,7-Dichloro-3-neopentyl-4oxo-3,4-dihydroquinazolin-2-yl)butyl)(methyl)carbamate (S3).

Compound S3 was synthesized according to general procedure 1 from 2-amino-4,5-dichloro-benzoic acid and 2a. Purification by flash chromatography (0–25% X/hexanes, X = 4:1 EtOAc/DCM) delivered S3 as a colorless oil (0.98 g, 54%).

¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.70 (s, 1H), 4.06 (s, 2H), 3.27–3.22 (m, 2H), 2.88–2.82 (m, 5H), 1.78–1.73 (m, 2H), 1.66–1.58 (m, 2H), 1.44 (s, 9H), 0.98 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 161.7, 159.5, 155.9, 146.1, 138.7, 130.6, 128.5, 128.4, 120.2, 79.4, 52.6, 48.7, 35.6, 34.8, 34.3, 28.8, 28.6, 27.4, 24.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₃H₃₄Cl₂N₃O₃, 470.1972. Found 470.1995.

Step 2: Synthesis of 6,7-Dichloro-2-(4-(methylamino)butyl)-3-neopentylquinazolin-4(3H)-one bis-hydrochloride Salt (5c).

Compound 5c was synthesized according to general procedure 2 from S3, which afforded 5c as a white solid (0.66 g, 91%). ¹H NMR (400 MHz, DMSO) δ 8.95 (s, 2H), 8.21 (s, 1H), 7.91 (s, 1H), 6.03 (s, 1H), 4.15–3.98 (m, 2H), 2.92 (dt, J = 12.4, 6.4 Hz, 4H), 2.51 (d, J = 2.0 Hz, 2H), 1.86–1.69 (m, 4H), 0.93 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 160.7, 159.9, 145.7, 137.2, 128.9, 128.2, 127.8, 120.0, 51.8, 47.9, 34.2, 34.1, 32.3, 28.3, 24.8, 23.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₂₆Cl₂N₃O, 370.1447. Found 370.1442.

Step 3: Synthesis of Benzyl (2-(3-(6,7-Dichloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-methylpiperidin-2-yl)ethyl)-carbamate (6c).

Compound 6c was synthesized according to general procedure 3 from 5c. Purification by reverse phase flash chromatography (10–90% MeOH/H₂O) which afforded 6c as an off-white solid (449 mg, 92%). 6c was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-10,11-Dichloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-21a and (±)-(4aR,13bS)-10,11-Dichloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-21b.

Both diastereomers of 21 were synthesized according to general procedure 5 starting from 6c Purified by flash chromatography: 15–50% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH). (±)-cis-21a and (±)-trans-21b: 165 mg (81%).

(±)-cis-21a, isolated as a yellow solid (60 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.72 (s, 1H), 4.11 (ddd, J = 14.2, 5.8, 3.8 Hz, 1H), 3.97 (ddd, J = 14.1, 11.4, 5.0 Hz, 1H), 2.97 (q, J = 3.5 Hz, 1H), 2.86–2.73 (m, 2H), 2.53 (dt, J = 5.9, 3.2 Hz, 1H), 2.35 (dq, J = 14.2, 4.7 Hz, 1H), 2.27 (s, 3H), 2.17 (td, J = 10.0, 9.4, 3.4 Hz, 1H), 1.94 (dddd, J = 14.4, 11.4, 5.8, 2.8 Hz, 1H), 1.68–1.47 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.0, 157.7, 146.7, 138.4, 130.2, 128.6, 127.8, 119.8, 58.0, 56.1, 42.9, 42.2, 39.9, 26.8, 25.1, 22.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₈Cl₂N₃O, 338.0821. Found 338.0809. HPLC purity: ≥97%, RT = 2.69 min.

(±)-trans-21b, isolated as a tan solid (105 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1H), 7.75 (s, 1H), 4.14 (ddt, J = 8.9, 5.9, 2.8 Hz, 2H), 3.03–2.94 (m, 1H), 2.68 (td, J = 11.5, 10.7, 3.6 Hz, 2H), 2.44 (dq, J = 13.4, 5.6 Hz, 1H), 2.37 (s, 3H), 2.12 (td, J = 12.0, 3.1 Hz, 1H), 1.99 (td, J = 10.1, 4.9 Hz, 1H), 1.94−1.70 (m, 3H), 1.45 (td, J = 13.2, 12.8, 4.0 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 157.3, 146.8, 138.7, 130.6, 128.8, 127.9, 119.8, 62.2, 56.9, 44.9, 42.8, 40.6, 27.2, 26.6, 25.1. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₈Cl₂N₃O, 338.0821. Found 338.0817. HPLC purity: ≥98%, RT = 2.75 min.

(4aR,13bS)-10,11-Dichloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, trans-21b-ent-1 and (4aS,13bR)-10,11-Dichloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-21b-ent-2.

70 mg of (±)-trans-21b was separated under the following conditions: Reflect I-Cellulose B5 μm (3,5-diphenylcarbamate), 25 cm × 21.1 mm. 2–15% IPA/hexanes, 1.5 mL injection, 23 mL/min.

(4aR,13bS)-10,11-Dichloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-21b-ent-1.

Absolute stereochemistry arbitrarily assigned. Twenty-eight mg isolated from the chiral separation (>99% ee, RT = 23.63 min). ¹H NMR (400 MHz, CDCl₃) δ 8.26 (s, 1H), 7.73 (s, 1H), 4.12 (t, J = 6.1 Hz, 2H), 2.97 (d, J = 11.4 Hz, 1H), 2.72–2.63 (m, 2H), 2.46–2.32 (m, 4H), 2.10 (td, J = 12.0, 3.1 Hz, 1H), 1.97 (td, J = 10.0, 4.8 Hz, 1H), 1.90–1.70 (m, 3H), 1.43 (qd, J = 13.2, 12.6, 3.8 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 157.3, 146.7, 138.6, 130.6, 128.8, 127.9, 119.8, 62.2, 56.9, 44.9, 42.7, 40.6, 27.1, 26.5, 25.1. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₈Cl₂N₃O, 338.0821. Found 338.0817. HPLC purity: ≥98%, RT = 2.75 min.

(4aS,13bR)-10,11-Dichloro-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-21b-ent-2.

Absolute stereochemistry arbitrarily assigned. Nineteen mg isolated from the chiral separation (>99% ee, RT = 27.42 min) ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.74 (s, 1H), 4.13 (t, J = 6.4 Hz, 2H), 2.98 (d, J = 11.6 Hz, 1H), 2.76–2.64 (m, 2H), 2.50–2.35 (m, 4H), 2.12 (td, J = 11.9, 3.2 Hz, 1H), 1.99 (td, J = 10.0, 4.9 Hz, 1H), 1.92–1.74 (m, 3H), 1.44 (qd, J = 12.9, 4.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 157.2, 146.8, 138.7, 130.7, 128.8, 127.9, 119.8, 62.3, 56.9, 44.8, 42.7, 40.5, 27.1, 26.5, 25.0. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₈Cl₂N₃O, 338.0821. Found 338.0817. HPLC purity: ≥98%, RT = 2.75 min.

(±)-(4aR,13bR)-10-Chloro-4-methyl-11-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-22a and (±)-(4aR,13bS)-10-Chloro-4-methyl-11-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-22b.

Step 1: Synthesis of tert-Butyl (4-(6-Chloro-3-neopentyl-4-oxo-7-(trifluoromethyl)-3,4-dihydroquinazolin-2-yl)butyl)(methyl)carbamate (S4).

Compound S4 was synthesized according to general procedure 1 from 2-amino-4-chloro-5-trifluoromethyl-benzoic acid and 2a. Purification by flash chromatography (5–20% X/hexanes, X = 4:1 EtOAc/DCM) delivered S4 as an orange oil (1.34 g, 32%). ¹H NMR (400 MHz, CDCl₃) δ 8.54 (s, 1H), 7.72 (s, 1H), 4.36–3.92 (m, 2H), 3.27 (t, J = 6.9 Hz, 2H), 2.86 (d, J = 18.7 Hz, 5H), 1.77 (d, J = 11.5 Hz, 2H), 1.68–1.58 (m, 2H), 1.45 (s, 9H), 0.99 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 162.0, 156.0, 149.7, 137.4, 129.5, 127.6, 126.4, 124.0, 121.3, 118.7, 115.5, 79.5, 52.7, 35.8, 34.8, 34.3, 28.8, 28.6, 27.4, 24.4. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.11. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₄H₃₄ClF₃N₃O₃, 504.2235. Found 504.2265.

Step 2: Synthesis of 6-Chloro-2-(4-(methylamino)butyl)-3-neopentyl-7-(trifluoromethyl)quinazolin-4(3H)-one bis-Hydrochloride Salt (5d).

Compound 5d was synthesized according to general procedure 2 from S4, which afforded 5d as a white solid (1.01 g, 86%). ¹H NMR (400 MHz, DMSO) δ 9.19 (s, 2H), 8.36 (s, 1H), 7.92 (s, 1H), 4.08 (s, 2H), 2.93 (dt, J = 29.4, 6.4 Hz, 4H), 2.48 (s, 4H), 1.79 (td, J = 10.6, 5.4 Hz, 4H), 0.92 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 162.4, 160.9, 149.3, 135.6, 129.0, 126.9 (q, J = 5.4 Hz), 124.0 (q, J = 31.7 Hz), 121.2, 118.5, 66.4, 52.0, 47.8, 34.3, 32.2, 28.3, 24.8, 23.5. ¹⁹F NMR (376 MHz, DMSO) δ − 61.47. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₆ClF₃N₃O, 404.1711. Found 404.1730.

Step 3: Synthesis of Benzyl (2-(3-(6-Chloro-3-neopentyl-4-oxo-7-(trifluoromethyl)-3,4-dihydroquinazolin-2-yl)-1-methylpiperidin-2-yl)ethyl)carbamate (6d).

Compound 6d was synthesized according to general procedure 3 from 5d. Purification by reverse phase flash chromatography (10–80% MeOH/H₂O) which afforded 6d as a yellow solid (872 mg, 92%). 6d was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-10-Chloro-4-methyl-11-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Cis-22a and (±)-(4aR,13bS)-10-Chloro-4-methyl-11-(trifluoromethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-22b.

Both diastereomers of 22 were synthesized according to general procedure 5 starting from 6d. Purified by flash chromatography: 15–50% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH). (±)-cis-22a and (±)-trans-22b: 411 mg (85%).

(±)-cis-22a, isolated as a yellow solid (197 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.56 (s, 1H), 7.74 (s, 1H), 4.14 (ddd, J = 14.2, 5.8, 3.6 Hz, 1H), 3.98 (ddd, J = 14.1, 11.6, 5.0 Hz, 1H), 3.00 (q, J = 3.6 Hz, 1H), 2.91–2.82 (m, 1H), 2.82–2.74 (m, 1H), 2.54 (dt, J = 5.9, 3.1 Hz, 1H), 2.38 (dtd, J = 14.0, 5.0, 3.5 Hz, 1H), 2.27 (s, 3H), 2.21–2.12 (m, 1H), 1.95 (dddd, J = 14.4, 11.6, 5.8, 2.7 Hz, 1H), 1.58 (dddd, J = 15.4, 13.3, 7.3, 4.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.3, 160.1, 150.3, 137.0, 137.0, 129.6, 127.16 (q, J = 5.6 Hz), 125.84 (q, J = 32.1 Hz), 122.75 (q, J = 273.0 Hz)., 118.3, 57.9, 56.2, 42.9, 42.4, 40.0, 26.8, 25.1, 22.5. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.02. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₆ClF₃N₃O, 404.1711. Found 404.1730. HPLC purity: ≥99%, RT = 2.73 min.

(±)-trans-22b, isolated as a yellow solid (214 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.54 (s, 1H), 7.74 (s, 1H), 4.22–4.12 (m, 1H), 4.15–4.05 (m, 1H), 2.97 (dq, J = 11.7, 2.0 Hz, 1H), 2.69 (tt, J = 10.4, 3.8 Hz, 2H), 2.44 (dq, J = 13.3, 5.4 Hz, 1H), 2.36 (s, 3H), 2.11 (td, J = 12.0, 3.1 Hz, 1H), 1.99 (td, J = 10.0, 4.8 Hz, 1H), 1.93–1.69 (m, 3H), 1.52–1.33 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.8, 159.5, 150.3, 137.3, 129.8, 127.17 (q, J = 5.5 Hz), 126.14 (q, J = 32.3 Hz), 122.65 (q, J = 273.1 Hz), 118.3, 62.1, 56.8, 45.1, 42.7, 40.7, 27.1, 26.5, 25.1. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.11. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₁₈ClF₃N₃O, 372.1066. Found 372.1099. HPLC purity: ≥97%, RT = 2.80 min.

(±)-(4aR, 13bR)-10-Chloro-4-methyl-11-phenyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-23a and (±)-(4aR,13bS)-10-Chloro-4-methyl-11-phenyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-23a.

Step 1: Synthesis of tert-Butyl (4-(7-Bromo-6-chloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)butyl)(methyl)carbamate (S5).

Compound S5 was synthesized according to general procedure 1 from 2-amino-4-bromo-5-chloro-benzoic acid and 2a. Purification by flash chromatography (0–5% EtOAc/DCM) delivered S5 as an orange oil (1.14 g, 50%). ¹H NMR (400 MHz, CDCl₃) δ 8.25 (s, 1H), 7.90 (s, 1H), 4.14–3.95 (m, 2H), 3.26 (t, J = 6.9 Hz, 2H), 2.85 (d, J = 10.3 Hz, 5H), 1.79–1.73 (m, 2H), 1.62 (p, J = 6.9 Hz, 2H), 1.44 (d, J = 0.7 Hz, 9H), 0.97 (d, J = 0.7 Hz, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 161.8, 159.4, 155.9, 146.0, 132.3, 132.1, 129.1, 128.0, 120.7, 79.4, 52.6, 47.9, 35.6, 34.8, 34.3, 28.8, 28.6, 27.3, 24.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₃H₃₄BrClN₃O₃, 514.1467. Found 514.1459.

Step 2: Synthesis of 7-Bromo-6-chloro-2-(4-(methylamino)-butyl)-3-neopentylquinazolin-4(3H)-one bis-Hydrochloride Salt (5e).

Compound 5e was synthesized according to general procedure 2 from S5, which afforded 5e as a yellowish white solid (0.61 g, 67%). ¹H NMR (400 MHz, DMSO) δ 9.11 (s, 2H), 8.17 (s, 1H), 8.05 (s, 1H), 6.79 (s, 1H), 4.06 (s, 2H), 2.92 (dt, J = 20.8, 6.5 Hz, 4H), 2.49 (d, J = 1.5 Hz, 3H), 1.77 (dq, J = 14.2, 7.8, 7.4 Hz, 4H), 0.92 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 160.7, 160.0, 145.1, 131.2, 130.9, 128.1, 127.4, 120.4, 51.9, 47.8, 34.2, 33.9, 32.2, 28.3, 24.7, 23.6. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₂₆BrClN₃O, 414.0942. Found 414.0930.

Step 3a: Synthesis of Benzyl (2-(3-(7-Bromo-6-chloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-methylpiperidin-2-yl)-ethyl)carbamate (6e).

