Abstract
The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described. This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC50 = 120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED90 values below 1 mg/kg when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism of action, inhibition of translation elongation factor 2 (PfEF2), led to progression of 2 (DDD107498) to preclinical development.
Introduction
Malaria is a devastating disease with over 214 million clinical cases in 2015.(1) In that year alone, the World Health Organization (WHO) estimated 438 000 deaths, mostly among children under five in Sub-Saharan Africa. The current malaria control programs that combine preventive measures with artemisinin combination therapy (ACT) treatment have proven very effective in reducing the malaria burden. Over the past decade, the number of deaths from malaria has fallen by 4% per year, and between 2000 and 2015 the number of clinical cases of malaria has been estimated to have decreased by 40% where the disease is endemic in Africa.(2) However, in recent years, parasite resistance to artemisinin has been detected in a number of countries in Southeast Asia. For example, in areas along the Cambodia–Thailand border, Plasmodium falciparum, the most deadly malaria parasite, has become resistant to most available antimalarial medicines and the spread of multidrug resistance is a major concern.(3)
The malaria drug discovery portfolio has dramatically improved over the past 10 years.(4) However, due to the constant battle against drug resistance and to achieve malaria elimination, new chemotypes with novel mechanisms of action are required. New drugs are needed: (1) that are not cross-resistant to existing drugs; (2) that can be given as a single dose; (3) that prevent transmission (active against the sexual stages of the parasite); (4) that can give chemoprotection (active against liver stages). Recently, we reported the discovery and profile of 2 (DDD107498),(5) a quinoline-4-carboxamide with excellent pharmacokinetic and antimalarial properties, including activity against multiple life-cycle stages of the parasite. Moreover, this compound acts through a novel mechanism of action for antimalarial chemotherapy, inhibition of translation elongation factor 2 (PfEF2), which is critical for protein synthesis.(5) In this paper we describe the medicinal chemistry program that led to the discovery of 2.
The quinoline-4-carboxamide series was identified from a phenotypic screen of the Dundee protein kinase library(6) against the blood stage of the P. falciparum 3D7 strain. The most active compound of the original hit series displayed good activity in vitro against a chloroquine sensitive P. falciparum strain (3D7) and good selectivity index (>100-fold) against a human cell line (MRC-5). However, hit compound 1 had a high clogP and poor aqueous solubility and was metabolically unstable with a high hepatic microsomal intrinsic clearance (Cli). (Figure 1 and Table 1).
Figure 1. Key data for screening hit 1 and preclinical candidate 2. Data reported previously.(5)
Table 1. Optimization the R1 and R2 Moieties
Table a
MLM: mouse liver microsomes.
Table b
Sol: kinetic aqueous solubility. Data for compounds 1, 11, and 19 reported previously.(5)
Results and Discussion
The initial aim of the hit to lead program was to improve potency (Pf EC50(3D7) < 0.1 μM), aqueous solubility (>100 μM), and metabolic stability (mouse liver microsomes Cli < 5 mL min–1 g–1) of compound 1. Iterative rounds of drug design, synthesis, and biological evaluation were driven by the Medicines for Malaria Venture (MMV) compound progression criteria.(7) Initial modifications were directed toward improving the physicochemical properties particularly reducing lipophilicity. The clogP of the hit was 4.3, which is higher than average for oral drugs and may contribute to the poor aqueous solubility and hepatic microsomal instability.(8) Several points for modification on the scaffold were identified that could address the high lipophilicity: the bromine atom (R1) significantly adds to lipophilicity, as do aromatic substituents in the carboxamide (R2) and quinoline (R3) moieties. High numbers of aromatic rings are associated with unfavorable lipophilicity and poor compound developability.(9)
The initial focus was on the R1 and R2 substituents. Quinoline-4-carboxamides 10–19 were prepared in two steps from the corresponding isatin (Scheme 1), employing the Pfitzinger reaction with 1-(p-tolyl)ethanone using potassium hydroxide as a base in a mixture of ethanol and water at 125 °C under microwave irradiation to afford the quinoline-4-carboxylic acid 3.(10) Coupling of 3 with the corresponding amine, using EDC and HOBt in DMF, led to compounds 10–19 (Scheme 1).
Scheme 1. a
aConditions: (a) KOH, EtOH/water, 125 °C, microwave, 20 min, 29–58% yield; (b) amine, EDC, HOBt, DMF, room temperature, 16 h, 22–43% yield.
Replacement of the bromine moiety at R1 with chlorine (10) or fluorine (11)(5) was tolerated without significant loss of activity while decreasing molecular weight and clogP (Table 1). However, removal of the halogen altogether in compound 12 led to an 8-fold drop in potency. Although fluorinated compound 11 was less lipophilic than initial hit 1, it still showed poor solubility and metabolic stability.
On the basis of these results, we next turned to the R2 position with the aim of reducing the number of aromatic rings. A range of nonaromatic amines able to duplicate the hydrogen bonding potential of the 3-pyridyl moiety were evaluated. Results demonstrated that basicity, lipophilicity, and linker length were all important for activity (Table 1). Compounds (17, 18, and 19) with an ethyl linked piperidine or pyrrolidine retained similar activities to the corresponding 3-pyridyl derivatives. The replacement of the cyclic amine for a dimethylamine (13) and the introduction of longer linker lengths (14) led to drop in potency. Compounds 18 and 19(5) showed enhanced hepatic microsomal stability and improved solubility. Furthermore, ligand-lipophilicity efficiency (LLE or LiPE)(11) improved for compound 19 (LLE = 3.2) compared with the initial hit 1 (LLE = 2.6). Subsequent analogues were optimized using the ethyl linked pyrrolidine substituent on the amide at R2, which displayed the best profile in terms of lipophilicity, activity, and hepatic microsomal stability.
After initial optimization of the R1 and R2 groups, we then turned our attention to the R3 substituent with the aim of improving potency while maintaining lipophilicity at a moderate level (clogP < 3.5). An efficient synthetic route (Scheme 2) was designed for rapid access to a variety of R3 analogues which involved reaction of 5-fluoroisatin or 5-chloroisatin with malonic acid in refluxing acetic acid to provide intermediate 4.(12) A one pot chlorination and amide formation was achieved by treating 2-hydroxyquinoline-4-carboxylic acid 4 with thionyl chloride in the presence of DMF followed by reaction of the intermediate acid chloride with 2-pyrrolidin-1-ylethanamine in THF at room temperature. Intermediate 5 underwent aromatic nucleophilic substitution with a range of amines under microwave irradiation in acetonitrile to afford compounds 21–30 (Table 2). This route was also used for the synthesis of derivatives with aromatic R3 substituents where a Suzuki coupling of intermediate 5 with the appropriate boronic acid or ester led to compounds 36–39.
Scheme 2. a
aConditions: (a) malonic acid, acetic acid, reflux, 16 h, 54%; (b) SOCl2, DMF, DCM, reflux, 3 h and then 2-pyrrolidin-1-ylethanamine, THF, room temperature, 16 h, 27–43% yield; (c) amine, acetonitrile, 170 °C, microwave, 1 h, 7–54% yield; (d) boronic acid or ester, potassium phosphate, Pd(PPh3)4, DMF/water 3/1, 130 °C, microwave, 30 min, 19–73%.
Table 2. SAR Study of the R3 Substituent: Amines
Table a
MLM: mouse liver microsomes.
Table b
Kinetic aqueous solubility.
In general, the replacement of the tolyl substituent by an array of primary and secondary amines drove lipophilicity down and also led to improved solubility and hepatic microsomal stability (Table 2). In terms of potency, small heterocyles like 4-amino-3-methyloxazole 20 and N-methylpiperazine 21 at the R3 position were not tolerated. However, introduction of the 4-morpholinopiperidine 24 (EC50 = 0.15 μM, LLE = 4.2) moiety improved both potency and ligand efficiency compared with previous lead compound 18. Compound 24 displayed good aqueous solubility and moderate mouse hepatic microsomal clearance, which is probably related to the reduction in lipophilicity (clogP = 2.9).
To improve the potency of compound 24, we investigated other aliphatic amines. The introduction of flexibility at R3 with an aminopropyl morpholine substituent (25) led to a further improvement in potency against P. falciparum (EC50 = 70 nM) and lipophilic ligand efficiency (LLE = 5.4), with excellent selectivity against mammalian cells. Compound 25 had good aqueous solubility and in vitro hepatic microsomal stability across a range of species (Cli (mL min–1 g–1): mouse 0.8; rat <0.5; human <0.5) and low plasma protein binding (59%). The good in vitro DMPK properties of compound 25 translated into reasonable in vivo pharmacokinetics in mouse (Table 7). Furthermore, compound 25 afforded oral in vivo activity (Table 8) in the P. berghei mouse model, with a 93% reduction of parasitemia when dosed orally at 30 mg/kg once a day for four consecutive days. An in vivo pharmacokinetic study in mice for compound 25 showed low clearance, with a moderate volume of distribution and a resultant good half-life. However, oral bioavailability was poor (F = 15%). The low systemic exposure of compound 25 was not attributed to high first-pass metabolism due to the low in vitro clearance in mouse microsomes and low in vivo blood clearance but was probably due to poor permeability as highlighted by results in a PAMPA assay (Table 6). Preliminary safety profiling of compound 25 showed a weak affinity to the hERG ion channel (16% inhibition at 11 μM) and an oral maximum tolerated dose (MTD) greater than 300 mg/kg b.i.d. for 4 days. With an attractive overall profile, compound 25 was identified as a key molecule to declare early lead status for this series, according to the MMV compound development criteria.(7)
Moving into lead optimization, our focus was to improve potency, permeability, and bioavailability through structural modifications while retaining good physicochemical properties. Reducing the flexibility of compound 25 by shortening the linker length of the aminoalkylmorpholine moiety at R3 was tolerated (26). More promising was the 17-fold improvement (EC50 = 4 nM) on antiplasmodial activity obtained when the linker was extended from three to four carbons (27). Compound 27 displayed excellent lipophilic ligand efficiency (LLE = 6.2). This improvement on in vitro potency led to enhanced in vivo efficacy (Table 8) with an ED90 of 2.6 mg/kg. In addition with compound 27, one out of three mice went to cure at 4 × 30 mg/kg (q.d. po). Mouse in vivo pharmacokinetics showed a longer half-life than the early lead 25 as a result of lower in vivo clearance and a slightly higher volume of distribution (Table 7). Despite having improved in vivo potency and half-life, oral bioavailability was still poor, presumably still due to poor permeability (PAMPA Pe = 2 nm/s).
Modifications of our lead compound (25) focused on modulation of basicity, with the aim of improving permeability. Specifically, we envisaged that lowering basicity would reduce protonation at physiological pH, increase passive permeability, and ultimately improve bioavailability. Early lead 25 has two basic groups, a pyrrolidine (R2) and a morpholine (R3) with calculated pKa values of 8.5 and 7.0 respectively. We first investigated the effect that modifications on the morpholine group at R3 had on in vitro activity and permeability. We found that the morpholine oxygen was crucial for antiparasitic activity and replacement of the morpholino group by piperidine (28) was not tolerated. In contrast, the morpholine nitrogen was not essential for potency and removal, as exemplified by compounds 29 and 30, was well tolerated leading to single digit nanomolar potency against P. falciparum. Moreover, the removal of the basic group at R3 had not only improved activity but increased permeability more than 20-fold (30, Pe = 48 nm/s). Furthermore, improved in vitro permeability also translated in vivo, with an increase in oral bioavailability in mice (F = 23%). Despite its shorter half-life, compound 30 was efficacious in the P. berghei mouse model with an ED90 of 1 mg/kg and achieved a level of in vivo efficacy that met the MMV late lead criteria. However, compound 30 showed very poor rat in vivo exposure that was explained by high intrinsic clearance in rat hepatocytes (Cli = 3 mL min–1 g–1) which was subsequently confirmed by high hepatic extraction (76%) in a rat hepatic portal vein study. As for other compounds in this series, in vitro hepatic microsomal clearance was consistently low across species (Table 6).