Compound 6e was synthesized according to general procedure 3 from 5e. Purification by reverse phase flash chromatography (10–90% MeOH/H₂O) which afforded 6e as a yellowish-white solid (611 mg, 67%). ¹H NMR (400 MHz, CDCl₃) δ 8.26 (d, J = 6.3 Hz, 1H), 8.14 (s, 1H), 7.94 (s, 0H), 7.40 (d, J = 7.3 Hz, 1H), 7.31 (ddt, J = 10.7, 9.1, 2.9 Hz, 4H), 6.13–5.70 (m, 0H), 5.20 (d, J = 12.4 Hz, 1H), 5.11 (d, J = 11.5 Hz, 1H), 5.07–4.93 (m, 1H), 4.39 (s, 1H), 3.80–3.52 (m, 1H), 3.30 (s, 2H), 3.15–3.04 (m, 1H), 2.98–2.90 (m, 1H), 2.85 (s, 1H), 2.83–2.66 (m, 1H), 2.52 (s, 1H), 2.45 (d, J = 13.3 Hz, 1H), 2.41 (s, 2H), 1.94 (d, J = 12.6 Hz, 1H), 1.88–1.68 (m, 2H), 1.67–1.50 (m, 1H), 1.42–1.26 (m, 0H), 0.98 (d, J = 9.1 Hz, 10H). ¹³C NMR (101 MHz, CDCl₃) δ 161.8, 161.6, 161.6, 159.5, 156.4, 156.4, 145.6, 145.4, 137.0, 136.7, 132.7, 132.5, 132.3, 131.9, 129.3, 129.0, 128.5, 128.5, 128.4, 128.2, 128.1, 128.1, 128.0, 120.7, 120.5, 69.5, 66.6, 63.1, 60.4, 56.7, 53.9, 52.4, 51.5, 46.5, 44.6, 42.3, 40.8, 40.5, 34.4, 32.2, 31.8, 31.6, 29.8, 29.4, 28.9, 28.4, 28.2, 23.5, 22.9, 21.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₉H₃₇BrClN₄O₃, 603.1702. Found 603.1734.

Step 3b: Synthesis of Benzyl (2-(3-(6-Chloro-3-neopentyl-4-oxo-7-phenyl-3,4-dihydroquinazolin-2-yl)-1-methylpiperidin-2-yl)-ethyl)carbamate (7).

Compound 7 was synthesized according to general procedure 4 from 6e and phenylboronic acid. Purification by flash chromatography (3–30% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH)) and scavenging afforded 7 as a tan solid (173 mg, 82%). 7 was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-10-Chloro-4-methyl-11-phenyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-23a and (±)-(4aR,13bS)-10-Chloro-4-methyl-11-phenyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-23b.

Both diastereomers of 23 were synthesized according to general procedure 5 starting from 7. Purified by flash chromatography: 10–60% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH). (±)-cis-23a and (±)-trans-23b = 89 mg (81%).

(±)-cis-23a, isolated as a waxy yellow solid (32 mg).

¹H NMR (400 MHz, CDCl₃) δ 8.32 (s, 1H), 7.61 (s, 1H), 7.50–7.39 (m, 5H), 4.09 (q, J = 9.8, 7.7 Hz, 2H), 3.04 (s, 1H), 2.80 (d, J = 10.7 Hz, 2H), 2.62 (t, J = 5.8 Hz, 1H), 2.42–2.18 (m, 5H), 1.99 (q, J = 10.9, 7.2 Hz, 1H), 1.61 (dd, J = 27.4, 12.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.4, 156.7, 146.3, 146.2, 138.6, 130.5, 129.7, 129.5, 128.3, 128.2, 127.3, 120.2, 58.1, 55.8, 42.8, 42.0, 39.9, 26.8, 24.7, 22.4. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₂₃ClN₃O, 380.1524. Found 380.1522. HPLC Purity: ≥97%, RT = 3.07 min.

(±)-trans-23b, isolated as a tan solid (57 mg).

¹H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 7.62 (s, 1H), 7.51–7.40 (m, 4H), 7.44–7.36 (m, 1H), 4.22–4.08 (m, 2H), 3.02–2.92 (m, 1H), 2.75–2.63 (m, 2H), 2.43 (dq, J = 13.4, 5.5 Hz, 1H), 2.37 (s, 3H), 2.11 (td, J = 11.9, 3.2 Hz, 1H), 1.99 (td, J = 10.0, 4.9 Hz, 1H), 1.92−1.81(m, 2H), 1.77 (qt, J = 13.2, 3.3 Hz, 1H), 1.53–1.38 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.9, 156.4, 146.5, 146.2, 138.4, 130.8, 129.8, 129.4, 128.4, 128.2, 127.4, 120.2, 62.4, 56.9, 44.8, 42.7, 40.5, 27.2, 26.6, 25.1. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₂₃ClN₃O, 380.1524. Found 380.1523. HPLC Purity: ≥99%, RT = 3.11 min.

(±)-(4aR,13bR)-10-Chloro-11-methoxy-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-24a and (±)-(4aR,13bS)-10-Chloro-11-methoxy-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-24b.

Step 1: Synthesis of tert-Butyl (4-(6-Chloro-7-methoxy-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)butyl)(methyl) carbamate (S6).

Compound S6 was synthesized according to general procedure 1 from 2-amino-5-chloro-4-methoxybenzoic acid and 2a. Purification by flash chromatography (3–25% X/Hexanes, X = 4:1 EtOAc/DCM) delivered S6 as a yellow oil (4.40 g, 86%). ¹H NMR (400 MHz, CDCl₃) δ 8.18 (s, 1H), 7.02 (s, 1H), 4.18–3.95 (m, 4H), 3.32–3.18 (m, 2H), 2.91–2.78 (m, 5H), 1.89–1.68 (m, 3H), 1.67–1.57 (m, 2H), 1.43 (s, 9H), 0.97 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 161.8, 159.7, 155.9, 147.7, 128.4, 128.2, 122.4, 114.6, 107.8, 100.0, 79.4, 56.6, 52.4, 35.8, 34.8, 34.3, 28.8, 28.6, 27.5, 24.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₄H₃₇ClN₃O₄, 466.2467. Found 466.2469.

Step 2: Synthesis of 6-Chloro-7-methoxy-2-(4-(methylamino)-butyl)-3-neopentylquinazolin-4(3H)-one bis-Hydrochloride Salt (5f).

Compound 5f was synthesized according to general procedure 2 from S6, which afforded 5f as a white solid (3.25 g, 79%). ¹H NMR (400 MHz, DMSO) δ 10.66 (s, 1H), 9.22 (q, J = 6.1 Hz, 2H), 8.07 (s, 1H), 7.42 (s, 1H), 4.09 (s, 2H), 4.02 (s, 3H), 3.04 (t, J = 7.1 Hz, 2H), 2.93 (p, J = 6.9 Hz, 2H), 2.53–2.51 (m, 3H), 1.84 (p, J = 7.3 Hz, 4H), 0.95 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 160.6, 160.0, 159.3, 127.4, 121.5, 113.4, 106.1, 66.4, 57.0, 52.0, 47.6, 34.2, 33.3, 32.2, 28.2, 24.8, 24.0. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₉ClN₃O₂, 366.1943. Found 366.1932.

Step 3: Synthesis of Benzyl (2-(3-(6-Chloro-7-methoxy-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-methylpiperidin-2-yl)-ethyl)carbamate (6f).

Compound 6f was synthesized according to general procedure 3 from 5f Purification by normal phase flash chromatography (3–25% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH)) afforded 6f as a an off-white solid (4.73 g, 82%). 6f was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-10-Chloro-11-methoxy-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-24a) and (±)-(4aR,13bS)-10-Chloro-11-methoxy-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-24b.

Both diastereomers of 24 were synthesized according to general procedure 5 starting from 6f with the following modification: the cyclization step was carried out at 95 °C for 18 h. Purified by flash chromatography: 10–50% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH). (±)-cis-24a and trans-24b: 2.26 g (80%).

(±)-cis-24a, isolated as a light yellow-white solid (0.68 g). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.03 (s, 1H), 4.08 (ddd, J = 14.0, 5.9, 4.3 Hz, 1H), 3.99 (s, 4H), 2.98 (q, J = 3.7 Hz, 1H), 2.77 (dq, J = 11.8, 4.4, 3.2 Hz, 2H), 2.56 (dt, J = 6.2, 3.2 Hz, 1H), 2.33 (dq, J = 14.7, 4.9 Hz, 1H), 2.28 (s, 3H), 2.19 (td, J = 10.5, 3.6 Hz, 1H), 1.94 (dddd, J = 14.1, 10.7, 5.9, 2.8 Hz, 1H), 1.69–1.51 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.2, 159.5, 157.2, 148.3, 127.6, 122.2, 114.3, 107.8, 58.0, 56.6, 55.7, 42.9, 42.1, 39.8, 27.0, 24.6, 22.7. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₂₁ClN₃O₂, 334.1312. Found 334.1313. HPLC Purity: ≥98%, RT = 2.03 min.

(±)-trans-24b was isolated as a light yellow-white solid (1.58 g). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.04 (s, 1H), 4.20–4.06 (m, 2H), 3.99 (s, 3H), 2.96 (dq, J = 11.7, 2.2 Hz, 1H), 2.73–2.62 (m, 2H), 2.41 (dq, J = 13.3, 5.5 Hz, 1H), 2.35 (s, 3H), 2.10 (td, J = 12.0, 3.1 Hz, 1H), 1.97 (td, J = 10.1, 4.8 Hz, 1H), 1.88–1.73 (m, 3H), 1.46 (qdd, J = 12.6, 3.5, 1.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.7, 159.6, 156.8, 148.4, 127.7, 122.5, 114.3, 108.0, 62.4, 56.9, 56.6, 44.9, 42.7, 40.4, 27.4, 26.6, 25.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₂₁ClN₃O₂, 334.1317. Found 334.1313. HPLC Purity: ≥99%, RT = 2.07 min.

(±)-(4aR,13bS)-11-Methoxy-4-methyl-8-oxo-2,3,4,4a,5,6,8,13b-octahydro-1H-[1,6]naphthyridino[5,6-b]quinazoline-10-carbonitrile, (±)-Trans-25b.

Steps 1–4: See Synthesis of (±)-Trans-24b.Step 5: Synthesis of (±)-(4aR,13bS)-11-Methoxy-4-methyl-8-oxo-2,3,4,4a,5,6,8,13b-octahydro-1H-[1,6]naphthyridino[5,6-b]-quinazoline-10-carbonitrile ((±)-Trans-25b).

(±)-trans-24b (1 equiv), Pd₂(dba)₃ (10 mol %), S-Phos (20 mol %), and Zn(CN)₂ (1.2 equiv) were combined in a 2–5 mL MWI tube. The tube was sealed, evacuated, and backfilled twice with Ar (g). To the tube was added DMF/H₂O (99:1, 0.11 M) and the reaction was heated under MWI 120 °C for 1 h. After cooling, the reaction mixture was concentrated and the residue was suspended in MeOH (20 mL) and absorbed to an Agilent 10 g HSCX column. After rinsing the column with an additional 80 mL of MeOH and set aside, the crude material was collected by eluting with 60 mL 7N NH₃/MeOH. After concentration, the residue was purified by flash chromatography (15–60% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-25b as an off-white solid (42.7 mg, 44%).¹H NMR (400 MHz, CDCl₃) δ 8.44 (s, 1H), 7.03 (s, 1H), 4.22–4.03 (m, 2H), 4.01 (s, 3H), 2.98 (dq, J = 12.0, 3.1, 2.4 Hz, 1H), 2.74–2.63 (m, 2H), 2.43 (dq, J = 13.5, 5.3 Hz, 1H), 2.36 (s, 3H), 2.11 (td, J = 12.0, 3.1 Hz, 1H), 1.99 (td, J = 10.2, 4.7 Hz, 1H), 1.92–1.73 (m, 3H), 1.45 (qd, J = 12.7, 3.2 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 164.1, 160.3, 159.4, 152.4, 134.3, 115.7, 114.1, 107.5, 101.8, 62.2, 56.8, 56.7, 45.1, 42.7, 40.6, 27.3, 26.5, 25.1. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₂₁ClN₄O₂, 325.1659. Found 325.1655. HPLC Purity: ≥99%, RT = 2.35 min.

(±)-(4aR,13bR)-10-Chloro-12-methoxy-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-26a and (±)-(4aR,13bS)-10-Chloro-12-methoxy-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-26b.

Step 1: Synthesis of tert-Butyl (4-(6-Chloro-8-methoxy-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)butyl)(methyl)carbamate (S7).

Compound S7 was synthesized according to general procedure 1 from 2-amino-3-methoxy-4-chloro benzoic acid and 2a. Purification by flash chromatography (5–20% X/hexanes, X = 4:1 EtOAc/DCM) delivered S7 as a yellow oil (1.91 g, 83%). ¹H NMR (400 MHz, CDCl₃) δ 7.77 (d, J = 2.2 Hz, 1H), 7.07 (d, J = 2.3 Hz, 1H), 4.40–3.75 (m, 5H), 3.22 (s, 2H), 2.94 (dd, J = 14.2, 6.6 Hz, 2H), 2.81 (s, 3H), 1.71 (d, J = 7.7 Hz, 2H), 1.61 (q, J = 7.2 Hz, 2H), 1.42 (s, 9H), 0.96 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 162.0, 155.9, 154.9, 136.6, 132.2, 122.3, 117.8, 114.8, 79.4, 56.7, 52.7, 52.0, 36.2, 34.8, 34.3, 32.1, 28.7, 28.6, 27.3, 25.3. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₄H₃₇ClN₃O₄, 466.2467. Found 466.2477.

Step 2: Synthesis of 6-Chloro-8-methoxy-2-(4-(methylamino)-butyl)-3-neopentylquinazolin-4(3H)-one bis-Hydrochloride Salt (5g).

Compound 5g was synthesized according to general procedure 2 from S7, which afforded 5g as a white solid (1.36 g, 81%). ¹H NMR (400 MHz, DMSO) δ 9.26–9.16 (m, 2H), 8.17 (s, 2H), 7.57 (d, J = 2.2 Hz, 1H), 7.38 (d, J = 2.2 Hz, 1H), 4.07 (s, 2H), 3.94 (s, 3H), 2.91 (dt, J = 21.8, 6.2 Hz, 4H), 2.47 (m, 2H), 1.76 (p, J = 3.2 Hz, 4H), 0.90 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 160.9, 157.9, 154.7, 135.2, 131.2, 121.6, 116.3, 115.3, 56.7, 51.9, 47.7, 34.3, 34.1, 32.1, 28.3, 24.8, 23.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₉ClN₃O₂, 366.1942. Found 366.1932.

Step 3: Synthesis of Benzyl (2-(3-(6-Chloro-8-methoxy-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-methylpiperidin-2-yl)-ethyl)carbamate (6g).

Compound 6g was synthesized according to general procedure 3 from 5g. Purification by reverse phase flash chromatography (10–90% MeOH/H₂O) which afforded 6g as an off-white solid (754 mg, 85%). 6g was used without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-10-Chloro-12-methoxy-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Cis-26a and (±)-(4aR,13bS)-10-Chloro-12-methoxy-4-methyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-26b.