After establishing a link between basicity, PAMPA permeability, and oral bioavailability for the series, we focused on modulating the pKa of the pyrrolidine group, the stronger of the two basic groups on early lead 25. Taking into account previous SAR showing that basicity and linker length were important for activity, we designed a short array of analogues with decreasing basicity.(13) Predicted pKa ranged from 5.0 for 3-difluoropyrrolidine 35 to 7.9 for 4-fluoropiperidine 31 compared with a predicted pKa of 8.5 for the lead pyrrolidine 25 (Table 3). As expected, a reduction of basicity resulted in up to 50-fold improvement in permeability. However, potency was dramatically reduced, highlighting the importance of the basic pyrrolidine nitrogen at the R2 position.
Table 3. Modulating Basicity of the Amide Substituent
Table a
Calculated pKa using ChemAxon software.
Table b
Controls: atenolol, 0.7 nm/s; propanolol, 159 nm/s.
Our attention then turned back to the R3 position, with the aim of further improving permeability and bioavailability across species. Previous SAR had shown that a methyl group at the para position of an aromatic ring at C-2 was tolerated, so we therefore expanded the range of substituents to include larger groups while maintaining moderate lipophilicity. This strategy also had the advantage of reducing the number of H-bond donors and molecular flexibility, two key factors that can modulate permeability. Early examples of substitution at R3 showed that introduction of amines, such as dimethylamine (36) and morpholine (37), reduced lipophilicity and was tolerated in terms of potency compared to compounds 11 and 19 (Table 4). Taking into account the leap in potency observed for 25 with a morpholine attached through a flexible linker at C-2, we prepared compound 2 with a carbon spacer between the phenyl ring and the morpholine to allow improved rotation. The introduction of the benzyl morpholine at R3 (compound 2) resulted in a 70-fold increase in potency against Pf (3D7) (EC50 = 1 nM) while retaining good ligand efficiency (LLE = 5.9), more than 30 fold increase in permeability (PAMPA Pe = 73 nm/s) and excellent bioavailability in mice (F = 74%). Furthermore, in vivo efficacy studies with compound 2 demonstrated complete cure at 4 × 30 mg/kg po q.d. in a P. berghei mouse model, with an ED90 of 0.1–0.3 mg/kg (Table 8).
Table 4. SAR Study of the R3 Substituent: Aromatic Groups
Table a
Calculated pKa using ChemAxon software. Experimental pKa using potentiometric titration is shown in parentheses.
Table b
Data for this compound reported previously.(5)
We developed short, four-step synthetic routes for synthesis of 2 and 42 which did not involve the use of palladium catalysis (Scheme 3). Two approaches were employed to synthesize the methyl ketone 8 depending on the availability of commercial starting materials. In the first route, nucleophilic displacement of commercially available 4-(bromomethyl)benzonitrile with morpholine using trimethylamine as a base in DCM gave intermediate 6 which was then converted into the desired methyl ketone 8 by reaction with methylmagnesium bromide followed by an acidic quench. In the second route, radical bromination of commercially available 1-(2-chloro-4-methylphenyl)ethanone with NBS using catalytic amounts of benzoyl peroxide in dichlorobenzene afforded compound 7, which was subsequently reacted with morpholine to yield the methyl ketone 8. As described earlier, a Pfitzinger reaction of 5-fluoroisatin with the appropriate methyl ketone led to acid 9. The final amides were prepared using 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) as coupling agent and N-methylmorpholine in DCM at room temperature. Details of other synthetic routes for individual compounds are described in the Supporting Information.
Scheme 3. a
aConditions: (a) morpholine, Et3N, DCM, 16 h, 72% yield; (b) MeMgBr, toluene, reflux, 4 h and then a 10% aqueous HCl, reflux, 1 h, 70% yield; (c) NBS, benzoyl peroxide, dichlorobenzene, 140 °C, 16 h, 70% yield; (d) morpholine, K2CO3, acetonitrile, 40 °C, 16 h, 64% yield; (e) 5-fluoroisatin, KOH, EtOH, 120 °C, microwave, 20 min, 30–76% yield; (f) amine, CDMT, N-methylmorpholine, DCM, 20–61% yield.
As highlighted above, a basic group is not required for activity at R3. Thus, derivatives of 2 with either reduced basicity (40, 41, and 42) or a nonbasic substituent (38, 43, 44) at R3 showed excellent potency with the exception of amide 39. The introduction of a conformationally restricted bridge amide as exemplified by compound 39 is detrimental for potency, possibly because it does not allow rotation of the morpholine group to adopt the optimal orientation for binding. The importance of the orientation of the morpholine substituent for activity is also highlighted by compound 45. In this case, a change of the methylmorpholine group from para to the meta position led to a weakly active compound.
On the basis of their excellent potency, good permeability, microsomal stability, and solubility (Table 6), compounds 2, 40, 41, 43, and 44 were progressed for efficacy studies in mice dosing at 1 mg/kg for 4 days (Table 8). Compounds 40, 43, and 44 showed excellent activity at this low dose, with reductions of parasitemia above 99%. Compound 41 showed the best survival time (14 days) comparable with 2.
Once an optimal R3 substituent had been identified, we turned our attention back to the R2 substituent to see if it was possible to improve the profile of compound 2 (Table 5). As highlighted before, lowering basicity at R2 results in a reduction in potency. The replacement of the pyrrolidine for a morpholine in compound 46 led to a 12-fold drop in potency. The amide NH is also important for activity, as capping with a methyl group resulted in an 87-fold decrease in potency against P. falciparum (3D7) (47, EC50 = 87 nM). Finally, it is possible to reduce the size of the ring on the R2 substituent and retain activity as shown by compounds 48 and 49. Compound 49 showed excellent in vivo activity in the P. berghei mouse model at 4 × 10 mg/kg and 4 × 3 mg/kg. (Table 8). However, none of these compounds offered a particular advantage to compound 2.
Table 5. SAR Study of the R2 Substituent
Table a
Calculated pKa using ChemAxon software.
Although in vitro DMPK data and in vivo efficacy in the P. berghei model were comparable for compound 2 and the fluorinated derivative 41, oral bioavailability in rat (33% for 41 vs 84% for 2) was lower and rat intravenous elimination half-life (4 h for 41 vs 10 h for 2) was shorter for 41 (Table 9). Therefore, compound 2 showed the best overall profile for further progression from this novel quinoline-4-carboxamide series (Table 9 and Figure 2). Compound 2 fulfilled the efficacy and DMPK requirements for a late lead according to the MMV criteria and, following further profiling, was selected as a preclinical candidate by MMV. The studies required to profile 2 for candidate selection have been described elsewhere.(5)
Table 6. Key in vitro DMPK Data for Selected Analogues
| compd | PAMPA Pe (nm/s)a | Sol. (μM) | MLMb Cli (mL min–1 g–1) | RLMc Cli (mL min–1 g–1) | HLMd Cli (mL min–1 g–1) | PPB (%)e | hERGf IC50 (μM) |
|---|---|---|---|---|---|---|---|
| 25 | 2.3 | 232 | 0.8 | <0.5 | <0.5 | 59 | >11 |
| 27 | 2 | 217 | 2.0 | <1 | 49 | >11 | |
| 30 | 48 | 232 | 2 | 0.8 | 1 | 77 | >11 |
| 2g | 73 | 216 | <0.5 | ≤0.8 | ≤1.1 | 63 | 16 |
| 38 | 29 | 109 | 1.9 | 75 | 11 | ||
| 40 | 63 | >208 | 1.0 | ||||
| 41 | 132 | 208 | <0.5 | <0.5 | 1.5 | ||
| 42 | >201 | 0.5 | 56 | ||||
| 43 | 14 | 171 | <0.5 | 0.8 | |||
| 44 | 49 | >217 | 2.0 | 0.6 | 2 | ||
| 46 | >210 | <0.5 | |||||
| 47 | 137 | >210 | <0.5 | ||||
| 48 | 49 | 2 | |||||
| 49 | 75 | 71 | 0.5 |
a
Controls: atenolol, 0.2–4.6 nm/s; propanolol, 103–159 nm/s.
b
MLM: mouse liver microsome.
c
RLM: rat liver microsome.
d
HLM: human liver microsome.
e
Mouse plasma protein binding.
f
Measured using IonWorks Patch Clamp electrophysiology.
g
Data for this compound reported previously.(5)
Table 7. In vivo Pharmacokinetic Profile in Mice of Key Compounds
| intravenous at 3 mg/kg | oral at 10 mg/kg | ||||||
|---|---|---|---|---|---|---|---|
| compd | Clb (mL min–1 kg–1) | Vdss (L/kg) | T1/2 (h) | Cmax (ng/mL) | AUC (ng·min/mL) | Tmax (h) | F (%) |
| 25 | 14 | 3 | 3.2 | 315 | 92922 | 2 | 15 |
| 27 | 4 | 3.5 | 12.5 | 579 | 176115 | 2 | 16 |
| 30 | 34 | 7.4 | 2.9 | 193 | 72728 | 0.5 | 23 |
| 2a | 12b | 15b | 16b | 90c | 179272c | 1c | 74 |
Table 8. In vivo Oral Activity in the P. berghei Mouse Model Peter’s Test
| 4 × 30 mg/kg | 4 × 10 mg/kg | 4 × 3 mg/kg | 4 × 1 mg/kg | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| compd | activity (%) | survival (days) | cure | activity (%) | survival (days) | cure | activity (%) | survival (days) | cure | activity (%) | survival (days) | cure |
| 25 | 93.0 | 7 | ||||||||||
| 27 | 99.8 | 22 | 1/3 | 99.7 | 15 | 96.0 | 9 | 48.0 | 6.0 | |||
| 30 | 99.9 | 10 | 99.9 | 8 | 98.0 | 6 | 90.0 | 7 | ||||
| 2 | 99.9 | >30 | 3/3 | 99.9 | >30 | 3/3 | 99.9 | 25 | 2/3 | 99.9 | 14 | |
| 40 | 99.8 | 11.0 | ||||||||||
| 41 | 99.9 | 14 | ||||||||||
| 43 | 99.0 | 7 | ||||||||||
| 44 | <40 | |||||||||||
| 49 | 99.1 | 20.3 | 1/3 | 99.2 | 9.3 | |||||||
Table 9. In vivo Pharmacokinetic Parameters in Male Sprague Dawley Rat
| intravenous at 1 mg/kg | oral at 3 mg/kg | ||||||
|---|---|---|---|---|---|---|---|
| compd | Clb (mL min–1 kg–1) | Vdss (L/kg) | T1/2 (h) | Cmax (ng/mL) | Tmax (h) | AUC (ng·min/mL) | F (%) |
| 2a | 18 | 15 | 10 | 180b | 4b | 200542b | 84 |
| 41 | 32 | 10 | 4 | 29 | 8 | 30400 | 33 |
a
Data for this compound reported previously.(5)
b
po dose: 5 mg/kg.
Figure 2. Mean blood concentration time profile of compounds 2 and 41 following intravenous or oral administration to male Sprague Dawley rats.
We also profiled key compounds of this series for their activity against different life stages of the malaria parasite life cycle (Table 10). Following a mosquito bite (blood meal), sporozoites are injected into the skin and migrate in the bloodstream to the liver, where they invade liver hepatocytes and then develop into liver schizonts. Compounds active against liver schizonts can potentially prevent disease development and be used in chemoprotection. Compounds 2, 27, 30, and 38 showed low nanomolar activity against the live schizont forms of Plasmodium yoelli.(14) Several compounds were also tested in vitro against P. falciparum late stage (IV–V) gametocytes. Stage V gametocytes, typically insensitive to antimalarial drugs, are infectious to mosquitoes and responsible for the transmission of the disease. 4-Quinolinecarboxamides, in particular compounds 2 and 30, are potent antigametocytocidal (stage IV–V) with nanomolar activities.(15) The ability of compounds of this series to block transmission was further tested in the P. berghei ookinete development assay, which simulates in vitro the first 24 h of parasite development in the mosquito midgut, from mature gametocyte transformation into gametes, through fertilization and to mature ookinete development. Compounds 2, 27, 30 and 38 showed nanomolar potency in this assay.(14b)
Table 10. Activity against Plasmodium Life Cycle Stages
| compd | Pf (3D7) EC50 (nM) | Py liver stage EC50 (nM) | Pf GAM IV–V EC50 (nM)a | Pb pokinete EC50 (nM) |
|---|---|---|---|---|
| 27 | 4 | 18 | 104 | 5 |
| 30 | 6 | 1 | 39 | 14 |
| 2 | 1 | 1b | 24 | 5b |
| 38 | 4 | 4 | 152 | 15 |
a
Run in duplicate. Reference controls: pyronaridine EC50 = 3108 nM, tafenoquine EC50 = 5250 nM, artemisinin EC50 = 0.8 nM.
b
Data reported previously.(5)
Conclusion
We have evolved a malaria phenotypic hit series, which started with moderate in vitro activity and selectivity but suboptimal metabolic stability and physicochemical properties, into an early lead compound 25 with improved potency, ligand efficiency, metabolic stability, and in vivo efficacy. The lead optimization phase focused on improving low oral bioavailability caused by the poor permeability of the early lead. The most advanced quinoline-4-carboxamides showed exceptional in vitro and in vivo activities, with a reduction of parasitemia of more than 99% when administered at low doses (4 × 1 mg/kg, 4 days, po) in the P. berghei mouse model. In addition to potent intraerythrocyte activity, compounds in this series showed similar potency against liver schizonts, gametocytes, and ookinetes in vitro. Furthermore, a combination of in vitro and in vivo activities across the different stages of the malaria parasite life cycle demonstrated the potential of this novel quinoline-4-carboxamide series to meet a number of the MMV’s malaria target candidate profiles (TCPs), as previously described.(7)
Compound 2 has been extensively profiled(5) in vitro and in vivo and displays activity against multiple life-cycle stages of the parasite with a long half-life in preclinical animal studies. With this profile, compound 2 has the potential for single dose treatment of malaria as part of a combination therapy, together with transmission blocking potential (TCP2 and TCP3b).(7) It also has the potential for chemoprotection (TCP4).(7) Compound 2 was active against parasites that show resistance to currently used antimalarials and has a novel mode of action through inhibition of elongation factor 2 (PfeEF2), which is involved in protein synthesis. The favorable potency, selectivity, DMPK properties, efficacy, safety profile, and novel mechanism of action support the progression of 2 toward clinical development.
Experimental Section
Chemistry. General
Solvents and reagents were purchased from commercial suppliers and used without further purification. Dry solvents were purchased in Sure/Seal bottles stored over molecular sieves. Reactions using microwave irradiation were carried out in a Biotage Initiator microwave. Normal phase TLCs were carried out on precoated silica plates (Kieselgel 60 F254, BDH) with visualization via UV light (UV 254/365 nm) and/or ninhydrin solution. Flash chromatography was performed using Combiflash Companion Rf (Teledyne ISCO) and prepacked silica gel columns purchased from Grace Davison Discovery Science or SiliCycle. Mass-directed preparative HPLC separations were performed using a Waters HPLC (2545 binary gradient pumps, 515 HPLC make-up pump, 2767 sample manager) connected to a Waters 2998 photodiode array and a Waters 3100 mass detector. Preparative HPLC separations were performed with a Gilson HPLC (321 pumps, 819 injection module, 215 liquid handler/injector) connected to a Gilson 155 UV/vis detector. On both instruments, HPLC chromatographic separations were conducted using Waters XBridge C18 columns, 19 mm × 100 mm, 5 μm particle size, using 0.1% ammonia in water (solvent A) and acetonitrile (solvent B) as mobile phase. 1H NMR, 19F NMR spectra were recorded on a Bruker Avance DPX 500 spectrometer (1H at 500.1 MHz, 19F at 470.5 MHz) or a Bruker Avance DPX 300 (1H at 300 MHz). Chemical shifts (δ) are expressed in ppm recorded using the residual solvent as the internal reference in all cases. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broadened (br), or a combination thereof. Coupling constants (J) are quoted to the nearest 0.1 Hz. Low resolution electrospray (ES) mass spectra were recorded on a Bruker Daltonics MicrOTOF mass spectrometer run in positive mode. High resolution mass spectroscopy (HRMS) was performed using a Bruker Daltonics MicroTof mass spectrometer. LC–MS analysis and chromatographic separation were conducted with a Bruker Daltonics MicrOTOF mass spectrometer or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC/MS, where both instruments were connected to an Agilent diode array detector. The column used was a Waters XBridge column (50 mm × 2.1 mm, 3.5 μm particle size), and the compounds were eluted with a gradient of 5–95% acetonitrile/water + 0.1% ammonia. All final compounds showed chemical purity of ≥95% as determined by the UV chromatogram (190–450 nm) obtained by LC–MS analysis. Unless otherwise stated herein reactions have not been optimized.
General Procedure A: Preparation of 2-(p-Tolyl)quinilone-4-carboxylic Acids (3)
To a mixture of the corresponding isatin (5 mmol) in ethanol (10 mL) were added the corresponding acetophenone (5 mmol), water (10 mL), and potassium hydroxide (2.80 g, 50 mmol). The reaction mixture was heated under microwave irradiation at 125 °C for 20 min. The resulting dark red colored solution was diluted with water (50 mL) and acidified by adding HCl (2 M, 30 mL). The resulting precipitate (yellow to ochre in color) was collected by filtration, washed with water (50 mL), ethyl acetate (100 mL), and dichloromethane (25 mL). The remaining solid was used in the next step without further purification.
6-Chloro-2-(p-tolyl)quinolone-4-carboxylic Acids (3, R1 = Cl)
Prepared using general procedure A. Yield, 58% (861 mg); 1H NMR (500 MHz, DMSO-d6) δ 8.82 (d, J = 2.3 Hz, 1H), 8.44 (s, 1H), 8.19 (d, J = 8.2 Hz, 2H), 8.13 (d, J = 9.0 Hz, 1H), 7.82 (dd, J = 2.3, 9.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 2.41 (s, 3H) ppm; LC–MS m/z 298 (M + H)+.
2-(p-Tolyl)quinolone-4-carboxylic Acids (3, R1 = H)
Prepared using general procedure A. Yield, 29% (384 mg); 1H NMR (500 MHz, DMSO-d6) δ 8.65 (d, 1H, J = 8.5 Hz), 8.45 (s, 1H), 8.22 (d, 2H, J = 8.2 Hz), 8.16 (d, 1H, J = 7.8 Hz), 7.85 (ddd, 1H, J = 1.4, 6.9, 8.4 Hz), 7.70 (ddd, 1H, J = 1.3, 6.9, 8.3 Hz), 7.39 (d, 2H, J = 7.9 Hz), 2.41 (s, 3H) ppm; LC–MS m/z 251 (M + H)+.
General Procedure B: Preparation of 2-(p-Tolyl)quinolone-4-carboxamides (10–18)
To a solution of quinolinecarboxylic acid 3 (0.5 mmol) in DMF (3 mL) was added diisopropylethylamine (0.07 mL, 0.75 mmol) followed by EDC (144 mg, 0.75 mmol) and HOBt (101 mg, 0.75 mmol). After 5 min the corresponding amine (1 mmol) was added followed by DMF (2 mL). The reaction mixture was stirred at room temperature overnight. The reaction was diluted with water (50 mL) and brine (10 mL) and extracted with ethyl acetate (30 mL). Solvents were removed, and the resulting off white solid was washed with dichloromethane and filtered. Amides that were soluble in dichloromethane were purified by column chromatography on silica.
6-Chloro-N-(pyridine-3-yl)-2-(p-tolyl)quinoline-4-carboxamide (10)
Prepared using general procedure B. Yield, 25% (47 mg); 1H NMR (500 MHz, DMSO-d6) δ 11.06 (br s, 1H), 8.96 (d, J = 2.0 Hz, 1H), 8.48 (s, 1H), 8.39 (dd, J = 1.4, 4.8 Hz, 1H), 8.30–8.25 (m, 4H), 8.18 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 2.4, 9.0 Hz, 1H), 7.47 (dd, J = 4.8, 8.3 Hz, 1H), 7.41 (d, J = 8.3 Hz, 2H), 2.41 (s, 3H) ppm; HRMS for C22H17ClN3O (M + H) + calcd 374.1055, found 374.1051.
N-(Pyridine-3-yl)-2-(p-tolyl)quinoline-4-carboxamide (12)
Prepared using general procedure B. Yield, 41% (69 mg); 1H NMR (500 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.02 (d, J = 1.9 Hz, 1H), 8.44–8.43 (m, 2H), 8.34–8.31 (m, 3H), 7.92–7.89 (m, 1H), 7.73–7.70 (m, 1H), 7.52 (dd, J = 4.8, 8.2 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 2.46 (s, 3H) ppm; LC–MS m/z 340 (M + H)+.
6-Chloro-N-(3-(dimethylamino)ethyl)-2-(p-tolyl)quinoline-4-carboxamide (13)
Prepared using general procedure B. Product was purified by column chromatography using a 4 g silica gel cartridge. Solvent A: DCM. Solvent B: 20% MeOH in DCM. Gradient: 3 min hold 100% A, 15 min ramp to 50% B, 2 min hold at 50% B. Fractions containing product were combined and concentrated to dryness under reduced pressure to obtain 13. Yield, 27% (50 mg); 1H NMR (500 MHz, CDCl3) 8.20 (d, J = 2.4 Hz, 1H), 8.08–8.05 (m, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.84 (s, 1H), 7.64 (dd, J = 2.2, 9.0 Hz, 1H), 7.33–7.28 (2H, m), 7.06 (1H, s), 3.65 (q, J = 5.6 Hz, 2H), 2.62 (t, J = 5.9 Hz, 2H), 2.45 (s, 3H), 2.33 (s, 6H); LC–MS m/z 368 (M + H)+.
6-Chloro-N-(3-(dimethylamino)propyl)-2-(p-tolyl)quinoline-4-carboxamide (14)
Prepared using general procedure B. Yield, 28% (53 mg); 1H NMR (500 MHz, CDCl3) δ 8.51 (br s, 1H), 8.34 (d, 1H, J = 2.4 Hz), 8.08 (d, 1H, J = 9.0 Hz), 8.04 (d, 2H, J = 8.2 Hz), 7.92 (s, 1H), 7.65 (dd, 1H, J = 2.1, 9.0 Hz), 7.30 (d, 2H, J = 8.2 Hz), 3.66 (q, 2H, J = 5.6 Hz), 2.51 (t, 2H, J = 5.6 Hz), 2.42 (s, 3H), 2.19 (s, 6H), 1.84–1.79 (m, 2H) ppm; HRMS for C22H25ClN3O (M + H) + calcd 382.1681, found 382.1673.