Both diastereomers of 26 were synthesized according to general procedure 5 starting from 6g. Purified by flash chromatography: 15–50% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH). (±)-cis-26a and (±)-trans-26b = 262 mg (79%).

(±)-cis-26a, isolated as a tan yellowish white solid (58 mg). ¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, J = 2.2 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 4.11 (ddd, J = 14.2, 5.8, 3.9 Hz, 1H), 4.05–3.93 (m, 4H), 3.10 (q, J = 4.2 Hz, 1H), 2.77 (ddt, J = 19.4, 11.2, 3.3 Hz, 2H), 2.57 (p, J = 2.8 Hz, 1H), 2.37 (dq, J = 14.5, 4.8 Hz, 1H), 2.29 (s, 3H), 2.20 (dt, J = 11.4, 6.8 Hz, 1H), 1.91 (dddd, J = 13.8, 11.0, 5.7, 2.2 Hz, 1H), 1.72–1.62 (m, 1H), 1.57 (dq, J = 10.0, 5.4, 4.6 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 161.4, 155.9, 155.0, 137.4, 131.8, 121.9, 117.2, 114.8, 57.7, 56.8, 55.4, 42.9, 42.3, 40.2, 27.5, 24.1, 22.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₂₁ClN₃O₂, 334.1317. Found 334.1326. HPLC purity: ≥99%, RT = 2.06 min.

(±)-trans-26b, isolated as a yellowish white solid (204 mg). ¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, J = 2.2 Hz, 1H), 7.07 (d, J = 2.3 Hz, 1H), 4.28 (ddd, J = 14.6, 6.0, 4.2 Hz, 1H), 3.98 (s, 4H), 2.96 (ddt, J = 11.6, 3.9, 1.9 Hz, 1H), 2.81 (ddt, J = 13.2, 4.0, 2.4 Hz, 1H), 2.73 (ddd, J = 11.5, 10.5, 3.7 Hz, 1H), 2.46 (ddt, J = 13.8, 5.8, 4.2 Hz, 1H), 2.36 (s, 3H), 2.11 (td, J = 11.9, 3.4 Hz, 1H), 1.98 (td, J = 10.4, 4.1 Hz, 1H), 1.91–1.70 (m, 3H), 1.47 (qd, J = 12.9, 4.7 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 161.0, 155.3, 155.1, 137.3, 132.2, 121.9, 117.3, 114.9, 62.5, 56.9, 56.8, 45.4, 42.7, 41.4, 27.8, 26.4, 25.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₂₁ClN₃O₂, 334.1317. Found 334.1332. HPLC purity: ≥99%, RT = 2.02 min.

(±)-(4aR,13bR)-4-Methyl-10-(trifluoromethyl)-3,4,4a,5,6,13b-hexahydro-[1,4]oxazino[2′,3′:3,4]pyrido[2,1-b]quinazolin-8(2H)-one, (±)-Cis-29a and (±)-(4aR,13bS)-4-Methyl-10-(trifluoromethyl)-3,4,4a,5,6,13b-hexahydro-[1,4]oxazino[2′,3′:3,4]pyrido[2,1-b]-quinazolin-8(2H)-one, (±)-Trans-29b.

Step 1: Synthesis of tert-Butyl Methyl(2-((3-neopentyl-4-oxo-6-(trifluoromethyl)-3,4-dihydroquinazolin-2-yl)methoxy)ethyl)carbamate (S8).

Compound S8 was synthesized according to general procedure 1 from 2-amino-5-trifluoromethyl-benzoic acid and 2b. Purification by flash chromatography (1–10% EtOAc/DCM) delivered S8 as a yellow oil (0.81 g, 39%). ¹H NMR (400 MHz, CDCl₃) δ 8.58–8.52 (m, 1H), 7.92 (dd, J = 8.6, 2.1 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 4.70 (s, 2H), 4.32–4.14 (m, 2H), 3.64 (s, 2H), 3.40 (s, 2H), 2.87 (s, 3H), 1.41 (s, 9H), 1.00 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 162.4, 155.7, 148.7, 130.5, 129.2 (q, J = 32.5 Hz), 128.4, 125.2 (q, J = 3.9 Hz), 122.4, 121.2, 119.7, 79.7, 73.1, 69.6, 52.6, 48.4, 35.8, 34.8, 28.9, 28.5. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.41. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₃H₃₃F₃N₃O₄, 472.2418. Found 472.2425.

Step 2: Synthesis of 2-((2-(Methylamino)ethoxy)methyl)-3-neopentyl-6-(trifluoromethyl)quinazolin-4(3H)-one bis-Hydrochloride Salt (5h).

Compound 5h was synthesized according to general procedure 2 from S8, which afforded 5h as an off-white solid (0.56 g, 78%). ¹H NMR (400 MHz, DMSO) δ 11.77 (s, 1H), 9.39–9.28 (m, 2H), 8.37 (d, J = 2.2 Hz, 1H), 8.16 (dd, J = 8.6, 2.2 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 4.82 (s, 2H), 4.08 (s, 2H), 3.90 (t, J = 5.1 Hz, 2H), 3.13 (p, J = 5.4 Hz, 2H), 2.55 (t, J = 5.4 Hz, 3H), 0.94 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 161.3, 157.1, 147.6, 130.8 (q, J = 3.3 Hz), 128.0, δ 127.36 (q, J = 32.5 Hz), 125.1, 124.0 (q, J = 4.2 Hz), 120.5, 70.7, 66.0, 52.0, 47.5, 34.2, 32.5, 28.3. ¹⁹F NMR (376 MHz, DMSO) δ − 61.53. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₂₆F₃N₃O₂, 372.1893. Found 372.1887.

Step 3: Synthesis of Benzyl (2-(4-Methyl-2-(3-neopentyl-4-oxo-6-(trifluoromethyl)-3,4-dihydroquinazolin-2-yl)-morpholin-3-yl)ethyl)carbamate (6h).

Compound 6h was synthesized according to general procedure 3 from 5h. Purification by reverse phase flash chromatography (10–85% MeOH/H₂O) which afforded 6h as a light yellow solid (527 mg, 84%). 6h was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-4-Methyl-10-(trifluoromethyl)-3,4,4a,5,6,13b-hexahydro-[1,4]oxazino[2′,3′:3,4]pyrido[2,1-b]-quinazolin-8(2H)-one, (±)-Cis-29a and (±)-(4aR,13bS)-4-Methyl-10-(trifluoromethyl)-3,4,4a,5,6,13b-hexahydro-[1,4]oxazino-[2′,3′:3,4]pyrido[2,1-b]quinazolin-8(2H)-one, (±)-Trans-29b.

Both diastereomers of 5x were synthesized according to general procedure 5 starting from 6h. Purified by flash chromatography: 15–50% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH). (±)-cis-29a + (±)-trans-29b: 180 mg (79%).

(±)-cis-29a, isolated as a brownish yellow solid (52 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.55 (d, J = 2.0 Hz, 1H), 7.96–7.86 (m, 2H), 4.71 (d, J = 3.7 Hz, 1H), 4.15 (ddd, J = 14.1, 5.6, 3.9 Hz, 1H), 3.98 (ddd, J = 14.0, 11.1, 4.7 Hz, 1H), 3.91–3.75 (m, 2H), 2.81 (dt, J = 6.2, 3.0 Hz, 1H), 2.66 (dt, J = 11.8, 3.0 Hz, 1H), 2.55–2.41 (m, 2H), 2.34 (s, 3H), 1.97 (dddd, J = 14.4, 11.2, 5.6, 2.6 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 161.5, 155.7, 149.5, 130.5 (q, J = 3.3 Hz), 128.5, 125.2, 124.6 (q, J = 4.2 Hz), 122.5, 120.4, 73.8, 63.6, 56.9, 53.5, 42.4, 39.6, 21.8. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.4. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₇F₃N₃O₂, 340.1267. Found 340.1267. HPLC purity: ≥99%, RT = 2.19 min.

(±)-trans-29b, isolated as a yellow solid (128 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.54 (dd, J = 2.0, 1.0 Hz, 1H), 7.96–7.88 (m, 2H), 4.35 (d, J = 10.1 Hz, 1H), 4.28–4.08 (m, 3H), 3.97 (td, J = 11.7, 2.5 Hz, 1H), 2.80 (ddd, J = 12.0, 2.5, 1.3 Hz, 1H), 2.55–2.40 (m, 2H), 2.38 (s, 3H), 2.27 (td, J = 10.1, 5.2 Hz, 1H), 1.88 (dddd, J = 13.7, 9.9, 8.8, 6.7 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.9, 154.3, 149.4, 130.6 (q, J = 3.3 Hz), 128.8, 125.1, 124.7 (q, J = 4.1 Hz), 122.4, 120.4, 76.7, 67.8, 60.7, 54.9, 42.2, 40.1, 23.7. ¹⁹F NMR (376 MHz, CDCl₃) δ − 62.4. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₇F₃N₃O₂, 340.1267. Found 340.1270. HPLC purity: ≥99%, RT = 2.02 min.

(±)-(4aR,13bR)-10-Chloro-4-methyl-3,4,4a,5,6,13b-hexahydro-[1,4]oxazino[2′,3′:3,4]pyrido[2,1-b]quinazolin-8(2H)-one, (±)-Cis-30a and (±)-(4aR,13bS)-10-Chloro-4-methyl-3,4,4a,5,6,13b-hexahydro-[1,4]oxazino[2′,3′:3,4]pyrido[2,1-b]quinazolin-8(2H)-one, (±)-Trans-30b.

Step 1: Synthesis of tert-Butyl (2-((6-Chloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)methoxy)ethyl)-(methyl)carbamate (S9).

Compound S9 was synthesized according to general procedure 1 from 2-amino-5-chloro-benzoic acid and 2b. Purification by flash chromatography (1–20% EtOAc/DCM) delivered S9 as a slight yellow oil (548.3 mg, 41%). ¹H NMR (400 MHz, Chloroform-d) δ 8.22 (d, J = 2.3 Hz, 1H), 7.67 (dd, J = 8.7, 2.4 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 4.67 (s, 2H), 4.21 (s, 2H), 3.63 (s, 2H), 3.40 (s, 2H), 2.87 (s, 3H), 1.42 (s, 9H), 0.99 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 162.2, 153.8, 145.1, 134.8, 133.1, 129.1, 126.6, 122.4, 79.7, 73.1, 53.6, 52.5, 34.8, 29.0, 28.6, 27.3. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₃₃ClN₃O₄: 438.2154; Found: 438.2148.

Step 2: Synthesis of 6-Chloro-2-((2-(methylamino)ethoxy)-methyl)-3-neopentylquinazolin-4(3H)-one bis Hydrochloride Salt (5i).

Compound 5i was synthesized according to general procedure 2 from S9, which afforded 5i as a white solid (392.5 mg, 79%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 2H), 8.09 (d, J = 2.2 Hz, 1H), 7.93–7.86 (m, 2H), 6.48 (s, 1H), 4.78 (s, 2H), 4.06 (s, 2H), 3.87 (t, J = 5.0 Hz, 2H), 3.12 (t, J = 5.6 Hz, 2H), 2.55 (t, J = 5.4 Hz, 3H), 0.94 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 160.8, 155.1, 143.7, 134.9, 131.8, 128.6, 125.5, 121.7, 70.7, 65.9, 51.9, 47.5, 34.2, 32.5, 28.3. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₂₄ClN₃O₂: 338.1630; Found: 338.1629.

Step 3: Synthesis of Benzyl (2-(2-(6-Chloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)-4-methylmorpholin-3-yl)ethyl) Carbamate (6i).

Compound 6i was synthesized according to general procedure 3 from 5i. Purification by reverse phase flash chromatography (10–75% MeOH/H₂O) which afforded 6i as a white solid (356 mg, 74%). 6i was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(4aR,13bR)-10-Chloro-4-methyl-3,4,4a,5,6,13b-hexahydro-[1,4]oxazino[2′,3′:3,4]pyrido[2,1-b]-quinazolin-8(2H)-one, (±)-Cis-30a and (±)-(4aR,13bS)-10-Chloro-4-methyl-3,4,4a,5,6,13b-hexahydro-[1,4]oxazino[2′,3′:3,4]pyrido-[2,1-b]quinazolin-8(2H)-one, (±)-Trans-30b.

Both diastereomers of 30 were synthesized according to general procedure 5 starting from 6i. Purified by flash chromatography: 10–60% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH). (±)-cis-30a and (±)-trans-30b: 127.6 mg (62%).

(±)-cis-30a, isolated as an off white solid (38.5 mg). ¹H NMR (400 MHz, Chloroform-d) δ 8.22 (d, J = 2.5 Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.66 (dd, J = 8.7, 2.4 Hz, 1H), 4.68 (d, J = 3.7 Hz, 1H), 4.12 (ddd, J = 14.1, 5.6, 4.2 Hz, 1H), 3.98 (ddd, J = 14.1, 10.9, 4.8 Hz, 1H), 3.90–3.75 (m, 2H), 2.81 (dt, J = 6.2, 3.1 Hz, 1H), 2.66 (dt, J = 11.8, 3.2 Hz, 1H), 2.54–2.37 (m, 2H), 2.34 (s, 3H), 1.95 (dddd, J = 14.8, 10.9, 5.6, 2.7 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 161.2, 153.6, 145.9, 134.8, 132.7, 129.2, 125.9, 121.6, 73.8, 63.6, 56.9, 53.3, 42.4, 39.7, 21.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₅H₁₇N₃O₂Cl⁺: 306.1004; Found: 306.1005. HPLC Purity: ≥98%, RT = 1.88 min.

(±)-trans-30b, isolated as an off white solid (89.1 mg). ¹H NMR (400 MHz, Chloroform-d) δ 8.20 (d, J = 2.4 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.65 (dd, J = 8.7, 2.5 Hz, 1H), 4.32 (d, J = 10.1 Hz, 1H), 4.22 (ddd, J = 11.6, 3.5, 1.3 Hz, 1H), 4.17–4.11 (m, 2H), 3.96 (td, J = 11.7, 2.5 Hz, 1H), 2.79 (ddd, J = 12.0, 2.6, 1.3 Hz, 1H), 2.49–2.41 (m, 2H), 2.37 (s, 3H), 2.24 (td, J = 10.1, 5.2 Hz, 1H), 1.85 (dddd, J = 13.8, 10.1, 8.7, 7.0 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 152.4, 145.7, 134.9, 132.9, 129.5, 126.0, 121.5, 76.7, 67.7, 60.8, 55.0, 42.2, 40.1, 23.7. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₅H₁₇N₃O₂Cl⁺: 306.1004; Found: 306.1003. HPLC purity: ≥98%, RT = 1.64 min.

(±)-(3aR,12bR)-9-Chloro-3-methyl-2,3,3a,4,5,12b-hexahydropyrrolo[3′,2′:3,4]pyrido[2,1-b]quinazolin-7(1H)-one, (±)-Cis-31a.

Step 1: Synthesis of tert-Butyl (3-(6-Chloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)propyl)(methyl)-carbamate (S10).