6-Chloro-N-(2-morpholinoethyl)-2-(p-tolyl)quinoline-4-carboxamide (15)
Prepared using general procedure B. Yield, 22% (45 mg); 1H NMR (500 MHz, DMSO-d6) δ 8.88 (t, J = 5.6 Hz, 1H), 8.31 (d, J = 2.4 Hz, 1H), 8.24–8.22 (m, 2H), 8.23 (s, 1H), 8.19 (s, 1H), 8.14 (d, J = 9.0 Hz, 1H), 7.85 (dd, J = 2.4, 9.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 3.65 (t, J = 4.5 Hz, 4H), 3.52 (q, J = 6.4 Hz, 2H), 2.57–2.54 (m, 2H), 2.41 (s, 3H) ppm; LC–MS m/z 410 (M + H)+.
6-Chloro-N-(2-(4-methylpiperazin-1-yl)ethyl)-2-(p-tolyl)quinoline-4-carboxamide (16)
Prepared using general procedure B. Yield, 27% (55 mg); 1H NMR (500 MHz, DMSO-d6) δ 8.30 (d, 1H, J = 2.4 Hz), 8.23 (d, J = 8.2 Hz, 2H), 8.18–8.13 (m, 2H), 7.85 (dd, J = 2.4, 9.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 3.54–3.48 (m, 2H), 2.55 (t, J = 6.4 Hz, 4H), 2.52–2.50 (m, 4H), 2.41 (s, 5H), 2.22 (s, 3H) ppm; LC–MS m/z 408 (M + H)+.
6-Chloro-N-(2-(piperidin-1-yl)ethyl-2-(p-tolyl)quinoline-4-carboxamide (17)
Prepared using general procedure B. Yield, 27% (55 mg); 1H NMR (500 MHz, DMSO-d6) δ 8.93 (t, J = 5.6 Hz, 1H), 8.39 (d, J = 2.4 Hz, 1H), 8.33–8.31 (m, 2H), 8.27 (s, 1H), 8.23 (d, J = 9.0 Hz, 1H), 7.94 (dd, J = 2.4, 9.0 Hz, 1H), 7.49 (d, J = 8.1 Hz, 2H), 3.60 (q, J = 6.4 Hz, 2H), 2.56–2.49 (m, 7H), 1.67–1.63 (m, 4H), 1.53–1.49 (m, 2H) ppm; LC–MS m/z 408 (M + H)+.
6-Chloro-N-(2-(pyrrolidin-1-yl)ethyl-2-(p-tolyl)quinoline-4-carboxamide (18)
Prepared using general procedure B. Yield, 43% (85 mg); 1H NMR (500 MHz, DMSO-d6) δ 8.87 (t, J = 5.7 Hz, 1H), 8.30 (d, J = 2.7 Hz, 1H), 8.22 (d, J = 8.8 Hz, 2H), 8.17 (s, 1H), 8.12 (d, J = 10.0 Hz, 1H), 7.82 (dd, J = 2.7, 8.8 Hz, 1H), 7.38 (d, J = 8.1 Hz, 2H), 3.50 (q, J = 5.7 Hz, 2H), 2.65 (t, J = 7.8 Hz, 2H), 2.56–2.54 (m, 4H), 2.30 (s, 3H), 1.73 (m, 4H) ppm; HRMS for C23H25ClN3O (M + H)+ calcd 394.1681, found 394.1663.
6-Fluoro-2-hydroxy-quinoline-4-carboxylic Acid (4, R1 = F)
A stirred suspension of 5-fluoroisatin (10.00 g, 61 mmol) and malonic acid (18.91 g, 182 mmol) in acetic acid (400 mL) was refluxed for 16 h. Acetic acid was removed under reduced pressure, and the residue was suspended in water (400 mL), filtered, and washed with water (300 mL) to give a brown solid. The solid was stirred in NaHCO3 saturated aqueous solution (800 mL), and the insoluble material was filtered off. The filtrate was acidified to pH 1–2 with concentrated HCl, and the resulting precipitate was filtered, washed with water (300 mL), and dried. The resulting pale yellow solid was used directly for synthesis of 4 without further purification. Yield, 54% (10 g); 1H NMR (500 MHz; DMSO-d6) δ 2.08 (s, 1.5 H), 7.01 (s, 1H), 7.35–7.37 (m, 0.5 H)*, 7.48–7.49 (m, 2H), 7.64–7.68 (m, 0.9H)*, 8.01–8.04 (m, 1H), 12.29 (brs, 1H), 13.38 (brs, 0.7H)* ppm; LC–MS purity 63%, m/z 208 (M + H)+; * corresponds to impurity.
6-Chloro-2-hydroxyquinoline-4-carboxylic Acid (4, R1 = Cl)
A stirred suspension of 6-chloroisatin (10.00 g, 55 mmol) and malonic acid (17.00 g, 165 mmol) in acetic acid (400 mL) was refluxed for 16 h. Acetic acid was removed under reduced pressure, and the residue was suspended in water (400 mL), filtered, and washed with water (300 mL) to give a gray solid. The solid was stirred in NaHCO3 saturated aqueous solution (800 mL), and the insoluble material was filtered off. The filtrate was acidified to pH 1–2 with concentrated HCl, and the precipitate was filtered, washed with water, and dried. The resulting pale yellow solid was used for the synthesis of 4 without further purification. Yield, 54% (9.5 g, 42 mmol); 1H NMR (500 MHz; DMSO-d6) δ 7.00 (s, 1H), 7.42 (d, 1H, J = 8.8 Hz), 7.58 (d, 0.3 H, J = 8.9 Hz)*, 7.60–7.62 (m, 1.3 H), 7.81 (dd, 0.3H, J = 2.3 Hz, J = 8.9 Hz)*, 8.29 (d, 1H, J = 2.4 Hz), 12.28 (brs, 1H), 13.22 (brs, 0.3 H)* ppm; LC–MS purity 65%, m/z 224 (M + H)+; * corresponds to impurity.
2-Chloro-6-fluoro-N-(2-pyrrolidin-1-ylethyl)quinoline-4-carboxamide (5, R1 = F)
To a stirred suspension of 6-fluoro-2-hydroxyquinoline-4-carboxylic acid (4, R1 = F) (10.00 g, 48 mmol) in anhydrous DCM (350 mL) were added anhydrous DMF (7 mL) and thionyl chloride (14 mL, 193 mmol) under argon at room temperature. The mixture was refluxed for 3 h and then allowed to cool to room temperature. The solvents were removed under reduced pressure, and the residue was dissolved in anhydrous THF (350 mL) under argon. 2-Pyrrolidin-1-ylethanamine (18 mL, 145 mmol) was added, and the reaction was stirred at room temperature for 16 h. Solvents were removed under vacuum and the residue was partitioned between NaHCO3 saturated aqueous solution (250 mL) and DCM (2 × 200 mL). The organic layers were combined, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography using a 120 g silica gel cartridge. Solvent A: DCM. Solvent B: 10% MeOH–NH3 in DCM. Gradient: 2 min hold 100% A followed by 18 min ramp to 30% B and then 15 min hold at 30% B. Fractions containing product were combined and concentrated to dryness under reduced pressure to obtain the desired compound as an off-white solid. Yield, 27% (4.59 g); 1H NMR (500 MHz; CDCl3) δ 8.07 (dd, J = 5.4, 9.2 Hz, 1H), 7.99 (dd, J = 2.8, 9.7 Hz, 1H), 7.56 (ddd, J = 2.8, 7.9, 9.2 Hz, 1H), 7.53 (s, 1H), 6.88 (brs, 1H), 3.65–3.69 (m, 2H), 2.81 (t, J = 5.5 Hz, 2H), 2.64 (brs, 4H), 1.83–1.85 (m, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −110.03 ppm; LC–MS m/z 322 (M + H)+.
2,6-Chloro-N-(2-pyrrolidin-1-ylethyl)quinoline-4-carboxamide (5, R1 = Cl)
To a stirred suspension of 6-chloro-2-hydroxyquinoline-4-carboxylic acid (3) (8.50 g, 38 mmol) in anhydrous DCM (250 mL) were added anhydrous DMF (2 mL) and thionyl chloride (11 mL, 152 mmol) at room temperature under argon. The mixture was refluxed for 3 h and then allowed to cool to room temperature. The solvents were removed under reduced pressure, and the residue was dissolved in anhydrous THF (300 mL) under argon. 2-Pyrrolidin-1-ylethanamine (14 mL, 114 mmol) was added, and the reaction was stirred at room temperature for 16 h. Solvents were removed under vacuum and the residue was partitioned between NaHCO3 saturated aqueous solution (250 mL) and DCM (2 × 250 mL). The organic layers were combined, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude was purified by column chromatography using a 120 g silica gel cartridge. Solvent A: DCM. Solvent B: 10% MeOH–NH3 in DCM. Gradient: 2 min hold 100% A followed by 18 min ramp to 30% B and then 15 min hold at 30% B. The relevant fractions were combined and concentrated to dryness under reduced pressure to obtain the desired product as an off-white solid. Yield, 43% (5.6 g); 1H NMR (500 MHz; CDCl3) δ 8.27 (d, J = 2.3 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.70 (dd, J = 2.3,9.0 Hz, 1H), 6.83 (brs, 1H), 7.48 (s, 1H), 3.64 (dt, J = 5.2, 11.6 Hz, 2H), 2.74–2.77 (m, 2H), 2.57–2.60 (m, 4H), 1.78–1.81 (m, 4H) ppm; LC–MS m/z 338 (M + H)+.
6-Fluoro-2-((3-methylisoxazol-5-yl)amino-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (20)
To a solution of 5 (R1 = F) (65 mg, 0.2 mmol) and 5-amino-3-methylisoxazole (39 mg, 0.4 mmol), BINAP (33 mg, 0.05 mmol) in dioxane (4 mL) were added sodium tert-butoxide (38 mg, 0.4 mmol) and palladium acetate (9 mg, 0.04 mmol). Reaction was heated at 115 °C overnight in a sealed tube. Reaction crude was filtered through Celite, and the filtrate was partitioned between water (5 mL) and DCM (25 mL). Organic phase was dried over MgSO4, and solvents were removed under reduce pressure. Product was purified by column chromatography on a 4 g silica cartridge using A (DCM) and B (10% MeOH–NH3 in DCM). Fractions containing product were pooled together to obtain 20 as a white solid. Yield, 13% (10 mg); 1H NMR (500 MHz, CDCl3) δ 7.94 (s, 1H), 7.62–7.57 (m, 2H), 7.32 (dd, J = 2.8, 8.2 Hz, 1H), 6.88 (s, 1H), 5.68–5.63 (m, 1H), 3.80 (d, J = 4.6 Hz, 2H), 2.95 (t, J = 5.3 Hz, 2H), 2.80 (s, 4H), 2.27 (s, 3H), 1.77 (s, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −117.06 ppm; LC–MS m/z 384 (M + 1).
General Procedure C: Preparation of 2-Amino-4-carboxamides (21–30)
A solution of 5 (1 equiv) and the corresponding amine (3 equiv) in acetonitrile (2.5 mL) in a microwave vial was heated at 170 °C for 1 h under microwave irradiation. Solvents were removed and product was purified by column chromatography on 4 g silica cartridges using A (DCM) and B (10% MeOH–NH3 in DCM).
6-Fluoro-2-(4-methylpiperazin-1-yl)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (21)
Prepared using general procedure C starting from 5 (R1 = F) (0.2 mmol, 65 mg), light yellow solid. Yield, 26% (20 mg); 1H NMR (500 MHz, CDCl3) δ 7.72–7.64 (m, 2H), 7.34–7.29 (m, 1H), 7.13 (s, 1H), 6.83 (s, 1H), 3.76 (dd, J = 5.0, 5.0 Hz, 4H), 3.68–3.62 (m, 2H), 2.78 (dd, J = 5.9, 5.9 Hz, 2H), 2.61–2.54 (m, 8H), 2.38 (s, 3H), 1.81–1.78 (m, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −118.04 ppm; LC–MS m/z 386 (M + 1).
2-(5,6-Dihydroimidazo[1,2-a]pyrazine-7(8H)-yl)-6-fluoro-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (22)
Prepared using general procedure C starting from 5 (R1 = F) (0.6 mmol, 96 mg), white solid. Yield, 18% (15 mg); 1H NMR (500 MHz, CDCl3) δ 7.78–7.71 (m, 2H), 7.39–7.34 (m, 1H), 7.23 (s, 1H), 7.13–7.11 (m, 1H), 7.04–7.03 (m, 1H), 6.88–6.87 (m, 1H), 4.88 (s, 2H), 4.28 (t, J = 5.4 Hz, 2H), 4.14 (t, J = 5.4 Hz, 2H), 3.67–3.62 (m, 2H), 2.77 (t, J = 6.0 Hz, 2H), 2.60–2.57 (m, 4H), 1.82–1.77 (m, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −117.16 ppm; LC–MS m/z 409 (M + 1).
6-Fluoro-2-(4-morpholinopiperidin-1-yl)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (23)
Prepared using general procedure C starting from 5 (R1 = F) (0.2 mmol, 65 mg), yellow solid. Yield, 22% (20 mg); 1H NMR (500 MHz, CDCl3) δ 7.71–7.64 (m, 2H), 7.35–7.30 (m, 1H), 7.14 (s, 1H), 6.77 (s, 1H), 4.55 (d, J = 13.2 Hz, 2H), 3.74 (t, J = 4.7 Hz, 4H), 3.66–3.62 (m, 2H), 2.98–2.93 (m, 2H), 2.77 (t, J = 6.0 Hz, 2H), 2.61–2.57 (m, 8H), 2.52–2.43 (m, 1H), 1.80 (t, J = 6.6 Hz, 4H), 1.61–1.50 (m, 2H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −118.31 ppm; LC–MS m/z 456 (M + 1).
6-Chloro-2-(4-morpholinopiperidin-1-yl)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (24)
Prepared using general procedure C starting from 5 (R1 = Cl) (0.2 mmol, 70 mg), yellow solid. Yield, 31% (28 mg); 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 2.2 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.50 (dd, J = 2.4, 8.8 Hz, 1H), 7.15 (s, 1H), 6.63–6.63 (m, 1H), 4.59 (d, J = 13.2 Hz, 2H), 3.75 (t, J = 4.6 Hz, 4H), 3.66 (q, J = 5.6 Hz, 2H), 3.05–2.98 (m, 2H), 2.78 (dd, J = 5.8, 5.8 Hz, 2H), 2.63- 2.58 (m, 8H), 2.53–2.47 (m, 1H), 2.00 (d, J = 12.5 Hz, 2H), 1.82 (dd, J = 3.2, 6.5 Hz, 4H), 1.65–1.53 (m, 2H) ppm; LC–MS m/z 473 (M + 1).
6-Fluoro-2-((3-morpholinopropyl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (25)
Prepared using general procedure C starting from 5 (R1 = F) (0.75 mmol, 241 mg), off white solid. Yield, 34% (110 mg); 1H NMR (500 MHz, CDCl3) δ 7.52–7.48 (m, 2H), 7.19–7.16 (m 1H), 6.36–6.35 (m 1H), 6.02 (s, 1H), 3.64 (br s, 4H), 3.56 (q, J = 5.6 Hz, 2H), 3.22 (br s, 2H), 2.70 (t, J = 5.9 Hz, 2H), 2.53 (br s, 4H), 2.35 (br s, 6H), 1.71 (br s, 4H), 1.65 (br s, 2H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −119.32 ppm; LC–MS m/z 430 (M + 1).
6-Fluoro-2-((3-morpholinoethyl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (26)
Prepared using general procedure C starting from 5 (R1 = F) (0.3 mmol, 88 mg), yellow solid. Yield, 29% (33 mg); 1H NMR (500 MHz, CDCl3) δ 7.69–7.65 (m, 2H), 7.31 (ddd, J = 2.9, 8.2, 9.1 Hz, 1H), 6.91–6.89 (m, 1H), 6.76 (s, 1H), 3.74 (dd, J = 4.6, 4.6 Hz, 4H), 3.65–3.53 (m, 4H), 2.75 (t, J = 5.9 Hz, 2H), 2.66–2.55 (m, 6H), 2.54–2.48 (m, 4H), 1.81–1.79 (m, 4H)ppm; 19F NMR (407.5 MHz; CDCl3) δ −118.87 ppm; LC–MS m/z 416 (M + 1).
6-Chloro-2-((4-morpholinobutyl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (27)
Prepared using general procedure C starting from 5 (R1 = Cl) (0.3 mmol, 100 mg), white solid. Yield, 54% (50 mg); 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 20.4 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.49 (dd, J = 2.4, 9.0 Hz, 1H), 6.82 (s, 1H), 6.66 (s, 1H), 3.75 (t, J = 4.7 Hz, 4H), 3.68–3.63 (m, 2H), 3.44 (q, J = 6.2 Hz, 2H), 2.78 (t, J = 5.9 Hz, 2H), 2.62 (br s, 4H), 2.48–2.39 (m, 8H), 1.84–1.79 (m, 4H), 1.73–1.61 (m, 2H) ppm; LC–MS m/z 460 (M + 1).
6-Fluoro-2-((2-(piperidin-1-yl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (28)
Prepared using general procedure C starting from 5 (R1 = F) (0.26 mmol, 84 mg), off white solid. Yield, 9% (10 mg); 1H NMR (500 MHz, CDCl3) δ 7.61–7.57 (m, 2H), 7.26 (ddd, J = 3.0, 8.6, 8.6 Hz, 1H), 6.70 (s, 1H), 6.67 (s, 1H), 5.44–5.42 (m, 1H), 3.57–3.44 (m, 4H), 2.66 (dd, J = 6.0, 6.0 Hz, 2H), 2.53–2.47 (m, 4H), 2.44–2.28 (m, 6H), 1.74–1.69 (m, 4H), 1.54–1.45 (m, 5H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −119.16 ppm; LC–MS m/z 414 (M + 1).
6-Fluoro-N-(2-(pyrrolidin-1-yl)ethyl)-2-((2-(tetrahydro-2H-pyran-4-yl)ethyl)amino)quinoline-4-carboxamide (29)
Prepared using general procedure C starting from 5 (R1 = F) (0.32 mmol, 100 mg), white solid. Yield, 7% (6 mg); 1H NMR (500 MHz, CDCl3) δ 7.61–7.55 (m, 2H), 7.23 (ddd, J = 2.4, 7.8, 9.7 Hz, 1H), 6.75 (br s, 1H), 6.59 (s, 1H), 4.74 (br s, 1H), 3.89 (dd, J = 3.8, 11.2 Hz, 2H), 3.55 (q, J = 5.6 Hz, 2H), 3.39–3.27 (m, 4H), 2.68 (t, J = 5.9 Hz, 2H), 2.50 (d, J = 5.4 Hz, 4H), 1.74–1.69 (m, 4H), 1.62–1.48 (m, 5H), 1.33–1.21 (m, 2H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −118.84 ppm; LC–MS m/z 415 (M + 1).
6-Chloro-N-(2-(pyrrolidin-1-yl)ethyl)-2-((2-(tetrahydro-2H-pyran-4-yl)ethyl)amino)quinoline-4-carboxamide (30)
Prepared using general procedure C starting from 5 (R1 = Cl) (1.5 mmol, 500 mg), yellow solid. Yield, 31% (200 mg); 1H NMR (500 MHz, CDCl3) δ 8.06 (br s, 1H), 8.05 (d, J = 2 0.4 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.37 (dd, J = 2.4, 8.8 Hz, 1H), 7.17 (m, 1H), 4.94 (t, J = 5.0 Hz, 1H), 3.88 (dd, J = 3.8, 11.2 Hz, 2H), 3.74 (q, J = 5.4 Hz, 2H), 3.46 (q, J = 6.6 Hz, 2H), 3.33–3.27 (m, 2H), 3.16–3.06 (m, 6H), 2.03–1.98 (m, 3H), 1.63–1.49 (m, 6H), 1.32–1.21 (m, 2H) ppm; LC–MS m/z 431 (M + 1).
General Procedure D: Suzuki Coupling on Intermediate 5
To a solution of 5 (1 equiv) in DMF/water 3/1 (4 mL) in a microwave vial were added potassium phosphate (3 equiv), the corresponding boronic acid (3 equiv), and Pd(PPh3)4 (3 mol %). The reaction was degassed by bubbling nitrogen through the mixture for 5 min and then heated under microwave irradiation at 130 °C for 30 min. Reaction mixture was filtered through Celite. Celite was washed with DCM. Filtrate was partitioned between water (5 mL) and DCM (25 mL × 2). Organic phase was dried over MgSO4, and solvents were evaporated under reduced pressure. Product was purified by column chromatography on 4 g silica cartridges using A (DCM) and B (20% MeOH–NH3 in DCM) and the following gradient: 3 min hold 100% A, 15 min ramp to 30% B, 4 min hold at 30% B.
2-(4-(Dimethylamino)phenyl)-6-fluoro-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (36)
Prepared using general procedure D starting from 5 (R1 = F) (0.2 mmol, 65 mg), yellow solid. Yield, 25% (20 mg); 1H NMR (500 MHz, CDCl3) δ 8.13–8.07 (m, 3H), 7.91–7.87 (m, 2H), 7.47 (ddd, J = 2.9, 8.1, 9.2 Hz, 1H), 6.96 (s, 1H), 6.85–6.82 (m, 2H), 3.70 (q, J = 5.7 Hz, 2H), 3.08 (s, 6H), 2.83 (t, J = 6.0 Hz, 2H), 2.67–2.64 (m, 4H), 1.86–1.81 (m, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −113.18 ppm; LC–MS m/z 407 (M + H)+.
6-Fluoro-2-(4-morpholinophenyl)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (37)
Prepared using general procedure D starting from 5 (R1 = F) (0.5 mmol, 150 mg), yellow solid. Yield, 19% (43 mg); 1H NMR (500 MHz, CDCl3) δ 8.15–8.07 (m, 3H), 7.91–7.87 (m, 2H), 7.52–7.47 (m, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.89 (t, J = 4.5 Hz, 1H), 3.92 (t, J = 4.8 Hz, 4H), 3.68 (q, J = 5.7 Hz, 2H), 3.30 (t, J = 4.8 Hz, 4H), 2.79 (t, J = 6.0 Hz, 2H), 2.60–2.62 (m, 4H), 1.81 (t, J = 3.3 Hz, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −112.39 ppm; LC–MS m/z 449 (M + H)+.