Compound S10 was synthesized according to general procedure 1 from 5-chloro-2-amino-benzoic acid and 2c. Purification by flash chromatography (5–20% X/hexanes, X = 4:1 EtOAc/DCM) delivered S10 as a yellow oil (4.36 g, 88%). ¹H NMR (400 MHz, CDCl₃) δ 8.18 (d, J = 2.4 Hz, 1H), 7.62 (dd, J = 8.7, 2.4 Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H), 4.06 (s, 2H), 3.36 (t, J = 6.8 Hz, 2H), 2.91–2.80 (m, 5H), 2.01 (p, J = 7.1 Hz, 2H), 1.44 (s, 9H), 0.98 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 162.2, 145.5, 134.6, 132.1, 128.5, 126.5, 121.8, 79.6, 52.6, 48.4, 47.9, 34.8, 34.3, 32.9, 28.8, 28.6, 27.5, 25.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₃₃ClN₃O₃, 422.2205. Found 422.2199.

Step 2: Synthesis of 6-Chloro-2-(3-(methylamino)propyl)-3-neopentylquinazolin-4(3H)-one bis-Hydrochloride Salt (5j).

Compound 5j was synthesized according to general procedure 2 from S10, which afforded 5j as a white solid (3.44 g, 86%). ¹H NMR (400 MHz, DMSO) δ 10.26 (s, 2H), 9.38 (q, J = 6.1 Hz, 2H), 8.03 (d, J = 2.5 Hz, 1H), 7.86 (dd, J = 8.7, 2.5 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 4.08 (s, 2H), 3.12 (t, J = 7.6 Hz, 2H), 3.07–2.96 (m, 2H), 2.50 (t, J = 5.6 Hz, 3H), 2.14 (p, J = 7.4 Hz, 2H), 0.92 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 160.6, 159.1, 142.7, 134.9, 131.2, 127.0, 125.6, 121.1, 52.1, 47.4, 34.3, 32.2, 31.4, 28.2, 23.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₂₅N₃O, 322.1681. Found 322.1673.

Step 3: Synthesis of Benzyl (2-(3-(6-Chloro-3-neopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-methylpyrrolidin-2-yl)ethyl)-carbamate (6j).

Compound 6j was synthesized according to general procedure 3 from 5j. Purification by normal phase flash chromatography (3–15% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH)) afforded 6j as a yellow solid (1.63 g, 63%). 6j was used directly in the next step without analysis.

Step 4: Synthesis of (±)-(3aR,12bR)-9-Chloro-3-methyl-2,3,3a,4,5,12b-hexahydropyrrolo[3′,2′:3,4]pyrido[2,1-b]quinazolin-7(1H)-one, (±)-Cis-31a.

(±)-cis-31a was synthesized according to general procedure 5 starting from 6j with the following modification: instead of heating at 80 °C for 8h, the reaction was heated to 150 °C for 4 h. Purified by flash chromatography: 3–20% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH). (±)-cis-31a was isolated as a yellow solid (186 mg, 55%). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J = 2.4 Hz, 1H), 7.62 (dd, J = 8.7, 2.4 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H), 4.50 (dddd, J = 13.7, 4.9, 3.5, 1.1 Hz, 1H), 3.87 (ddd, J = 13.8, 11.1, 2.8 Hz, 1H), 3.54 (q, J = 9.1 Hz, 1H), 3.11–3.06 (m, 1H), 2.76 (dt, J = 8.6, 3.8 Hz, 1H), 2.42–2.32 (m, 5H), 2.04–1.90 (m, 2H), 1.79 (tt, J = 10.6, 4.0 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.9, 157.7, 146.3, 134.6, 131.9, 128.5, 126.2, 121.2, 61.7, 55.9, 44.6, 40.1, 37.3, 31.8, 27.0. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₅H₁₇N₃O, 290.1040. Found 290.1047. HPLC purity: ≥98%, RT = 1.60 min.

(±)-(4aR,13bS)-10,11-Dichloro-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one ((±)-Trans-32b). Steps 1–4: Synthesis of (±)-Trans-21b

Step 5: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-32b.

Compound (±)-trans-32b was synthesized according to general procedure 6 from (±)-trans-21b. Purification by flash chromatography: 40–75% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-32b as a light-yellow solid (134 mg, 70%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.74 (s, 1H), 4.18 (ddd, J = 14.4, 6.3, 4.7 Hz, 1H), 4.08 (ddd, J = 14.7, 9.5, 5.9 Hz, 1H), 3.15 (ddt, J = 11.9, 4.0, 1.9 Hz, 1H), 2.70 (ddt, J = 12.1, 9.1, 4.3 Hz, 3H), 2.47 (td, J = 11.0, 3.6 Hz, 1H), 2.19 (dq, J = 13.6, 5.2 Hz, 1H), 1.85 (ddt, J = 16.8, 9.6, 3.2 Hz, 2H), 1.73–1.57 (m, 2H), 1.47 (qd, J = 13.2, 3.9 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.7, 157.2, 146.7, 138.7, 130.6, 128.8, 127.9, 119.9, 55.3, 46.6, 45.9, 41.2, 29.3, 27.3, 26.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₅H₁₆Cl₂N₃O, 324.0665. Found 324.0672. HPLC purity: ≥99%, RT = 2.71 min.

(±)-(4aR,13bS)-10-Chloro-11-methoxy-1,2,3,4,4a,5,6,13b-octa-hydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one ((±)-Trans-33b).

Steps 1–4:

See synthesis of (±)-Trans-25b

Step 5: Synthesis of (±)-(4aR,13bS)-10-Chloro-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-33b.

Compound (±)-trans-33b was synthesized according to general procedure 6 from (±)-trans-25b. Purification by flash chromatography 55–80% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-33b as a tan solid (583 mg, 61%). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.04 (s, 1H), 4.18 (ddd, J = 14.3, 6.2, 4.8 Hz, 1H), 4.07 (ddd, J = 14.2, 9.6, 5.8 Hz, 1H), 3.99 (s, 3H), 3.15 (ddt, J = 11.9, 3.9, 1.8 Hz, 1H), 2.76–2.65 (m, 3H), 2.47 (td, J = 11.0, 3.6 Hz, 1H), 2.24–2.14 (m, 1H), 1.91–1.72 (m, 3H), 1.64 (qt, J = 12.9, 4.0 Hz, 1H), 1.49 (tdd, J = 13.2, 11.3, 3.8 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.9, 159.6, 156.7, 148.3, 127.7, 122.5, 114.4, 108.0, 56.7, 55.4, 46.6, 45.8, 40.9, 29.4, 27.5, 26.3. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₆H₁₉ClN₃O₂, 320.1160. Found 320.1159. HPLC purity: ≥99%, RT = 2.14 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-isopropyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-34b. Steps 1–5: See synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-isopropyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-34b.

Compound (±)-trans-34b was prepared by suspending (±)-trans-32b (1 equiv), isopropyl iodide (9.25 equiv), and Et₃N (4 equiv) in DMF (0.25 M) and heating to 80 °C for 20 h. Purification by flash chromatography 1–20% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-34b as a light-yellow solid (27 mg, 48%). ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.74 (s, 1H), 4.16–4.10 (m, 2H), 3.40 (q, J = 6.6 Hz, 1H), 3.02–2.97 (m, 1H), 2.72–2.64 (m, 2H), 2.48–2.39 (m, 2H), 2.11–2.05 (m, 1H), 1.93–1.78 (m, 2H), 1.68–1.58 (m, 1H), 1.47–1.37 (m, 1H), 1.17 (d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 157.9, 146.8, 138.6, 130.5, 128.8, 127.9, 119.8, 57.6, 47.2, 45.4, 43.4, 40.6, 27.7, 25.8, 25.3, 21.9, 12.4. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₂₂Cl₂N₃O, 366.1134. Found 366.1152. HPLC purity: ≥99%, RT = 2.78 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-isobutyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-35b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-isobutyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-35b.

Compound (±)-trans-35b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), isobutyl bromide (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 80 °C for 18 h. Purification by flash chromatography 5–25% X/hexanes (X = 4:1 EtOAc/DCM) afforded (±)-trans-35b as a yellowish white solid (40 mg, 50%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.75 (s, 1H), 4.20 (dt, J = 14.7, 7.1 Hz, 1H), 4.04 (dt, J = 14.4, 6.2 Hz, 1H), 3.07 (d, J = 11.4 Hz, 1H), 2.73–2.56 (m, 2H), 2.54–2.33 (m, 2H), 2.15 (q, J = 8.9 Hz, 1H), 2.03–1.87 (m, 2H), 1.75 (d, J = 37.4 Hz, 4H), 1.45 (qd, J = 13.0, 4.0 Hz, 1H), 0.92 (dd, J = 11.0, 6.5 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 157.9, 146.9, 138.6, 130.6, 128.9, 127.9, 119.9, 61.8, 60.7, 53.7, 45.0, 40.3, 27.3, 27.2, 26.9, 24.9, 21.4, 20.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₄Cl₂N₄O, 380.1272. Found 380.1277. HPLC purity: ≥99%, RT = 2.96 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(cyclopropylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-36b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(cyclopropylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-36b.

Compound (±)-trans-36b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), (bromomethyl)-cyclopropane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 80 °C for 18 h. Purification by flash chromatography 0.5–6% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-36b as a light-yellow solid (52.3 mg, 65%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.75 (s, 1H), 4.20 (ddd, J = 14.5, 8.3, 6.2 Hz, 1H), 4.06 (dt, J = 14.4, 6.1 Hz, 1H), 3.30 (ddt, J = 11.9, 4.0, 1.8 Hz, 1H), 2.77 (dd, J = 13.4, 6.1 Hz, 1H), 2.74–2.62 (m, 2H), 2.43 (dq, J = 13.4, 6.0 Hz, 1H), 2.35–2.19 (m, 3H), 1.91–1.68 (m, 3H), 1.46 (tdd, J = 12.9, 11.2, 3.8 Hz, 1H), 0.93–0.82 (m, 1H), 0.68–0.48 (m, 2H), 0.18–0.09 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 157.6, 146.8, 138.6, 130.6, 128.9, 127.9, 119.9, 59.5, 58.3, 53.1, 44.9, 40.3, 27.1, 26.7, 25.0, 7.4, 5.0, 3.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₂Cl₂N₃O, 378.1134. Found 378.1140. HPLC purity: ≥99%, RT = 2.75 min.

(±)-(4aR,13bS)-4-Benzyl-10,11-dichloro-1,2,3,4,4a,5,6,13b-octa-hydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-37b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-4-Benzyl-10,11-dichloro-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-37b.

Compound (±)-trans-37b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), benzyl bromide (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 18 h. Purification by flash chromatography 0.5–8% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-37b as a white solid (74.5 mg, 74%). HPLC Purity: ≥ 99%. ¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 7.76 (s, 1H), 7.33 (d, J = 4.4 Hz, 4H), 7.30–7.24 (m, 1H), 4.29 (ddd, J = 14.4, 8.0, 6.3 Hz, 1H), 4.17–4.04 (m, 2H), 3.24 (d, J = 13.7 Hz, 1H), 2.95 (dq, J = 13.3, 3.5, 2.7 Hz, 1H), 2.77 (td, J = 11.1, 3.7 Hz, 1H), 2.67–2.60 (m, 1H), 2.55 (dq, J = 13.3, 6.1 Hz, 1H), 2.30 (ddd, J = 10.6, 9.1, 5.4 Hz, 1H), 2.05–1.91 (m, 2H), 1.82–1.75 (m, 1H), 1.66 (qt, J = 12.9, 3.7 Hz, 1H), 1.47 (tdd, J = 13.2, 11.5, 4.1 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.7, 146.8, 138.7, 138.6, 130.6, 129.0, 128.9, 128.5, 127.9, 127.2, 119.9, 60.2, 58.0, 53.1, 45.0, 40.1, 27.3, 27.1, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₂₂Cl₂N₃O, 414.1134. Found 414.1136. HPLC purity: ≥99%, RT = 2.92 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(pyridin-2-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-38b.

Steps 1–5:

See synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(pyridin-2-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-38b.

Compound (±)-trans-38b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 2-(chloromethyl)-pyridine hydrochloride (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 18 h. Purification by flash chromatography 3–30% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-38b as an off white solid (72.3 mg, 81%). ¹H NMR (400 MHz, CDCl₃) δ 8.55 (dd, J = 5.0, 1.7 Hz, 1H), 8.27 (s, 1H), 7.73 (s, 1H), 7.65 (td, J = 7.7, 1.8 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.20–7.12 (m, 1H), 4.25–4.03 (m, 3H), 3.56 (d, J = 14.4 Hz, 1H), 2.95 (dt, J = 11.6, 3.4 Hz, 1H), 2.76 (td, J = 11.2, 3.7 Hz, 1H), 2.70–2.54 (m, 2H), 2.37 (td, J = 10.0, 5.1 Hz, 1H), 2.18 (td, J = 11.8, 3.2 Hz, 1H), 1.97–1.64 (m, 3H), 1.45 (qd, J = 12.9, 4.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 158.9, 157.5, 149.4, 146.8, 138.6, 136.5, 130.6, 128.8, 127.9, 123.3, 122.2, 119.8, 60.2, 59.8, 53.7, 45.0, 40.4, 27.1, 27.1, 24.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₁Cl₂N₄O, 415.1087. Found 415.1090. HPLC purity: ≥99%, RT = 2.96 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(pyridin-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-39b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(pyridin-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-39b.

Compound (±)-trans-39b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 3-(bromomethyl)-pyridine hydrobromide (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 20 h. Purification by flash chromatography 5–40% X/Hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-39b as a tan solid (28.1 mg, 31%). ¹H NMR (400 MHz, CDCl₃) δ 8.57 (d, J = 2.2 Hz, 1H), 8.52 (dd, J = 4.8, 1.6 Hz, 1H), 8.30 (s, 1H), 7.76 (s, 1H), 7.68 (dt, J = 7.8, 2.0 Hz, 1H), 7.27 (dd, J = 7.8, 4.8 Hz, 1H), 4.32–4.24 (m, 1H), 4.17–4.05 (m, 2H), 3.22 (d, J = 14.0 Hz, 1H), 2.89 (dt, J = 11.6, 3.5 Hz, 1H), 2.76 (td, J = 11.1, 3.7 Hz, 1H), 2.68–2.61 (m, 1H), 2.58–2.48 (m, 1H), 2.33 (ddd, J = 10.7, 9.1, 5.4 Hz, 1H), 2.07–1.90 (m, 2H), 1.79 (dt, J = 13.3, 3.4 Hz, 1H), 1.66 (dddd, J = 16.7, 13.0, 8.4, 3.8 Hz, 1H), 1.48 (qd, J = 13.1, 3.9 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.4, 150.3, 148.8, 146.8, 138.7, 136.4, 134.2, 130.7, 128.9, 127.9, 123.5, 119.9, 60.4, 55.3, 53.2, 44.9, 40.0, 27.3, 27.0, 24.7. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₁Cl₂N₄O, 415.1087. Found 415.1100. HPLC purity: ≥99%, RT = 2.90 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(pyridin-4-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-40b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(pyridin-4-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one ((±)-Trans-40b).

Compound (±)-trans-40b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 2–4-(bromomethyl)-pyridine hydrobromide (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 20 h. Purification by flash chromatography 5–30% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-40b as a tan solid (38 mg, 42%). ¹H NMR (400 MHz, CDCl₃) δ 8.57 (d, J = 5.1 Hz, 2H), 8.30 (s, 1H), 7.77 (s, 1H), 7.35–7.30 (m, 2H), 4.25 (ddd, J = 14.5, 8.3, 6.2 Hz, 1H), 4.15–4.06 (m, 2H), 3.22 (d, J = 14.7 Hz, 1H), 2.89 (dq, J = 11.7, 2.1 Hz, 1H), 2.81–2.75 (m, 1H), 2.71–2.65 (m, 1H), 2.44 (dt, J = 13.2, 5.8 Hz, 1H), 2.35 (ddd, J = 10.6, 9.0, 5.3 Hz, 1H), 2.07 (td, J = 11.9, 2.9 Hz, 1H), 1.94–1.86 (m, 1H), 1.85–1.78 (m, 1H), 1.70 (qt, J = 13.0, 3.7 Hz, 1H), 1.50 (tdd, J = 13.2, 11.5, 4.1 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.3, 150.0, 148.6, 146.8, 138.7, 130.7, 128.9, 127.9, 123.6, 119.9, 60.5, 57.0, 53.8, 45.0, 40.1, 27.3, 27.0, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₁Cl₂N₄O, 415.1087. Found 415.1071. HPLC purity: ≥ 99%, 3.48 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(pyrazin-2-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-41b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(pyrazin-2-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-41b.

Compound (±)-trans-41b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 2-(bromomethyl)-pyrazine hydrobromide (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 20 h. Purification by flash chromatography 1–25% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-41b as an off-white solid (75.7 mg, 84%).¹H NMR (400 MHz, CDCl₃) δ 8.69 (d, J = 1.5 Hz, 1H), 8.53 (dd, J = 2.6, 1.5 Hz, 1H), 8.47 (d, J = 2.5 Hz, 1H), 8.28 (s, 1H), 7.74 (s, 1H), 4.26–4.07 (m, 3H), 3.64 (d, J = 14.8 Hz, 1H), 2.94 (ddt, J = 11.4, 4.0, 2.0 Hz, 1H), 2.77 (td, J = 11.2, 3.7 Hz, 1H), 2.70–2.57 (m, 2H), 2.40 (ddd, J = 10.7, 9.4, 5.1 Hz, 1H), 2.21 (td, J = 11.9, 3.1 Hz, 1H), 1.92 (dtd, J = 13.5, 8.9, 6.3 Hz, 1H), 1.84–1.68 (m, 2H), 1.51–1.39 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 157.3, 154.4, 146.8, 145.2, 144.2, 143.4, 138.7, 130.7, 128.9, 127.9, 119.8, 60.0, 57.3, 53.8, 45.0, 40.3, 27.1, 27.1, 24.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₀Cl₂N₅O, 416.1039. Found 416.1040. HPLC purity: ≥99%, RT = 2.97 min.

(±)-(4aR,13bS)-4-Benzoyl-10,11-dichloro-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-42b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-4-Benzoyl-10,11-dichloro-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one ((±)-Trans-42b).

Compound (±)-trans-34b was prepared according to general procedure 8 from (±)-trans-32b (1 equiv), benzoyl chloride (2.6 equiv), Et₃N (2 equiv), and DMAP (0.9 equiv) in DCM (0.25 M) with stirring at rt for 48 h. Purification by flash chromatography (5–55% EtOAc/hexanes) afforded (±)-trans-42b as a white solid (44.5 mg, 48%). ¹H NMR (400 MHz, chloroform-d) δ 8.33 (s, 1H), 7.78 (s, 1H), 7.56–7.41 (m, 5H), 4.70 (dt, J = 13.9, 5.3 Hz, 1H), 4.24 (ddd, J = 13.8, 9.0, 4.5 Hz, 1H), 3.84 (dt, J = 13.7, 4.2 Hz, 1H), 3.48 (ddd, J = 11.1, 8.1, 6.0 Hz, 1H), 3.25 (ddd, J = 13.7, 10.8, 2.8 Hz, 2H), 2.71−2.53 (m, 2H), 2.36 (dtd, J = 14.0, 8.6, 5.1 Hz, 1H), 1.87–1.71 (m, 2H), 1.64–1.54 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 173.9, 160.1, 157.2, 146.7, 138.7, 136.3, 130.8, 130.8, 128.9, 128.8, 128.2, 128.0, 120.1, 58.1, 50.6, 40.7, 40.4, 26.6, 26.3, 25.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₂₀Cl₂N₃O₂⁺: 428.0927; Found: 428.0920. HPLC purity: ≥99%, RT = 3.93 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-pivaloyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-43b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-pivaloyl-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-43b.

Compound (±)-trans-43b was prepared according to general procedure 8 from (±)-trans-32b (1 equiv), pivaloyl chloride (1.3 equiv) and Et₃N (2 equiv) in DCM (0.25 M). Purification by flash chromatography 5–50% EtOAc/Hexanes afforded (±)-trans-43b as a white solid (77 mg, 87%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.73 (s, 1H), 4.65 (dt, J = 13.9, 5.4 Hz, 1H), 4.09 (ddd, J = 13.9, 8.3, 5.4 Hz, 1H), 3.99–3.88 (m, 1H), 3.47–3.31 (m, 2H), 3.08 (td, J = 11.3, 3.5 Hz, 1H), 2.67–2.55 (m, 1H), 2.28–2.14 (m, 2H), 2.00–1.89 (m, 1H), 1.80–1.64 (m, 2H), 1.29 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 180.2, 160.1, 157.5, 146.6, 138.5, 130.7, 128.8, 128.1, 120.1, 58.2, 46.8, 40.4, 40.1, 39.9, 28.5, 26.8, 25.4, 25.0. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₄Cl₂N₃O₂, 408.1240. Found 408.1052. HPLC purity: ≥ 98%, RT = 3.98 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(tetrahydro-2H-pyran-4-carbonyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-44b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(tetrahydro-2H-pyran-4-carbonyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-44b.

Compound (±)-trans-44b was prepared according to general procedure 8 from (±)-trans-32b (1 equiv), tetrahydro-2H-pyran-4-carbonyl chloride (1.3 equiv), and Et₃N (2 equiv) in DCM (0.25 M) and stirring at room temperature for 24h. Purification by flash chromatography 50–100% EtOAc/hexanes afforded (±)-trans-44b as an off-white solid (78 mg, 90%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.73 (s, 1H), 4.54 (dt, J = 14.1, 6.0 Hz, 1H), 4.12 (dt, J = 13.6, 6.5 Hz, 1H), 4.02 (dtd, J = 11.2, 4.3, 2.3 Hz, 2H), 3.75 (dt, J = 14.2, 4.8 Hz, 1H), 3.52–3.36 (m, 4H), 3.02 (td, J = 11.4, 3.8 Hz, 1H), 2.78–2.63 (m, 2H), 2.35 (q, J = 6.6 Hz, 2H), 2.02–1.85 (m, 3H), 1.78–1.60 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 175.7, 160.1, 156.9, 146.4, 138.5, 130.7, 128.7, 128.0, 119.9, 67.3, 67.2, 57.1, 45.5, 40.7, 40.5, 39.2, 29.6, 29.0, 26.7, 25.3, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₄Cl₂N₃O₃, 436.1189. Found 436.1184. HPLC purity: ≥99%, RT = 3.65 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(morpholine-4-carbonyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-45b).

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)- (4aR,13bS)-10,11-Dichloro-4-(morpholine-4-carbonyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-45b.

Compound (±)-trans-45b was prepared according to general procedure 8 from (±)-trans-32b (1 equiv), morpholine-4-carbonyl chloride (2.6 equiv), and DMAP (0.25 equiv) in DCM (0.25 M) and heating to 40 °C for 16 h. Purification by flash chromatography 5–40% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-45b as an off-white solid (87 mg, 93%).). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.74 (s, 1H), 4.49 (dt, J = 14.0, 5.9 Hz, 1H), 4.02 (ddd, J = 13.8, 8.3, 5.1 Hz, 1H), 3.68–3.65 (m, 4H), 3.47 (q, J = 4.5 Hz, 3H), 3.39–3.33 (m, 1H), 3.27–3.24 (m, 1H), 3.12–2.98 (m, 2H), 2.82 (td, J = 12.5, 2.8 Hz, 1H), 2.68−2.59 (m, 1H), 2.37 (dddd, J = 13.9, 8.2, 6.9, 5.6 Hz, 1H), 1.93–1.82 (m, 2H), 1.71–1.57 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 163.5, 160.1, 157.4, 146.6, 138.5, 130.5, 128.7, 127.9, 119.9, 66.8, 66.6, 56.4, 51.6, 47.2, 45.8, 41.9, 40.0, 27.3, 26.7, 25.0. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₃Cl₂N₄O₃, 437.1120. Found 437.1132. HPLC purity: ≥ 99%, RT = 3.80 min.

(±)- (4aR,13bS)-10,11-Dichloro-4-(dimethylglycyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-46b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)- (4aR,13bS)-10,11-Dichloro-4-(dimethyl-glycyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-46b.

Compound (±)-trans-46b was prepared according to general procedure 8 from (±)-trans-32b (1 equiv), dimethylglycinoyl chloride (1.3 equiv), and Et₃N (2 equiv) in DCM (0.25 M) and stirring at rt for 23 h. Purification by flash chromatography 5–50% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-46b as an off-white solid (73 mg, 83%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.72 (s, 1H), 4.50 (dt, J = 14.2, 6.1 Hz, 1H), 4.12 (ddd, J = 14.1, 7.3, 5.6 Hz, 1H), 3.81 (dt, J = 14.3, 5.4 Hz, 1H), 3.45 (ddt, J = 21.9, 7.7, 6.0 Hz, 2H), 3.17–2.95 (m, 3H), 2.65 (ddt, J = 8.8, 5.8, 3.2 Hz, 1H), 2.41 (dq, J = 7.4, 6.1 Hz, 2H), 2.29 (s, 6H), 2.03–1.91 (m, 1H), 1.75–1.63 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 171.4, 160.2, 157.1, 146.5, 138.6, 130.8, 128.8, 128.1, 120.0, 63.7, 57.3, 45.8, 45.6, 41.1, 40.7, 26.6, 24.9, 24.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₃Cl₂N₄O₃, 409.1193. Found 409.1188. HPLC purity: ≥97%, RT = 2.94 min.

(±)- 2-((4aR,13bS)-10,11-Dichloro-8-oxo-2,3,5,6,8,13b-hexahydro-1H-[1,6]naphthyridino[5,6-b]quinazolin-4(4aH)-yl)-N,N-dimethylacetamide ((±)-Trans-47b).

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)- 2-((4aR,13bS)-10,11-Dichloro-8-oxo-2,3,5,6,8,13b-hexahydro-1H-[1,6]naphthyridino[5,6-b]quinazolin-4(4aH)-yl)-N,N-dimethylacetamide ((±)-Trans-47b).

Compound (±)-trans-47b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 2-bromo-N,N-dimethylacetamide (1.4 equiv), and Et₃N (2 equiv) in DMF (0.25 M) and heating to 50 °C for 24 h. Purification by flash chromatography 10–50% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-47b as a tan solid (79 mg, 90%). ¹H NMR (400 MHz, CDCl₃) δ 8.26 (s, 1H), 7.72 (s, 1H), 4.22–4.16 (m, 1H), 4.00 (ddd, J = 14.5, 9.8, 5.9 Hz, 1H), 3.63 (d, J = 15.2 Hz, 1H), 3.27 (d, J = 15.2 Hz, 1H), 3.07 (s, 3H), 2.94 (s, 4H), 2.73–2.61 (m, 3H), 2.50 (td, J = 11.9, 3.0 Hz, 1H), 2.45–2.38 (m, 1H), 1.86–1.67 (m, 3H), 1.44 (tdd, J = 13.6, 11.0, 4.5 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 170.2, 160.5, 157.3, 146.8, 138.6, 130.5, 128.8, 127.8, 119.8, 59.4, 55.4, 53.6, 45.4, 40.9, 37.1, 35.7, 27.4, 26.3, 25.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₃Cl₂N₄O₂, 409.1193. Found 409.1221. HPLC purity: ≥99%, RT = 2.75 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(2-hydroxyethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one ((±)-Trans-48b).

Steps 1–5:

See synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(2-hydroxyethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-48b.

Compound (±)-trans-48b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 2-bromoethanol (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 24 h. Purification by flash chromatography 10–60% X/Hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-48b as a yellow solid (54 mg, 68%). ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1H), 7.76 (s, 1H), 4.29–4.14 (m, 1H), 4.07 (dt, J = 14.0, 6.0 Hz, 1H), 3.75 (ddd, J = 12.4, 9.6, 3.7 Hz, 1H), 3.66–3.57 (m, 1H), 3.20–3.06 (m, 2H), 2.74–2.63 (m, 2H), 2.54–2.29 (m, 4H), 2.17 (td, J = 12.2, 2.7 Hz, 1H), 1.93–1.80 (m, 2H), 1.77–1.67 (m, 1H), 1.59–1.43 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.2, 146.7, 138.7, 130.7, 128.9, 127.9, 119.8, 60.3, 58.7, 54.2, 53.0, 45.0, 40.1, 27.2, 27.0, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₇H₂₀Cl₂N₃O₂, 368.0909. Found 368.0930. HPLC purity: ≥99%, RT = 2.75 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(2-methoxyethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-49b.

Steps 1–5:

See Synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(2-methoxyethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-49b.

Compound (±)-trans-49b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 1-bromo-2-methoxyethane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 22 h. Purification by flash chromatography 3–20% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-49b as a yellow solid (62 mg, 75%). ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.74 (s, 1H), 4.20 (ddd, J = 14.4, 8.2, 6.2 Hz, 1H), 4.05 (dt, J = 14.4, 6.2 Hz, 1H), 3.52 (dd, J = 6.2, 5.1 Hz, 2H), 3.35 (s, 3H), 3.13–2.99 (m, 2H), 2.76–2.52 (m, 3H), 2.46 (dq, J = 13.3, 5.9 Hz, 1H), 2.39−2.18 (m, 2H), 1.91–1.69 (m, 3H), 1.50–1.39 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.6, 146.8, 138.6, 130.6, 128.8, 127.9, 119.9, 70.1, 60.0, 59.1, 53.5, 52.9, 44.8, 40.3, 27.0, 26.9, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₂₂Cl₂N₃O₂, 382.1084. Found 382.1070. HPLC purity: ≥99%, RT = 2.94.

(±)-(4aR,13bS)-10,11-dichloro-4-(2-isopropoxyethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one ((±)-trans-50b).

Steps 1–5:

See synthesis of (±)-trans-32b.

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(2-isopropoxyethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino-[5,6-b]quinazolin-8-one, (±)-Trans-50b.

Compound (±)-trans-50b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 2-(2-bromoethoxy)propane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 20 h. Purification by flash chromatography 3–20% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-50b as a tan solid (67.9 mg, 77%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.74 (s, 1H), 4.18–4.08 (m, 2H), 3.65–3.46 (m, 3H), 3.07–3.00 (m, 1H), 2.96 (dt, J = 13.9, 5.8 Hz, 1H), 2.75 (dt, J = 13.5, 6.4 Hz, 1H), 2.70–2.61 (m, 2H), 2.55 (dq, J = 13.3, 5.5 Hz, 1H), 2.38 (dtd, J = 23.7, 12.0, 10.9, 3.9 Hz, 2H), 1.90–1.65 (m, 3H), 1.50–1.38 (m, 1H), 1.15 (dd, J = 6.1, 1.2 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 157.6, 146.8, 138.6, 130.6, 128.8, 127.9, 119.9, 72.1, 65.3, 59.8, 53.5, 53.0, 45.2, 40.7, 27.3, 26.8, 25.1, 22.3, 22.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₆Cl₂N₃O₂, 410.1397. Found 410.1397. HPLC purity: ≥99%, RT = 2.91 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(2-(dimethylamino)ethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-51b.