6-Fluoro-2-(4-(morpholinosulfonyl)phenyl)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (38)
Prepared using general procedure D starting from 5 (R1 = F) (0.23 mmol, 75 mg), white solid. Yield, 20% (24 mg); 1H NMR (500 MHz, CDCl3) δ 8.36–8.33 (m, 2H), 8.22 (dd, J = 5.4, 9.2 Hz, 1H), 8.04–7.99 (m, 2H), 7.91 (d, J = 8.5 Hz, 2H), 7.58 (ddd, J = 2.8, 8.0, 9.2 Hz, 1H), 6.98 (s, 1H), 3.79–3.69 (m, 6H), 3.07 (t, J = 4.7 Hz, 4H), 2.81 (t, J = 5.9 Hz, 2H), 2.62 (d, J = 5.4 Hz, 4H), 1.84–1.79 (m, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −109.76 ppm; LC–MS m/z 513 (M + H)+.
6-Fluoro-2-(4-(morpholine-4-carbonyl)phenyl)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (39)
Prepared using general procedure D starting from 5 (R1 = F) (0.25 mmol, 80 mg), yellow solid. Yield, 73% (87 mg); 1H NMR (500 MHz, CDCl3) δ 8.18–8.14 (m, 3H), 7.96–7.94 (m, 2H), 7.54–7.50 (m, 3H), 7.10 (dd, J = 4.9, 4.9 Hz, 1H), 3.76–3.72 (m, 6H), 3.69–3.66 (m, 4H), 2.78 (t, J = 6.0 Hz, 2H), 2.61–2.59 (m, 4H), 1.81–1.79 (m, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −110.76 ppm; LC–MS m/z 477 (M + H)+.
1-(4-(Bromomethyl)-2-chlorophenyl)ethanone (7)
A mixture of 1-(2-chloro-4-methylphenyl)ethanone (1.6 g, 9.50 mmol) and chlorobenzene (60 mL) was prepared at rt and N-bromosuccinimide (NBS) (1.86 g, 10.44 mmol) added followed by benzoyl peroxide (∼1.5 mg, 0.005 mmol, catalytic amount), and the mixture was heated to 140–145 °C for 16 h. The mixture was then cooled to rt, diluted with toluene (50 mL), and filtered through a Celite pad. The pad was washed with toluene (2 × 50 mL) and the filtrate concentrated under reduced pressure and purified by column chromatography (0–10% EtOAc/hexanes) to afford 1-(4-(bromomethyl)-2-chlorophenyl)ethanone (1.65 g, 6.65 mmol, 70%) as a yellow oil. 1H NMR (500 MHz; CDCl3) δ 2.65 (s, 3H), 4.43 (s, 2H), 7.34 (dd, 1H, J = 1.3, 8.0 Hz), 7.46 (s, 1H), 7.54 (d, 1H, J = 7.9 Hz) ppm.
1-(2-Chloro-4-(morpholinomethyl)phenyl)ethanone (8, X = Cl)
A mixture of 1-(4-(bromomethyl)-2-chlorophenyl)ethanone (1.65 g, 6.65 mmol) and acetonitrile (25 mL) was prepared at rt and stirred under nitrogen. Potassium carbonate (1.10g 7.98 mmol) was then added followed by morpholine (0.695 mL, 695 mg, 7.98 mmol), and the mixture was stirred at rt. After 2 h, TLC showed presence of product and starting material. Mixture was then heated under nitrogen to 40 °C for 16 h, cooled to room temp, filtered to remove excess carbonate and filtrate concentrated under reduced pressure. Mixture was then diluted in DCM (30 mL), washed with water (2 × 10 mL), filtered through a phase separator and filtrate concentrated under reduced pressure. Mixture was purified by column chromatography (40–100% ethyl acetate/hexane) to afford 1-(2-chloro-4-(morpholinomethyl)phenyl)ethanone (1.08 g, 4.25 mmol, 64%) as a yellow oil. 1H NMR (500 MHz; CDCl3) δ 2.44 (brs, 4H), 2.65 (s, 3H), 3.49 (s, 2H), 3.72 (t, J = 4.6 Hz, 4H), 7.29 (dd, J = 1.5, 7.9 Hz, 1H,), 7.43 (brs, 1H), 7.55 (d, J = 7.9 Hz, 1H) ppm; LCMS m/z 254 [M + H]+
2-(2-Chloro-4-(morpholinomethyl)phenyl)-6-fluoroquinoline-4-carboxylic Acid (9, X = Cl)
A mixture of 5-fluoroisatin (7.02 mg, 4.25 mmol) and 1-(2-chloro-4-(morpholinomethyl)phenyl)ethanone (1.08 g, 4.25 mmol) was prepared in EtOH/water (1:1) (10 mL) and then KOH (2.40 g, 42.50 mmol) added and the mixture heated in microwave, 125 °C, 20 min. The mixture was then diluted with water (10 mL), acidified to pH 3 with 2 M HCl, stirred for 16 h at rt and the resulting precipitate filtered, washed with water (2 × 10 mL), and concentrated under reduced pressure to afford 2-(2-chloro-4-(morpholinomethyl)phenyl)-6-fluoroquinoline-4-carboxylic acid (503 mg, 1.25 mmol, 30%) as an orange solid. 1H NMR (500 MHz; CDCl3) δ 2.54 (brs, 4H), 3.64 (s, 4H), 3.70 (brs, 2H), 7.50 (d, J = 8.1 Hz, 1H), 7.62 (s, 1H), 7.73 (bd, J = 7.9 Hz, 1H), 7.84 (dt, J = 2.9, 8.2 Hz, 1H,), 8.24 (dd, J = 5.8, 9.2 Hz, 1H), 8.56 (dd, J = 2.9, 11.0 Hz, 1H) ppm; LCMS m/z 399 [M – H]−.
2-(2-Chloro-4-(morpholinomethyl)phenyl)-6-fluoro-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (42)
A mixture of 2-(2-chloro-4-(morpholinomethyl)phenyl)-6-fluoroquinoline-4-carboxylic acid (303 mg, 0.76 mmol) in DCM (6 mL) was prepared at rt, and N-methylmorpholine (0.166 mL, 153 mg, 1.51 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (159 mg, 0.91 mmol) were added, and the mixture was stirred for 1 h in a sealed vial. 2-(Pyrrolidin-1-yl)ethanamine (0.143 mL, 130 mg, 1.13 mmol) was then added and the mixture stirred in a sealed vial for 17 h. The mixture was then diluted with DCM (5 mL), and the organic layers were washed with water (2 × 3 mL) and filtered through a phase separator and the organic layers concentrated under reduced pressure and purified by column chromatography (0–10% 7 M NH3 in MeOH/DCM) to afford an off white solid. Analysis by 1H NMR showed impurities, and the mixture was further purified by MDAP to produce 2-(2-chloro-4-(morpholinomethyl)phenyl)-6-fluoro-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (228 mg, 0.46 mmol, 61%) as an off-white solid. 1H NMR (500 MHz; CDCl3) δ 1.76–1.79 (m, 4H), 2.49 (brs, 4H), 2.57 (brs, 4H), 2.75 (t, J = 6.0 Hz, 2H), 3.55 (s, 2H), 3.65 (q, J = 5.3 Hz, 2H), 3.74 (t, J = 4.6 Hz, 4H), 6.82 (brs, 1H), 7.40 (dd, J = 1.6, 7.9 Hz, 1H), 7.52–7.57 (m, 2H), 7.68 (d, J = 7.9 Hz, 1H), 7.89 (s, 1H), 8.05 (dd, J = 2.8, 10.0 Hz, 1H), 8.19 (dd, J = 5.5, 9.2 Hz, 1H) ppm; LCMS m/z 497 [M + H]+.
6-Fluoro-N-(2-morpholinoethyl)-2-(4-morpholinomethyl)phenyl)quinoline-4-carboxamide (46)
A solution of 6-fluoro-2-(4-(morpholinomethyl)phenyl)quinoline-4-carboxylic acid (9, X = H)(5) (0.1 g, 0.27 mmol), 2-chloro-4,6-dimethoxy-1,3,5-triazine CDMT (57 mg, 0.32 mmol, 1.2 equiv), and N-methylmorpholine (0.06 mL, 0.54 mmol, 2 equiv) in DCM (10 mL) was stirred at room temperature for 1 h. 2-Morpholinoethanamide (0.054 mL, 0.41 mmol, 1.5 equiv) was added, and the reaction mixture was stirred at room temperature overnight. Reaction was partitioned between DCM (50 mL) and NaHCO3 sat. aq solution (10 mL). Organic phase was dried over MgSO4, and solvents were removed under reduce pressure. Product precipitated from acetonitrile (1.5 mL) as a white solid. Yield, 63% (83 mg); 1H NMR (500 MHz, CDCl3) δ 8.22 (dd, J = 5.4, 9.2 Hz, 1H), 8.12 (d, J = 8.2 Hz, 2H), 8.00–7.97 (m, 2H), 7.58–7.52 (m, 3H), 6.67 (t, J = 4.8 Hz, 1H), 3.77–3.70 (m, 10H), 3.61 (s, 2H), 2.70 (t, J = 5.9 Hz, 2H), 2.57 (br s, 4H), 2.51 (t, J = 4.3 Hz, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −111.23; LC–MS m/z 479 (M + H)+.
6-Fluoro-N-methyl-2-(4-(morpholinomethyl)phenyl)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (47)
To a suspension of 6-fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-carboxylic acid (9, X = H)(5) (100 mg, 0.27 mmol) in anhydrous DCM (10 mL) were added N-methylmorpholine (0.06 mL, 0.54 mmol, 2 equiv) and 2-chloro-4,6-dimethoxy-1,3,5-triazine CDMT (57 mg, 0.32 mmol, 1.2 equiv). The reaction mixture was stirred at room temperature for 1 h. N-Methyl-2-pyrrolidin-1-ylethanamine (0.034 g, 0.27 mmol) was added and the reaction mixture stirred at room temperature overnight. The mixture was then diluted with DCM (10 mL), water (10 mL) was added, and the layers were separated. The aqueous portion was extracted with further DCM (10 mL). The combined DCM extracts were evaporated in vacuo. The residue was dissolved in DMF and purified by mass directed autoprep 5–95% MeCN, basic, to afford impure product. The sample was dissolved in DMF and purified using mass directed autoprep 25–75% MeCN, basic, and the product obtained was freeze-dried to afford 47 as a cream colored solid (24 mg, 17% yield); 1H NMR (500 MHz, CDCl3) δ 8.15–8.08 (m, 3H), 8.04–8.01 (m, 1H), 7.48–7.42 (m, 3H), 7.36 (dd, J = 2.8, 9.2 Hz, 1H), 4.06–4.03 (m, 1H), 3.67 (dd, J = 4.3, 4.3 Hz, 4H), 3.53 (s, 2H), 3.27 (s, 2H), 3.22 (s, 1H), 2.90 (s, 3H), 2.43 (s, 4H), 2.11- 2.06 (m, 4H), 1.81–1.51 (m, 4H) ppm; LC–MS m/z 477 (M + H)+.