Steps 1–6:

See Synthesis of (±)-trans-48b

Step 7: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(2-(dimethylamino)ethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-trans-51b.

Compound (±)-trans-51b was prepared according to general procedure 9 by suspending (±)-trans-48b (1 equiv) and Et₃N (2 equiv) in anhydrous DCE (0.25 M) and cooled to 0 °C. At 0 °C, MsCl (1.2 equiv) was added dropwise. Then allowed to warm to room temperature and stir for 2 h. After 2 h, dimethylamine (2 M in THF, 6 equiv) was added dropwise. The reaction was allowed to stir at room temperature for 22 h. Purification by flash chromatography 3–25% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-51b as an orange solid (33.2 mg, 44%).¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.74 (s, 1H), 4.24–4.04 (m, 2H), 3.10 (ddd, J = 12.1, 5.8, 3.4 Hz, 2H), 2.72–2.53 (m, 4H), 2.50–2.41 (m, 2H), 2.38 (s, 6H), 2.27 (td, J = 10.0, 5.1 Hz, 1H), 2.17 (td, J = 12.0, 2.9 Hz, 1H), 1.94–1.80 (m, 2H), 1.72 (qt, J = 13.0, 3.8 Hz, 1H), 1.50–1.37 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.4, 146.8, 138.6, 130.6, 128.8, 128.8, 127.9, 119.8, 60.1, 56.6, 53.5, 51.0, 45.7, 44.9, 40.4, 27.1, 26.9, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₅Cl₂N₄O, 395.1400. Found 395.1415. HPLC Purity: ≥98%, RT = 2.99 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(2-(isopropylamino)ethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-52b.

Steps 1–6:

See Synthesis of (±)-Trans-48b

Step 7: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(2-(isopropylamino)ethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-52b.

Compound (±)-trans-52b was prepared according to procedure 11 by suspending (±)-trans-48b (1 equiv) and Et₃N (2 equiv) in anhydrous DCE (0.25 M) and cooled to 0 °C. At 0 °C, MsCl (1.2 equiv) was added dropwise. Then allowed to warm to rt, stirred for 2 h, and then isopropylamine (6 equiv) was added dropwise. The reaction was allowed to stir at rt for 22 h. Purification by flash chromatography 75–100% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-52b as an orange solid (38.1 mg, 49%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.75 (s, 1H), 4.20 (ddd, J = 14.4, 8.4, 6.3 Hz, 1H), 4.05 (ddd, J = 14.9, 6.8, 5.4 Hz, 1H), 3.12–2.98 (m, 2H), 2.86–2.60 (m, 5H), 2.48–2.40 (m, 1H), 2.34–2.21 (m, 2H), 2.08 (td, J = 12.1, 2.7 Hz, 1H), 1.91–1.77 (m, 3H), 1.70 (ddt, J = 16.6, 12.9, 6.6 Hz, 1H), 1.52–1.40 (m, 1H), 1.09 (dd, J = 6.4, 3.4 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.6, 146.8, 138.6, 130.6, 128.9, 127.9, 119.9, 60.3, 53.0, 52.9, 49.2, 44.9, 44.6, 40.3, 27.1, 24.9, 23.1, 22.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₇Cl₂N₄O, 409.1556. Found 409.1576. HPLC purity: ≥99%, RT = 3.02 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-53b.

Steps 1–5:

See synthesis of (±)-trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-53b.

Compound (±)-trans-53b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 3-(bromomethyl)-oxetane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 80 °C for 19 h. Purification by flash chromatography 3–30% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-53b as a tan solid (60.3 mg, 71%). ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.73 (s, 1H), 4.81 (q, J = 7.7, 7.3 Hz, 2H), 4.44 (t, J = 5.9 Hz, 1H), 4.37 (t, J = 5.8 Hz, 1H), 4.21 (dt, J = 14.6, 7.1 Hz, 1H), 4.07 (dt, J = 14.0, 6.2 Hz, 1H), 3.24 (q, J = 8.2 Hz, 2H), 2.88 (d, J = 11.3 Hz, 1H), 2.64 (d, J = 12.3 Hz, 2H), 2.46 (dq, J = 19.2, 6.3, 5.8 Hz, 2H), 2.21 (s, 1H), 2.01 (t, J = 12.0 Hz, 1H), 1.85 (t, J = 14.3 Hz, 2H), 1.67 (d, J = 13.3 Hz, 1H), 1.49 (d, J = 3.8 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.3, 146.7, 138.7, 130.7, 128.9, 127.9, 119.8, 76.4, 76.4, 60.3, 56.8, 53.0, 45.9, 44.6, 40.1, 33.3, 26.9, 24.7. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₉H₂₂Cl₂N₄O₂, 394.1064. Found 394.1070. HPLC purity: ≥99%, RT = 2.85 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-((tetrahydro-2H-pyran-4-yl)-methyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-54b.

Steps 1–5:

See synthesis of (±)-Trans-32b

Step 6: Synthesis of (±)- (4aR,13bS)-10,11-Dichloro-4-((tetrahydro-2H-pyran-4-yl)methyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-54b.

Compound (±)-trans-54b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 4-bromomethyl-tetrahydropyran (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 80 °C for 20 h. Purification by flash chromatography 1–12% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-54b as a yellowish tan solid (109 mg, 70%).¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.75 (s, 1H), 4.20 (ddd, J = 14.4, 8.3, 6.2 Hz, 1H), 4.10–3.94 (m, 3H), 3.39 (tdd, J = 11.7, 9.2, 2.2 Hz, 2H), 3.10–3.04 (m, 1H), 2.67–2.57 (m, 3H), 2.38 (dq, J = 13.4, 5.9 Hz, 1H), 2.17 (td, J = 9.8, 5.2 Hz, 1H), 2.00 (ddd, J = 12.5, 10.5, 7.0 Hz, 2H), 1.84–1.56 (m, 6H), 1.50–1.39 (m, 1H), 1.32–1.18 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 157.7, 146.8, 138.6, 130.6, 128.9, 127.9, 119.9, 68.1, 68.0, 60.6, 59.8, 53.9, 45.0, 40.3, 34.0, 32.0, 32.0, 27.3, 27.1, 24.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₆Cl₂N₃O, 422.1397. Found 422.1418. HPLC Purity: ≥98%, RT = 2.76 min.

(±)-(4aR,13bS)-10,11-Dichloro-4-(2-morpholinoethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-55b.

Steps 1–5:

See synthesis of (±)-trans-32b

Step 6: Synthesis of (±)-(4aR,13bS)-10,11-Dichloro-4-(2-morpholinoethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino-[5,6-b]quinazolin-8-one ((±)-Trans-55b).

Compound (±)-trans-55b was prepared according to general procedure 7 by suspending (±)-trans-32b (1 equiv), 4-(2-chloroethyl)morpholine hydrochloride (8 equiv), and Et₃N (12 equiv) in DMF/MeOH (1.7:1 DMF:MeOH, 0.08 M) and heating to 80 °C for 24 h. Purification by flash chromatography 30–80% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-55b as a tan solid (55.8 mg, 59%). ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.74 (s, 1H), 4.19 (ddd, J = 14.5, 8.4, 6.1 Hz, 1H), 4.07 (dt, J = 14.4, 6.0 Hz, 1H), 3.70 (t, J = 4.7 Hz, 4H), 3.10–3.04 (m, 1H), 3.02–2.91 (m, 1H), 2.65 (ddt, J = 12.0, 9.5, 3.6 Hz, 2H), 2.55–2.41 (m, 8H), 2.28 (td, J = 10.5, 5.2 Hz, 1H), 2.21 (td, J = 12.0, 2.9 Hz, 1H), 1.89–1.80 (m, 2H), 1.70 (qt, J = 13.1, 3.8 Hz, 1H), 1.45 (tdd, J = 14.0, 10.8, 6.6 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 157.4, 146.8, 138.7, 130.6, 128.9, 127.9, 119.8, 67.0, 59.9, 56.3, 54.4, 53.6, 50.6, 45.0, 40.3, 27.1, 26.9, 24.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₇Cl₂N₄O₂, 437.1506. Found 437.1509. HPLC Purity: ≥98%, RT = 2.85 min.

(±)-(4aR,13bS)-10-Chloro-4-(2-hydroxyethyl)-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-56b.

Steps 1–5:

See Synthesis of (±)-Trans-33b

Step 6: Synthesis of (±)-(4aR,13bS)-10-Chloro-4-(2-hydroxyethyl)-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-56b.

Compound (±)-trans-56b was prepared according to general procedure 7 by suspending (±)-trans-33b (1 equiv), 2-bromoethanol (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 24 h. Purification by flash chromatography 0.5–10% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-56b as a tan solid (484 mg, 73%). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.04 (s, 1H), 4.20 (ddd, J = 14.3, 8.2, 5.9 Hz, 1H), 4.05 (dt, J = 14.4, 5.9 Hz, 1H), 3.99 (s, 3H), 3.73 (ddd, J = 10.9, 9.2, 3.8 Hz, 1H), 3.59 (dt, J = 10.9, 4.4 Hz, 1H), 3.12 (ddt, J = 18.2, 9.2, 4.2 Hz, 2H), 2.71–2.60 (m, 3H), 2.46–2.33 (m, 2H), 2.29 (dt, J = 13.2, 3.7 Hz, 1H), 2.15 (td, J = 12.1, 2.8 Hz, 1H), 1.92–1.77 (m, 2H), 1.70 (qt, J = 13.0, 3.7 Hz, 1H), 1.51 (qd, J = 12.7, 3.5 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 159.6, 156.8, 148.4, 127.7, 122.5, 114.3, 108.1, 60.4, 58.7, 56.7, 54.1, 53.0, 45.0, 39.9, 27.2, 27.2, 24.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₈H₂₃ClN₃O₃, 364.1422. Found 364.1414. HPLC Purity: ≥97%, RT = 2.09 min.

(±)-(4aR,13bS)-10-Chloro-11-methoxy-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-57b.

Steps 1–5:

See synthesis of (±)-trans-33b

Step 6: Synthesis of (±)-(4aR,13bS)-10-Chloro-11-methoxy-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-57b.

Compound (±)-trans-57b was prepared according to general procedure 7 by suspending (±)-trans-33b (1 equiv), 3-(bromomethyl)-oxetane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 80 °C for 20 h. Purification by flash chromatography 20–40% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-57b as a white solid (48.1 mg, 57%). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.05 (s, 1H), 4.81 (ddd, J = 9.1, 7.4, 6.0 Hz, 2H), 4.44 (t, J = 6.0 Hz, 1H), 4.38 (t, J = 5.9 Hz, 1H), 4.20 (ddd, J = 14.5, 8.4, 6.1 Hz, 1H), 4.08 (dt, J = 14.4, 6.1 Hz, 1H), 3.99 (s, 3H), 3.31–3.19 (m, 2H), 2.88 (dt, J = 11.4, 3.8 Hz, 1H), 2.70–2.59 (m, 2H), 2.53–2.40 (m, 2H), 2.19 (td, J = 9.9, 5.2 Hz, 1H), 2.00 (td, J = 11.9, 2.8 Hz, 1H), 1.88–1.77 (m, 2H), 1.67 (qt, J = 13.0, 3.6 Hz, 1H), 1.47 (qd, J = 13.9, 13.5, 3.5 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 156.9, 148.4, 127.7, 122.5, 114.3, 108.1, 76.5, 76.5, 60.4, 56.8, 56.7, 53.1, 44.7, 40.0, 33.4, 27.2, 27.1, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₅ClN₃O₃, 390.1579. Found 390.1589. HPLC Purity: ≥99%, RT = 2.10 min.

(4aR,13bS)-10-Chloro-11-methoxy-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, Trans-57b-ent-1 and (4SR,13bR)-10-Chloro-11-methoxy-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-57b-ent-2.

500 mg of (±)-trans-57b was separated by Lotus Separations (Princeton University, Princeton, NJ) using chiral supercritical fluid chromatography. Analytical SFC Conditions: Agilent 1260 SFC equipped with a Diacel ChirralCel OJ-H, 250 (L) × 4.6 (ID) mm column. Temperature-Ambient. Mobile Phase CO₂ Modifier: 30% EtOH with 0.1% diethylamine. Flow rate: 2 mL/min. Back Pressure: 100 bar. UV wavelength: 220 nm.

Preparative SFC Conditions.

Berger Multi-Gram-II Preparative-SFC equipped with a Diacel ChiralCel OJ-H, 250 (L) × 20 (ID) mm. Temperature: 35 °C. CO₂ Modifier: 25% EtOH with 0.1% diethylamine. Flow rate: 45 mL/min. Back Pressure: 100 bar. UV wavelength: 220 nm.

(4aR,13bS)-10-Chloro-11-methoxy-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, Trans-57b-ent-1.

235 mg isolated from the chiral separation (>99% ee, RT = 4.68 min). Absolute stereochemistry determined by X-ray crystallography. ¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.05 (s, 1H), 4.81 (ddd, J = 9.1, 7.4, 6.0 Hz, 2H), 4.44 (t, J = 6.0 Hz, 1H), 4.38 (t, J = 5.9 Hz, 1H), 4.20 (ddd, J = 14.5, 8.4, 6.1 Hz, 1H), 4.08 (dt, J = 14.4, 6.1 Hz, 1H), 3.99 (s, 3H), 3.31–3.19 (m, 2H), 2.88 (dt, J = 11.4, 3.8 Hz, 1H), 2.70–2.59 (m, 2H), 2.53–2.40 (m, 2H), 2.19 (td, J = 9.9, 5.2 Hz, 1H), 2.00 (td, J = 11.9, 2.8 Hz, 1H), 1.88–1.77 (m, 2H), 1.67 (qt, J = 13.0, 3.6 Hz, 1H1.46 (qd, J = 14.0, 13.6, 3.7 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 156.9, 148.4, 127.7, 122.5, 114.3, 108.1, 76.5, 76.5, 60.4, 56.8, 56.7, 53.1, 44.7, 40.0, 33.4, 27.2, 27.1, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₅ClN₃O₃, 390.1579. Found 390.1589. HPLC Purity: ≥99%, RT = 2.10 min.

(4SR,13bR)-10-Chloro-11-methoxy-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, Trans-57b-ent-2.

225 mg isolated from the chiral separation (>99% ee, RT = 6.67 min). Absolute stereochemistry determined by X-ray crystallography. ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.04 (s, 1H), 4.81 (ddd, J = 9.1, 7.4, 6.0 Hz, 2H), 4.44 (t, J = 6.0 Hz, 1H), 4.37 (t, J = 5.9 Hz, 1H), 4.20 (ddd, J = 14.5, 8.4, 6.1 Hz, 1H), 4.08 (dt, J = 14.4, 6.1 Hz, 1H), 3.99 (s, 3H), 3.30–3.19 (m, 2H), 2.87 (dt, J = 11.7, 3.6 Hz, 1H), 2.68–2.59 (m, 2H), 2.53–2.39 (m, 2H), 2.19 (td, J = 9.9, 5.2 Hz, 1H), 2.00 (td, J = 11.9, 2.8 Hz, 1H), 1.89–1.77 (m, 2H), 1.66 (qt, J = 13.1, 3.9 Hz, 1H), 1.46 (qd, J = 14.2, 13.7, 3.8 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 159.6, 156.8, 148.4, 127.7, 122.5, 114.3, 108.0, 76.5, 76.5, 60.4, 56.8, 56.7, 53.0, 44.7, 40.0, 33.4, 27.2, 27.1, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₅ClN₃O₃, 390.1579. Found 390.1587. HPLC purity: ≥99%, RT = 2.10 min.

(±)-(4aR,13bS)-11-Methoxy-4-(oxetan-3-ylmethyl)-8-oxo-2,3,4,4a,5,6,8,13b-octahydro-1H-[1,6]naphthyridino[5,6-b]-quinazoline-10-carbonitrile ((±)-Trans-58b).

Steps 1–6:

See synthesis of (±)-Trans-57

Step 7: Synthesis of (±)-(4aR,13bS)-11-Methoxy-4-(oxetan-3-ylmethyl)-8-oxo-2,3,4,4a,5,6,8,13b-octahydro-1H-[1,6]-naphthyridino[5,6-b]quinazoline-10-carbonitrile, (±)-Trans-58b.

Compound (±)-trans-58b was prepared from (±)-trans-57b. (±)-trans-57b (1 equiv), Pd₂(dba)₃ (10 mol %), S-Phos (20 mol %), and Zn(CN)₂ (1.2 equiv) were combined in a 2–5 mL MWI tube which was sealed, evacuated, and backfilled with Ar(g) two times. Then DMF/H₂O (99:1, 0.11 M) was added and the tube was heated MWI 120 °C for 1.5 h. The reaction mixture was concentrated, and the residue was suspended MeOH (20 mL) and absorbed to a 10 g HSCX column (Agilent). After rinsing the column with an additional 80 mL of MeOH, the crude material was then collected by flushing the column with 60 mL 7N NH₃/MeOH. Purification by flash chromatography 0.5–10% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-58b as a yellow-white solid (72.9 mg, 50%). ¹H NMR (400 MHz, CDCl₃) δ 8.43 (s, 1H), 7.03 (s, 1H), 4.81 (dt, J = 9.5, 6.5 Hz, 2H), 4.43 (t, J = 5.8 Hz, 1H), 4.37 (t, J = 5.8 Hz, 1H), 4.18 (ddd, J = 14.5, 8.6, 6.2 Hz, 1H), 4.13–4.05 (m, 1H), 4.01 (s, 3H), 3.29–3.18 (m, 2H), 2.88 (d, J = 11.3 Hz, 1H), 2.72–2.61 (m, 2H), 2.55–2.41 (m, 2H), 2.22 (q, J = 8.9 Hz, 1H), 2.01 (t, J = 11.8 Hz, 1H), 1.91–1.78 (m, 2H), 1.67 (q, J = 13.7 Hz, 1H), 1.46 (qd, J = 15.5, 14.4, 5.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 164.1, 160.1, 159.5, 152.4, 134.3, 115.6, 114.1, 107.5, 101.8, 76.4, 76.4, 60.2, 56.8, 56.7, 53.0, 44.9, 40.1, 33.4, 27.1, 26.9, 24.7. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₅ClN₄O₃, 381.1921. Found 381.1911. HPLC purity: ≥99%, RT = 2.49 min.

(±)-(4aR,13bS)-10-Chloro-4-(cyclobutylmethyl)-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-59b.

Steps 1–5:

See synthesis of (±)-trans-33b.

Step 6: Synthesis of (±)-(4aR,13bS)-10-Chloro-4-(cyclobutyl-methyl)-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-59b.

Compound (±)-trans-59b was prepared according to general procedure 7 by suspending (±)-trans-33b (1 equiv), (bromomethyl)cyclobutane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 80 °C for 20 h. Purification by flash chromatography 0.5–15% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-59b as a yellow-white solid (217 mg, 72%). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.04 (s, 1H), 4.18 (ddd, J = 14.5, 8.4, 6.1 Hz, 1H), 4.06 (dt, J = 14.3, 6.0 Hz, 1H), 3.98 (s, 3H), 3.00 (dt, J = 11.6, 3.6 Hz, 1H), 2.84 (dd, J = 13.1, 7.0 Hz, 1H), 2.70–2.50 (m, 3H), 2.47–2.29 (m, 2H), 2.19 (td, J = 9.9, 5.2 Hz, 1H), 2.06 (qdd, J = 12.8, 11.7, 6.8, 3.5 Hz, 3H), 1.98–1.60 (m, 7H), 1.45 (dd, J = 13.3, 3.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 157.2, 148.4, 127.7, 122.4, 114.3, 108.0, 60.0, 59.8, 56.6, 53.1, 44.8, 40.1, 33.6, 28.7, 28.0, 27.3, 26.9, 25.0, 18.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₇ClN₃O₂, 388.1786. Found 388.1781. HPLC purity: ≥99%, RT = 2.77 min.

(±)-(4aR,13bS)-10-Chloro-4-(cyclopropylmethyl)-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-60b.

Steps 1–5:

See Synthesis of (±)-Trans-33b

Step 6: Synthesis of (±)-(4aR,13bS)-10-Chloro-4-(cyclopropyl-methyl)-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-60b.

Compound (±)-trans-60b was prepared according to general procedure 7 by suspending (±)-trans-33b (1 equiv), (bromomethyl)-cyclopropane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 80 °C for 20 h. Purification by flash chromatography 2–20% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-60b as a yellow solid (358 mg, 70%).¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.05 (s, 1H), 4.19 (ddd, J = 14.5, 8.4, 6.1 Hz, 1H), 4.07 (dt, J = 14.4, 6.1 Hz, 1H), 3.99 (s, 3H), 3.29 (dt, J = 11.9, 2.8 Hz, 1H), 2.77 (dd, J = 13.4, 6.1 Hz, 1H), 2.69 (td, J = 12.3, 11.6, 3.7 Hz, 2H), 2.42 (dq, J = 13.3, 5.9 Hz, 1H), 2.33–2.20 (m, 3H), 1.95–1.67 (m, 3H), 1.48 (qd, J = 13.6, 13.1, 3.8 Hz, 1H), 0.95–0.83 (m, 1H), 0.60–0.47 (m, 2H), 0.16–0.08 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 157.2, 148.4, 127.7, 122.4, 114.3, 108.1, 59.6, 58.4, 56.7, 53.1, 44.9, 40.1, 27.3, 26.8, 25.1, 7.4, 4.9, 3.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₅ClN₃O₂, 374.1611. Found 374.1622. HPLC purity: ≥99%, RT = 2.43 min.

(±)-(4aR,13bS)-10-Chloro-4-(2-isopropoxyethyl)-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-61b.

Steps 1–5:

See synthesis of (±)-Trans-33b

Step 6: Synthesis of (±)-(4aR,13bS)-10-Chloro-4-(2-isopropoxyethyl)-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-61b.

Compound (±)-trans-61b was prepared according to general procedure 7 by suspending (±)-trans-33b (1 equiv), 1-bromo-2-methylpropane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 50 °C for 20 h. Purification by flash chromatography 0.5–10% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-61b as yellow solid (193 mg, 61%).¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.04 (s, 1H), 4.17–4.06 (m, 2H), 3.98 (s, 3H), 3.65–3.45 (m, 3H), 3.07–3.01 (m, 1H), 3.01–2.90 (m, 1H), 2.78–2.60 (m, 3H), 2.53 (dq, J = 13.2, 5.5 Hz, 1H), 2.37 (dtd, J = 19.6, 11.9, 10.9, 3.9 Hz, 2H), 1.89–1.65 (m, 3H), 1.45 (qd, J = 13.0, 4.2 Hz, 1H), 1.14 (dd, J = 6.1, 1.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 160.7, 159.6, 157.0, 148.4, 127.7, 122.4, 114.3, 108.0, 72.0, 65.3, 59.9, 56.6, 53.5, 53.0, 45.1, 40.5, 27.4, 26.8, 25.1, 22.3, 22.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₁H₂₉ClN₃O₃, 406.1892. Found 406.1886. HPLC purity: ≥99%, RT = 3.25 min.

(±)-(4aR,13bS)-10-Chloro-4-isobutyl-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-62b.

Steps 1–5:

See Synthesis of (±)-Trans-33b

Step 6: Synthesis of (±)-(4aR,13bS)-10-Chloro-4-isobutyl-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6b]quinazolin-8-one, (±)-Trans-62b.

Compound (±)-trans-62b was prepared according to general procedure 7 by suspending (±)-trans-33b (1 equiv), 1-bromo-2-methylpropane (4 equiv), and Et₃N (6 equiv) in DMF (0.25 M) and heating to 80 °C for 20 h. Purification by flash chromatography 0.5–10% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-62b as an off-white solid (205 mg, 70%). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.06 (s, 1H), 4.20 (ddd, J = 14.4, 8.3, 6.2 Hz, 1H), 4.05 (dt, J = 14.3, 6.1 Hz, 1H), 4.00 (s, 3H), 3.06 (d, J = 11.5 Hz, 1H), 2.70–2.60 (m, 2H), 2.47 (dd, J = 12.6, 9.2 Hz, 1H), 2.37 (dq, J = 13.4, 5.9 Hz, 1H), 2.15 (td, J = 9.9, 5.3 Hz, 1H), 2.02–1.87 (m, 2H), 1.87–1.61 (m, 4H), 1.47 (qd, J = 13.7, 13.2, 4.3 Hz, 1H), 0.91 (dd, J = 10.9, 6.5 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 157.4, 148.5, 127.8, 122.4, 114.4, 108.1, 61.8, 60.9, 56.7, 53.7, 44.9, 40.1, 27.4, 26.9, 25.0, 21.4, 20.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₇ClN₃O₂, 376.1786. Found 376.1784. HPLC purity: ≥99%, RT = 3.05 min.

(4aR,13bS)-10-Chloro-4-isobutyl-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, trans-62b-ent-1 and (4aS,13bR)-10-chloro-4-isobutyl-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, Trans-62b-ent-2.

100 mg of (±)-trans-62b was separated under the following conditions: (R,R)-Welk-O 1 5 μm Kromasil, 25 cm × 21.1 mm: 5% isocratic gradient B/A (B = 2% IPA/DCM; A = 25% B in hexanes), 1.5 mL injection, 23 mL/min.

(4aR,13bS)-10-Chloro-4-isobutyl-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-62b-ent-1.

Absolute stereochemistry arbitrarily assigned. 42.1 mg isolated from the chiral separation (>99% ee, RT = 19.24 min). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.06 (s, 1H), 4.20 (ddd, J = 14.4, 8.3, 6.2 Hz, 1H), 4.05 (dt, J = 14.3, 6.1 Hz, 1H), 4.00 (s, 3H), 3.06 (d, J = 11.5 Hz, 1H), 2.70–2.60 (m, 2H), 2.47 (dd, J = 12.6, 9.2 Hz, 1H), 2.37 (dq, J = 13.4, 5.9 Hz, 1H), 2.15 (td, J = 9.9, 5.3 Hz, 1H), 2.02–1.87 (m, 2H), 1.87–1.61 (m, 4H), 1.47 (qd, J = 13.7, 13.2, 4.3 Hz, 1H), 0.91 (dd, J = 10.9, 6.5 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 157.4, 148.5, 127.8, 122.4, 114.4, 108.1, 61.8, 60.9, 56.7, 53.7, 44.9, 40.1, 27.4, 26.9, 25.0, 21.4, 20.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₇ClN₃O₂, 376.1786. Found 376.1784. HPLC purity: ≥99%, RT = 3.05 min.

(4aS,13bR)-10-Chloro-4-isobutyl-11-methoxy-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one, Trans-62b-ent-2.

Absolute stereochemistry arbitrarily assigned. 34.6 mg isolated from the chiral separation (>99% ee, RT = 20.86 min). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.06 (s, 1H), 4.20 (ddd, J = 14.4, 8.3, 6.1 Hz, 1H), 4.05 (dt, J = 14.3, 6.1 Hz, 1H), 4.00 (s, 3H), 3.07 (dd, J = 10.9, 4.1 Hz, 1H), 2.71–2.58 (m, 2H), 2.47 (dd, J = 12.6, 9.3 Hz, 1H), 2.37 (dq, J = 13.6, 5.9 Hz, 1H), 2.15 (td, J = 9.8, 5.2 Hz, 1H), 2.04–1.87 (m, 2H), 1.86–1.73 (m, 3H), 1.73–1.61 (m, 1H), 1.55–1.39 (m, 1H), 0.91 (dd, J = 11.0, 6.5 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 157.4, 148.5, 127.8, 122.4, 114.4, 108.1, 61.8, 60.9, 56.7, 53.7, 44.9, 40.1, 27.4, 26.9, 25.0, 21.4, 20.9. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₀H₂₇ClN₃O₂, 376.1786. Found 376.1782. HPLC purity: ≥99%, RT = 3.05 min.

(±)-(4aR,13bS)-10-Chloro-11-methoxy-4-(2-morpholinoethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-63b.

Steps 1–6:

See Synthesis of (±)-Trans-56b

Step 7: Synthesis of (±)-(4aR,13bS)-10-Chloro-11-methoxy-4-(2-morpholinoethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-63b.

Compound (±)-trans-63b was prepared according to procedure 11 by suspending (±)-trans-56b (1 equiv) and Et₃N (2 equiv) in anhydrous DCE (0.25 M) and cooled to 0 °C. At 0 °C, MsCl (1.2 equiv) was added dropwise. The reaction was allowed to warm to rt and stirred for 2 h. Morpholine (8 equiv) was added dropwise and the mixture was stirred at rt for 23h and then heated to reflux for 7 h. Purification by flash chromatography 50–75% X/hexanes (X = 2% NEt₃ 3:1 EtOAc/EtOH) afforded (±)-trans-63b as a tan solid (143 mg, 40%). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.03 (s, 1H), 4.17 (ddd, J = 14.4, 8.4, 6.0 Hz, 1H), 4.07 (dt, J = 14.3, 6.0 Hz, 1H), 3.98 (s, 3H), 3.69 (q, J = 3.9, 3.2 Hz, 4H), 3.08 (dt, J = 11.7, 3.4 Hz, 1H), 3.02–2.92 (m, 1H), 2.70–2.60 (m, 2H), 2.55–2.39 (m, 8H), 2.25 (dtd, J = 27.4, 11.9, 10.8, 4.0 Hz, 2H), 1.89−1.79 (m, 2H), 1.71 (qt, J = 13.1, 3.7 Hz, 1H), 1.46 (qd, J = 13.4, 3.9 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 156.9, 148.4, 127.7, 122.5, 114.3, 108.0, 67.0, 60.1, 56.7, 56.3, 56.2, 54.3, 54.2, 53.6, 50.6, 44.9, 40.1, 27.3, 27.0, 25.0. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₃₀ClN₄O₃, 433.2001. Found 433.1998. HPLC purity: ≥98%, RT = 2.26 min.