N-(2-(Cyclopropylamino)ethyl)-6-fluoro-2-(4-(morpholinomethyl)phenyl)quinoline-4-carboxamide (48)
To a suspension of 6-fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-carboxylic acid (9, X = H)(5) (0.1 g, 0.27 mmol) in anhydrous DCM (10 mL) were added N-methylmorpholine (0.06 mL, 0.54 mmol, 2 equiv) and 2-chloro-4,6-dimethoxy-1,3,5-triazine CDMT (57 mg, 0.32 mmol, 1.2 equiv). The reaction mixture was stirred at room temperature for 1 h. N′-Cyclopropylethane-1,2-diamine (0.027 g,0.27 mmol) was added and the reaction mixture stirred at room temperature for 2.5 days. The mixture was then diluted with DCM (10 mL), water (10 mL) was added, and the layers were separated. The aqueous portion was extracted with further DCM (10 mL). The combined DCM extracts were evaporated in vacuo. The residue was dissolved in DMF and purified by mass directed autoprep 5–95% MeCN, basic, method to afford 48 as a yellow solid (25 mg, 18% yield); 1H NMR (500 MHz, CDCl3) δ 8.18 (dd, J = 5.4, 9.2 Hz, 1H), 8.09 (d, J = 8.2 Hz, 2H), 7.94–7.91 (m, 2H), 7.55–7.49 (m, 3H), 6.74–6.72 (m, 1H), 3.75–3.66 (m, 6H), 3.59 (s, 2H), 3.06 (t, J = 5.8 Hz, 2H), 2.49 (s, 4H), 2.23–2.18 (m, 1H), 0.54–0.49 (m, 2H), 0.39–0.35 (m, 2H); LC–MS m/z 449 (M + H)+.
N-(2-(Azetidin-1-yl)ethyl)-6-fluoro-2-(4-(morpholinomethyl)phenyl)quinoline-4-carboxamide (49)
To a suspension of 6-fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-carboxylic acid (9, X = H)(5) (100 mg,0.27 mmol) in anhydrous DCM (10 mL) were added N-methylmorpholine (0.06 mL, 0.54 mmol, 2 equiv) and 2-chloro-4,6-dimethoxy-1,3,5-triazine CDMT (57 mg, 0.32 mmol, 1.2 equiv). The reaction mixture was stirred at room temperature for 1 h. 2,2-Dimethoxyethanamine (28 mg,0.27 mmol) was added and the reaction mixture stirred at room temperature for 2.5 days. The mixture was then diluted with DCM (10 mL), water (10 mL) was added, and the layers were separated. The aqueous portion was extracted with further DCM (10 mL). The combined DCM extracts were evaporated in vacuo. The residue was dissolved in DCM and purified by silica (12 g), eluting with 0–100% EtOAc/hexane and then 0–50% (10% MeOH in DCM/DCM) to afford N-(2,2-dimethoxyethyl)-6-fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-carboxamide as a yellow gum (109 mg, 79% yield). A solution of N-(2,2-dimethoxyethyl)-6-fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-carboxamide (109 mg,0.24 mmol) in 1,4-dioxane (5 mL) was treated with conc. HCl (1 mL) and stirred at room temperature for 1.5 h. The mixture was neutralized with saturated sodium bicarbonate portionwise and then exracted with EtOAc (2 × 20 mL). The combined EtOAc extracts were evaporated in vacuo to afford 6-fluoro-2-[4-(morpholinomethyl)phenyl]-N-(2-oxoethyl)quinoline-4-carboxamide as a yellow gum (78 mg, 71% yield). A mixture of 6-fluoro-2-[4-(morpholinomethyl)phenyl]-N-(2-oxoethyl)quinoline-4-carboxamide (78 mg,0.19 mmol), azetidine (32 mg,0.57 mmol) in DCM (5 mL) was stirred for 15 min in a stoppered flask at room temperature. Sodium triacetoxyborohydride (56 mg,0.26 mmol) was then added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned between water (10 mL) and DCM (10 mL) and the aqueous extracted with further DCM (10 mL). The combined DCM extracts were evaporated in vacuo. The residue was dissolved in DMF and purified by mass directed autoprep, 5–95% MeCN, basic, to afford a yellow gum. The sample was freeze-dried to afford 49 as a cream colored solid (18 mg, 14% yield); 1H NMR (500 MHz, CDCl3) δ 8.21 (dd, J = 5.4, 9.2 Hz, 1H), 8.13 (d, J = 8.4 Hz, 2H), 8.00–7.97 (m, 2H), 7.58–7.52 (m, 3H), 6.73–6.72 (m, 1H), 3.76 (t, J = 4.6 Hz, 4H), 3.61 (s, 2H), 3.57–3.53 (m, 2H), 3.28 (t, J = 7.0 Hz, 4H), 2.72 (t, J = 5.8 Hz, 2H), 2.51 (s, 4H), 2.14–2.07 (m, 2H); LC–MS m/z 449 (M + H)+.
Details of other synthetic routes for individual compounds are described in the Supporting Information.
Biology Materials and Methods
This is included in the Supporting Information.
Ethical Statements
In vivo antimalarial efficacy studies in P. berghei carried out at the Swiss Tropical and Public Health Institute (Basel, Switzerland) adhere to local and national regulations of laboratory animal welfare in Switzerland (awarded Permission No. 1731). Protocols are regularly reviewed and revised following approval by the local authority (Veterinäramt Basel Stadt).
Mouse and rat pharmacokinetics were carried out at the University of Dundee. All regulated procedures on living animals were carried out under the authority of a license issued by the Home Office under the Animals (Scientific Procedures) Act 1986, as amended in 2012 (and in compliance with EU Directive EU/2010/63). License applications will have been approved by the University’s Ethical Review Committee (ERC) before submission to the Home Office. The ERC has a general remit to develop and oversee policy on all aspects of the use of animals on University premises and is a subcommittee of the University Court, its highest governing body.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00723.
The authors declare no competing financial interest.
Acknowledgment
We acknowledge Medicines for Malaria Venture for financial support. The University of Dundee also acknowledges support from the Wellcome Trust (Grant 100476 and a Principal Research Fellowship to A.H.F.). We thank Christian Scheurer and Jolanda Kamber from the Swiss TPH for assistance in performing assays against the drug-resistant parasites and the in vivo (Pb) antimalarial assays.
| Abbreviations Used | |
| ACT | artemisinin combination therapy |
| CDMT | 2-chloro-4,6-dimethoxy-1,3,5-triazine |
| Cli | intrinsic clearance |
| EDC | N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride |
| HOBt | hydroxybenzotriazole |
| LELP | ligand-efficiency-dependent lipophilicity |
| LLE | lipophilic ligand efficiency |
| MMV | Medicines for Malaria Venture |
| Pe | permeability |
| TCP | target candidate profile |
| WHO | World Health Organization |
References
This article references 15 other publications.
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, K. M.; Imwong, M.; Lwin, K. M.; Win, A. A.; Hlaing, T. M.; Hlaing, T.; Lin, K.; Kyaw, M. P.; Plewes, K.; Faiz, M. A.; Dhorda, M.; Cheah, P. Y.; Pukrittayakamee, S.; Ashley, E. A.; Anderson, T. J.; Nair, S.; McDew-White, M.; Flegg, J. A.; Grist, E. P.; Guerin, P.; Maude, R. J.; Smithuis, F.; Dondorp, A. M.; Day, N. P.; Nosten, F.; White, N. J.; Woodrow, C. J.Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker Lancet Infect. Dis. 2015, 15, 415– 421, DOI: 10.1016/S1473-3099(15)70032-0[CrossRef], [PubMed], [CAS]3a.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFShtr8%253D&md5=2a8f63354ba261347cd0d8b95ed03743Tun, Kyaw M.; Imwong, Mallika; Lwin, Khin M.; Win, Aye A.; Hlaing, Tin M.; Hlaing, Thaung; Lin, Khin; Kyaw, Myat P.; Plewes, Katherine; Abul Faiz, M.; Dhorda, Mehul; Cheah, Phaik Yeong; Pukrittayakamee, Sasithon; Ashley, Elizabeth A.; Anderson, Tim J. C.; Nair, Shalini; McDew-White, Marina; Flegg, Jennifer A.; Grist, Eric P. M.; Guerin, Philippe; Maude, Richard J.; Smithuis, Frank; Dondorp, Arjen M.; Day, Nicholas P. J.; Nosten, Francois; White, Nicholas J.; Woodrow, Charles J.Lancet Infectious Diseases (2015),Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of mol. surveillance in remote areas through anal. of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by detg. the relative prevalence of P falciparum parasites carrying K13-propeller mutations. We did this cross-sectional survey at malaria treatment centers at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between Jan., 2013, and Sept., 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estd. prevalence of mutations of the K13 propeller region across Myanmar. Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations. Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided. Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Program and the Bill & Melinda Gates Foundation.(b) Phyo, A. P.; Nkhoma, S.; Stepniewska, K.; Ashley, E. A.; Nair, S.; McGready, R.; ler Moo, C.; Al-Saai, S.; Dondorp, A. M.; Lwin, K. M.; Singhasivanon, P.; Day, N. P.; White, N. J.; Anderson, T. J.; Nosten, F.Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study Lancet 2012, 379, 1960– 1966, DOI: 10.1016/S0140-6736(12)60484-X[CrossRef], [PubMed], [CAS]3b.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38rjtFehsg%253D%253D&md5=9de4b3f2862332e0bf33d1d6612b06d0Phyo Aung Pyae; Nkhoma Standwell; Stepniewska Kasia; Ashley Elizabeth A; Nair Shalini; McGready Rose; ler Moo Carit; Al-Saai Salma; Dondorp Arjen M; Lwin Khin Maung; Singhasivanon Pratap; Day Nicholas P J; White Nicholas J; Anderson Tim J C; Nosten FrancoisLancet (London, England) (2012),BACKGROUND: Artemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international effort is underway to contain artemisinin-resistant Plasmodium falciparum, but containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand-Myanmar (Burma) border. METHODS: In malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≥4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms. FINDINGS: 3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5-2·7) in 2001, to 3·7 h (3·6-3·8) in 2010, compared with a mean of 5·5 h (5·2-5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≥6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identified, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010. INTERPRETATION: Genetically determined artemisinin resistance in P falciparum emerged along the Thailand-Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2-6 years. FUNDING: The Wellcome Trust and National Institutes of Health.(c) Takala-Harrison, S.; Jacob, C. G.; Arze, C.; Cummings, M. P.; Silva, J. C.; Dondorp, A. M.; Fukuda, M. M.; Hien, T. T.; Mayxay, M.; Noedl, H.; Nosten, F.; Kyaw, M. P.; Nhien, N. T.; Imwong, M.; Bethell, D.; Se, Y.; Lon, C.; Tyner, S. D.; Saunders, D. L.; Ariey, F.; Mercereau-Puijalon, O.; Menard, D.; Newton, P. N.; Khanthavong, M.; Hongvanthong, B.; Starzengruber, P.; Fuehrer, H. P.; Swoboda, P.; Khan, W. A.; Phyo, A. P.; Nyunt, M. M.; Nyunt, M. H.; Brown, T. S.; Adams, M.; Pepin, C. S.; Bailey, J.; Tan, J. C.; Ferdig, M. T.; Clark, T. G.; Miotto, O.; MacInnis, B.; Kwiatkowski, D. P.; White, N. J.; Ringwald, P.; Plowe, C. V.Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia J. Infect. Dis. 2015, 211, 670– 679, DOI: 10.1093/infdis/jiu491[CrossRef], [PubMed], [CAS]3c.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M%252FntFGgsg%253D%253D&md5=f951db8da7bc72877f04d63184e295b2Takala-Harrison Shannon; Jacob Christopher G; Nyunt Myaing M; Brown Tyler S; Adams Matthew; Pepin Christopher S; Bailey Jason; Plowe Christopher V; Arze Cesar; Silva Joana C; Cummings Michael P; Dondorp Arjen M; White Nicholas J; Fukuda Mark M; Bethell Delia; Tyner Stuart D; Saunders David L; Hien Tran Tinh; Nhien Nguyen Thanh Thuy; Mayxay Mayfong; Noedl Harald; Starzengruber Peter; Fuehrer Hans-Peter; Swoboda Paul; Nosten Francois; Kyaw Myat P; Nyunt Myat H; Imwong Mallika; Se Youry; Lon Chanthap; Ariey Frederic; Mercereau-Puijalon Odile; Menard Didier; Newton Paul N; Khanthavong Maniphone; Hongvanthong Bouasy; Khan Wasif A; Phyo Aung Pyae; Tan John C; Ferdig Michael T; Clark Taane G; Miotto Olivo; MacInnis Bronwyn; Kwiatkowski Dominic P; Ringwald PascalThe Journal of infectious diseases (2015),BACKGROUND: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. METHODS: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. RESULTS: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas. CONCLUSIONS: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar. - 4.Wells