(±)-(4aR,13bS)-10-Chloro-11-methoxy-4-(2-(pyrrolidin-1-yl)-ethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-64b.

Steps 1–6:

See synthesis of (±)-Trans-56b

Step 7: Synthesis of (±)-(4aR,13bS)-10-Chloro-11-methoxy-4-(2-(pyrrolidin-1-yl)ethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-64b.

Compound (±)-trans-64b was prepared according to general procedure 9 by suspending (±)-trans-56b (1 equiv) and Et₃N (2 equiv) in anhydrous DCE (0.25 M). After cooling to 0 °C, MsCl (1.2 equiv) was added dropwise and then the reaction was allowed to warm to rt and stirred for 2 h. Pyrrolidine (8 equiv) was added dropwise. The reaction was allowed to stir at rt for 23 h and then heated to reflux for 7 h. Purification by flash chromatography 50–80% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-64b as a yellow-orange solid (24.7 mg, 17%). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.03 (s, 1H), 4.22–4.02 (m, 2H), 3.98 (s, 3H), 3.12–2.97 (m, 2H), 2.73–2.40 (m, 10H), 2.32–2.17 (m, 2H), 1.91–1.63 (m, 7H), 1.46 (qd, J = 13.2, 4.1 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 159.6, 157.0, 148.4, 127.7, 122.4, 114.3, 108.0, 60.1, 60.0, 56.6, 54.7, 53.7, 53.6, 52.3, 44.9, 40.2, 27.3, 27.2, 27.0, 25.0, 23.5. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₃₉ClN₄O₂, 417.2052. Found 417.2053. HPLC purity: ≥99%, RT = 2.36 min.

(±)-(4aR,13bS)-10-Chloro-11-methoxy-4-(pyridin-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]-quinazolin-8-one, (±)-Trans-65b.

Steps 1–5:

See Synthesis of (±)-Trans-56b

Step 6: Synthesis of (±)-(4aR,13bS)-10-Chloro-11-methoxy-4-(pyridin-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]-naphthyridino[5,6-b]quinazolin-8-one, (±)-Trans-65b.

Compound (±)-trans-65b was prepared according to general procedure 7 by suspending (±)-trans-33b (1 equiv), 3-(bromomethyl)pyridine hydrobromide (1.2 equiv), and Et₃N (3 equiv) in DMF (0.25 M) and heating to 50 °C for 24 h. Purification by flash chromatography 20–40% X/hexanes (X = 2% NEt₃, 3:1 EtOAc/EtOH) afforded (±)-trans-65b as an off-white solid (143 mg, 37%). ¹H NMR (400 MHz, CDCl₃) δ 8.58 (d, J = 2.2 Hz, 1H), 8.53 (dd, J = 4.8, 1.6 Hz, 1H), 8.23 (s, 1H), 7.69 (dt, J = 7.9, 2.0 Hz, 1H), 7.32–7.24 (m, 1H), 7.07 (s, 1H), 4.30 (ddd, J = 14.3, 8.1, 6.3 Hz, 1H), 4.20–4.04 (m, 2H), 4.01 (s, 3H), 3.22 (d, J = 13.9 Hz, 1H), 2.90 (dt, J = 11.4, 3.7 Hz, 1H), 2.76 (td, J = 11.1, 3.7 Hz, 1H), 2.72–2.63 (m, 1H), 2.52 (dq, J = 12.3, 6.0 Hz, 1H), 2.33 (ddd, J = 10.7, 9.1, 5.4 Hz, 1H), 2.08–1.87 (m, 2H), 1.86–1.75 (m, 1H), 1.67 (qt, J = 12.8, 3.7 Hz, 1H), 1.52 (qd, J = 13.1, 3.9 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 160.5, 159.6, 156.9, 150.3, 148.8, 148.4, 136.4, 134.3, 127.7, 123.5, 122.5, 114.3, 108.1, 60.5, 56.7, 55.3, 53.2, 44.9, 39.8, 27.4, 27.2, 24.8. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₂H₂₄ClN₄O₂, 411.1561. Found 411.1567. HPLC purity: ≥99%, RT = 3.21 min.

X-ray Crystallography.

CCDC 2370879 and CCDC 2370880 contain the supporting crystallographic data for compounds (4aR,13bS)-57b-ent-1 (BDGR-20236) and (4aS,13bR)-57b-ent-2 (BDGR-20237), respectively. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures. Data collection and refinement statistics are reported in the Supporting Information.

In Vitro Assay Experimental Protocols.

Amoeba Strains.

N. fowleri strain Nf69 (ATCC 30215) trophozoites, which were originally isolated from a patient in Australia in 1969, were chosen to perform dose response evaluations. N. fowleri strain TY (ATCC 30107) trophozoites, isolated from a patient in Virginia, USA in 1969, were used to score compounds for potential geographic differences in strain susceptibility. Both strains were generously donated by Dr. Dennis Kyle at UGA.

Determination of EC₅₀ Values in the N. fowleri Assay.

N. fowleri strain Nf69 (ATCC 30215) trophozoites were cultured axenically as previously described.²⁹ Briefly, amoeba were seeded at 1 × 10⁴ cells/mL in 100 μL of the indicated medium in white TC-treated 96-well plates (Thermo Scientific Nunc Microwell 136101). Cells were seeded into Nelson’s Complete Media (NCM, 0.17% liver infusion broth (BD Difco, Franklin Lakes, NJ), 0.17% glucose, 0.012% sodium chloride, 0.0136% potassium phosphate monobasic, 0.0142% sodium phosphate dibasic, 0.0004% calcium chloride, 0.0002% magnesium sulfate, 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin) at 37 °C in 96-well tissue culture plates. Vehicle or compound at concentrations initially ranging from 20 to 0.375 μM were added to cells, followed by culture for 48 h (37 °C, 5% CO₂). Plates were equilibrated to rt for 15 min and CellTiter Glo reagent was added followed by orbital shaking for 2 min. After an additional incubation (rt, 10 min), luminescence was scored on a BioTek Synergy H1 microplate reader. Luminescence values were used to calculate percent cell growth inhibition (based on controls) for each concentration of compound tested. Averages of triplicates were calculated and fit to dose–response curves for the determination of EC₅₀ values, using Prism 9.0 (GraphPad Software, San Diego, CA). Miltefosine (Sigma-Aldrich) was used a positive control (N. fowleri EC₅₀ = 35.7 ± 9.10 μM). Other controls included media alone, cells and media, and vehicle and media. For very potent compounds, concentration of reagent in the dose–response assays were reduced to provide an appropriate range for EC₅₀ value resolution.

Determination of CC₅₀ Values in the SH-SY5Y Cytotoxicity Assay.

Dry powder compound was dissolved in DMSO to a final concentration of 20 mM. Serial dilutions were performed to provide a starting concentration of 20 μM in the assay. SH-SY5Y cells (human neuroblastoma cell line, ATCC CRL-2266) were plated at 2000 cells per well into a Corning 3765 plate using a ThermoFisher multidrop reagent dispenser in 50 μL of media and allowed to attach overnight at 37 °C. Using an Echo acoustic dispenser (Labcyte), 50 nL of the diluted compound (20, 1 μM or a series dilution to afford a dose response curve) was added in triplicate to the target cell plates. Doxorubicin at the same concentrations as the test compound was used as a positive control. After incubating for 72 h, cell viability was evaluated by adding 25 μL of Cell Titer-Glo reagent (Promega) per well, followed by incubation (10 min), and detection of the luminescent signal using a Pherostar plate reader (BMG). Percent viability was determined by comparing the percent signal reduction as compared to DMSO negative control. Data was analyzed by Collaborative Drug Discovery (CDD) data management software to generate data points and dose response curves.

ADME Experimental Protocols.

Log D Determination.

Compound (2 μL of a 10 mM DMSO stock solution) was transferred into tubes in duplicate, and to each tube was added phosphate buffer (pH 7.4)-1-octanol solution (pH 7.4). After shaking of the tubes for 1 h at rt, contents were centrifuged, and buffer layer samples and 1-octanol layer samples were aliquoted. The samples were diluted with equal volumes of labetalol and tolbutamide in 50% methanol) and analyzed by LC-MS/MS.

Kinetic Solubility Determination.

To the lower chamber of a mini-uniprep vial was added separate dilutions of compound prepared from DMSO stock solutions, followed by the appropriate volume of phosphate buffer (pH 7.4). After shaking for 24 h at rt, the sample was centrifuged for 30 min, and mini-unipreps were compressed to collect filtrates which were analyzed by HPLC.

Liver Microsomal Stability.

To incubation plates containing mouse microsomes (CD-1 mouse, Corning, cat. no. 452413) in phosphate buffer was added acetonitrile-diluted DMSO stock solution of compound with mixing. Samples were with and without NADPH cofactor. Plates were incubated at 37 °C for 60 min while shaking. At incremental time points ranging from 5 to 60 min, samples were quenched using equal concentrations of tolbutamide and labetalol. Samples were centrifuged, diluted with water, and supernatant was sampled for LC-MS/MS analysis. Controls included: testosterone, diclofenac, and propafenone.

Mouse Plasma Stability.

DMSO stock solutions of test compound and controls (enalapril maleate, bisacodyl, and procaine HCl) were prepared. To a 96-well plate containing plasma (CD-1 mouse, EDTA-K2 anticoagulant, minimum of 20 male donors) was added compound or control, and the mixtures were incubated at 37 °C for 0, 10, 30, 60, or 120 min. At the indicated incubation time, tolbutamide and labetalol in acetonitrile were added to precipitate protein. After shaking for 20 min and centrifuging for 20 min, supernatant was added to water, shaken for 10 min, and then samples were analyzed by LCMS using an internal standard.

Plasma Protein Binding.

A 96-well equilibrium dialysis device (HTDialysis LLC, cat. No. 1101) was employed using CD-1 male mouse plasma with EDTA-K2 anticoagulant. Warfarin was used as a positive control. An aliquot of the compound in phosphate buffer was transferred to the donor side of each dialysis well in triplicate, and the dialysis buffer was loaded to the receiver side of the well. The dialysis plate was incubated at 37 °C with 5% CO₂ on a slow shaking platform for 4 h. Samples from each side of the dialysis device were transferred to new 96-well plates and diluted with a solution of tolbutamide, labetalol, and metformin in acetonitrile which contained an internal standard. After mixing and centrifuging samples for 20 min, supernatants were analyzed by LCMS/MS.

BBB Parallel Artificial Membrane Permeation Assay.²⁷

A DMSO stock solution of test compound was diluted in PBS (pH 7.4) and then added to a 96-well donor plate. The positive controls, verapamil, clonidine, hydrocortisone and theophylline were purchased from Sigma-Aldrich. The filter membrane was coated with dodecane-diluted porcine polar brain lipid (PBL, Avanti Polar Lipids, cat no. 141101P), The filter plate was filled with PBS buffer, and then the filter plate was carefully put on the donor plate to form a “sandwich” with test compound solution on the bottom, artificial lipid membrane in the middle, and the acceptor PBS on the top. After incubating at 18 h at rt, the donor plate was removed, and the solution from donor and filter wells were transferred into the vials from which the concentration ratios were determined by HPLC in triplicate.

In Vivo Assay Experimental Protocols.

Ethics Statement.

All procedures were carried out in accordance with the PHS Policy on the Care and Use of Laboratory Animals and in accord with the CU PHS Assurance Number D16–00435 (A3737–01) under the approval of the Clemson University Institutional Animal Care and Use Committee (IACUC). All animal research programs and facilities have full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). Euthanasia by heavy anesthetization followed by bilateral pneumothorax was used based on recommendations from the AVAM Guidelines for the Euthanasia of Animals.

Mouse Pharmacokinetics Protocol.

For single bolus 12 mg/kg dosing, six female CD-1 mice (Charles River, 24.6–28.4 g) were split into two groups of three mice each. The group receiving an oral dose of compound was fasted. Compound was dissolved in 10% NMP/20% cremophor EL/70% water to give a clear solution. Mice administered compound by IV received 11.1 mg/kg while mice administered compound by PO received 10.7 mg/kg. Plasma concentrations were analyzed at 0.5, 2, 4, 8, and 12 h in each group. Brains were collected from mice in each group at 12 h, tissue was homogenized with MeOH/PBS, and the supernatant of processed samples were analyzed by LCMS/MS (ACQUITY UPLC BEH C18 1.7 μm 2.1 mm × 50 mm column), flow rate: 0.55 mL/min, mobile phase A:0.1% NH₃·H₂O and 2 mM NH₄OAc in water/ACN (v/v, 95:5) and mobile phase B: 0.1% NH₃·H₂O and 2 mM NH₄OAc in ACN/water (v/v, 95:5). Plasma LLOQ: 1 ng/mL; brain homogenate LLOQ: 10 ng/g. Verapamil was used as an internal standard. For single bolus 50 mg/kg dosing, three, fasted, female CD-1 mice (Charles River, 27 g) were administered compound by IP injection (43.8 mg compound as 5 mg/mL in 10% DMSO/30% cremophor EL/60% water, homogeneous opaque suspension). Plasma samples were obtained at 2 and 4 h. Brain tissue was analyzed at 4 h. Identical conditions for LCMS/MS analysis of samples were used as described above. Data from each cohort (n = 3) was averaged. Clinical observations were normal during both studies.

Mouse Efficacy Protocol.

Eighteen CD-1 female mice (aged 35–40 days, Charles River) were infected by nasal instillation with 5 × 10⁴ N. fowleri Nf69 strain trophozoites that had been isolated after passage through mice to ensure virulence. Mice were provided unlimited food and water throughout the duration of the experiment. The three groups consisted of equal numbers of rodents. Groups received either vehicle (10% 1-methyl-2-pyrrolidinone (NMP), 20% Cremophor) or test agent delivered by IP injection for 5 days starting 48 h after infection. BDGR-20237 (11 mg/kg) was administered BID (12 h apart), while amphotericin B (5 mg/kg) was injected QD. Rodents were monitored carefully and euthanized when symptoms of infection, including failure to respond to stimuli or seizures, were evident. After 14 days, surviving animals were sacrificed and brains were removed and analyzed to assess cure. Mice were considered cured if amoebae from isolated brains failed to grow in culture after 2 weeks. Median survival of vehicle treated animals was 5.4 days, compared to 6.1 days for BDGR-20237 treated mice. The p value for the analysis was 0.09.

ABBREVIATIONS USED

ADME

absorption-distribution-metabolism-excretion

BBB

blood–brain barrier

BID

twice daily dosing

B/P

brain-to-plasma ratio

CNS

central nervous system

HLM

human liver microsomes

IACUC

Institutional Animal Care and Use Committee

IN

intranasal

IP

intraperitoneal

IV

intravenous

LLOQ

lower limit of quantification

MLM

mouse liver microsomes

MTD

maximum tolerated dose

N. fowleri

Naegleria fowleri

PAM

primary amoebic meningoencephalitis

PAMPA

parallel artificial membrane permeability assay

PO

oral administration

QD

once daily dosing

SAR

structure–activity relationship

SFC

supercritical fluid chromatography

SI

selectivity index

SPR

structure–property relationship

Tx

treatment