, T. N.; Hooft van Huijsduijnen, R.; Van Voorhis, W. C.Malaria medicines: a glass half full? Nat. Rev. Drug Discovery 2015, 14, 424– 442, DOI: 10.1038/nrd4573[CrossRef], [PubMed], [CAS]4.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFemsbvM&md5=43f35590f86c240a4150b5cb4f5670adWells, Timothy N. C.; Hooft van Huijsduijnen, Rob; Van Voorhis, Wesley C.Nature Reviews Drug Discovery (2015),A review. Despite substantial scientific progress over the past two decades, malaria remains a worldwide burden that causes hundreds of thousands of deaths every year. New, affordable and safe drugs are required to overcome increasing resistance against artemisinin-based treatments, treat vulnerable populations, interrupt the parasite life cycle by blocking transmission to the vectors, prevent infection and target malaria species that transiently remain dormant in the liver. In this Review, we discuss how the antimalarial drug discovery pipeline has changed over the past 10 years, grouped by the various target compd. or product profiles, to assess progress and gaps, and to recommend priorities. - 5.Baragana
, B.; Hallyburton, I.; Lee, M. C. S.; Norcross, N. R.; Grimaldi, R.; Otto, T. D.; Proto, W. R.; Blagborough, A. M.; Meister, S.; Wirjanata, G.; Ruecker, A.; Upton, L. M.; Abraham, T. S.; Almeida, M. J.; Pradhan, A.; Porzelle, A.; Martinez, M. S.; Bolscher, J. M.; Woodland, A.; Norval, S.; Zuccotto, F.; Thomas, J.; Simeons, F.; Stojanovski, L.; Osuna-Cabello, M.; Brock, P. M.; Churcher, T. S.; Sala, K. A.; Zakutansky, S. E.; Jimenez-Diaz, M. B.; Sanz, L. M.; Riley, J.; Basak, R.; Campbell, M.; Avery, V. M.; Sauerwein, R. W.; Dechering, K. J.; Noviyanti, R.; Campo, B.; Frearson, J. A.; Angulo-Barturen, I.; Ferrer-Bazaga, S.; Gamo, F. J.; Wyatt, P. G.; Leroy, D.; Siegl, P.; Delves, M. J.; Kyle, D. E.; Wittlin, S.; Marfurt, J.; Price, R. N.; Sinden, R. E.; Winzeler, E. A.; Charman, S. A.; Bebrevska, L.; Gray, D. W.; Campbell, S.; Fairlamb, A. H.; Willis, P. A.; Rayner, J. C.; Fidock, D. A.; Read, K. D.; Gilbert, I. H.A novel multiple-stage antimalarial agent that inhibits protein synthesis Nature 2015, 522, 315– 320, DOI: 10.1038/nature14451[CrossRef], [PubMed], [CAS]5.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFeiurzE&md5=bd5f1dd3c4389aa8a6f193df0aa068a1Baragana, Beatriz; Hallyburton, Irene; Lee, Marcus C. S.; Norcross, Neil R.; Grimaldi, Raffaella; Otto, Thomas D.; Proto, William R.; Blagborough, Andrew M.; Meister, Stephan; Wirjanata, Grennady; Ruecker, Andrea; Upton, Leanna M.; Abraham, Tara S.; Almeida, Mariana J.; Pradhan, Anupam; Porzelle, Achim; Martinez, Maria Santos; Bolscher, Judith M.; Woodland, Andrew; Norval, Suzanne; Zuccotto, Fabio; Thomas, John; Simeons, Frederick; Stojanovski, Laste; Osuna-Cabello, Maria; Brock, Paddy M.; Churcher, Tom S.; Sala, Katarzyna A.; Zakutansky, Sara E.; Jimenez-Diaz, Maria Belen; Sanz, Laura Maria; Riley, Jennifer; Basak, Rajshekhar; Campbell, Michael; Avery, Vicky M.; Sauerwein, Robert W.; Dechering, Koen J.; Noviyanti, Rintis; Campo, Brice; Frearson, Julie A.; Angulo-Barturen, Inigo; Ferrer-Bazaga, Santiago; Gamo, Francisco Javier; Wyatt, Paul G.; Leroy, Didier; Siegl, Peter; Delves, Michael J.; Kyle, Dennis E.; Wittlin, Sergio; Marfurt, Jutta; Price, Ric N.; Sinden, Robert E.; Winzeler, Elizabeth A.; Charman, Susan A.; Bebrevska, Lidiya; Gray, David W.; Campbell, Simon; Fairlamb, Alan H.; Willis, Paul A.; Rayner, Julian C.; Fidock, David A.; Read, Kevin D.; Gilbert, Ian H.Nature (London, United Kingdom) (2015),There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compd. with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clin. needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening program against blood-stage malaria parasites; its mol. target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along mRNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery. - 6.Brenk
, R.; Schipani, A.; James, D.; Krasowski, A.; Gilbert, I. H.; Frearson, J.; Wyatt, P. G.Lessons learnt from assembling screening libraries for drug discovery for neglected diseases ChemMedChem 2008, 3, 435– 444, DOI: 10.1002/cmdc.200700139[CrossRef], [PubMed], [CAS]6.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXlt1Krtb4%253D&md5=b804d0ac8766e954391798e24aa7fbffBrenk, Ruth; Schipani, Alessandro; James, Daniel; Krasowski, Agata; Gilbert, Ian Hugh; Frearson, Julie; Wyatt, Paul GrahamChemMedChem (2008),To enable the establishment of a drug discovery operation for neglected diseases, out of 2.3 million com. available compds. 222 552 compds. were selected for an in silico library, 57 438 for a diverse general screening library, and 1 697 compds. for a focused kinase set. Compiling these libraries required a robust strategy for compd. selection. Rules for unwanted groups were defined and selection criteria to enrich for lead-like compds. which facilitate straightforward structure-activity relationship exploration were established. Further, a literature and patent review was undertaken to ext. key recognition elements of kinase inhibitors ("core fragments") to assemble a focused library for hit discovery for kinases. Computational and exptl. characterization of the general screening library revealed that the selected compds. (1) span a broad range of lead-like space, (2) show a high degree of structural integrity and purity, and (3) demonstrate appropriate soly. for the purposes of biochem. screening. The implications of this study for compd. selection, esp. in an academic environment with limited resources, are considered. - 7.Burrows
, J. N.; Hooft van Huijsduijnen, R. H.; Mohrle, J. J.; Oeuvray, C.; Wells, T. N.Designing the next generation of medicines for malaria control and eradication Malar. J. 2013, 12, 187, DOI: 10.1186/1475-2875-12-187[CrossRef], [PubMed], [CAS]7.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3sjgsVSisA%253D%253D&md5=c4283af83b04bc217f40528dde196e23Burrows Jeremy N; van Huijsduijnen Rob Hooft; Mohrle Jorg J; Oeuvray Claude; Wells Timothy N CMalaria journal (2013),In the fight against malaria new medicines are an essential weapon. For the parts of the world where the current gold standard artemisinin combination therapies are active, significant improvements can still be made: for example combination medicines which allow for single dose regimens, cheaper, safer and more effective medicines, or improved stability under field conditions. For those parts of the world where the existing combinations show less than optimal activity, the priority is to have activity against emerging resistant strains, and other criteria take a secondary role. For new medicines to be optimal in malaria control they must also be able to reduce transmission and prevent relapse of dormant forms: additional constraints on a combination medicine. In the absence of a highly effective vaccine, new medicines are also needed to protect patient populations. In this paper, an outline definition of the ideal and minimally acceptable characteristics of the types of clinical candidate molecule which are needed (target candidate profiles) is suggested. In addition, the optimal and minimally acceptable characteristics of combination medicines are outlined (target product profiles). MMV presents now a suggested framework for combining the new candidates to produce the new medicines. Sustained investment over the next decade in discovery and development of new molecules is essential to enable the long-term delivery of the medicines needed to combat malaria. - 8.(a) Leeson
, P. D.; Young, R. J.Molecular property design: does everyone get it? ACS Med. Chem. Lett. 2015, 6, 722– 725, DOI: 10.1021/acsmedchemlett.5b00157[ACS Full Text
], [CAS]8a.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptVGksr4%253D&md5=5a807a8d049ce8cf4adc186891a9234dLeeson, Paul D.; Young, Robert J.ACS Medicinal Chemistry Letters (2015),A review. The principles of mol. property optimization in drug design have been understood for decades, yet much drug discovery activity today is conducted at the periphery of historical druglike property space. Lead optimization trajectories aimed at reducing physicochem. risk, assisted by ligand efficiency metrics, could help to reduce clin. attrition rates.(b) Hann, M. M.Molecular obesity, potency and other addictions in drug discovery MedChemComm 2011, 2, 349– 355, DOI: 10.1039/c1md00017a[CrossRef], [CAS]8b.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXls1Kht7g%253D&md5=954870db4b1d9e846612c3bb256582f7Hann, Michael M.MedChemComm (2011),A review. Despite the increase in global biol. and chem. knowledge the discovery of effective and safe new drugs seems to become harder rather than easier. Some of this challenge is due to increasing demands for safety and novelty, but some of the risk involved in this should be controllable if we had more effectively learned from our failures. This perspective reflects on some of the learnings of recent years in relation to the causes of attrition. The term Mol. Obesity is introduced to describe our tendency to build potency into mols. by the inappropriate use of lipophilicity which leads to the premature demise of drug candidates. - 9.(a) Ritchie
, T. J.; Macdonald, S. J. F.The impact of aromatic ring count on compound developability - are too many aromatic rings a liability in drug design? Drug Discovery Today 2009, 14, 1011– 1020, DOI: 10.1016/j.drudis.2009.07.014[CrossRef], [PubMed], [CAS]9a.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlCgt7nP&md5=48454c9809e8225b4336ea76aad3f3baRitchie, Timothy J.; MacDonald, Simon J. F.Drug Discovery Today (2009),A review. The impact of arom. ring count (the no. of arom. and heteroarom. rings) in mols. has been analyzed against various developability parameters, aq. soly., lipophilicity, serum albumin binding, CyP450 inhibition and hERG inhibition. On the basis of this anal., it was concluded that the fewer arom. rings contained in an oral drug candidate, the more developable that candidate is probably to be; in addn., more than three arom. rings in a mol. correlates with poorer compd. developability and, thus, an increased risk of attrition in development. Data are also presented that demonstrate that even within a defined lipophilicity range, increased arom. ring count leads to decreased aq. soly.(b) Ritchie, T. J.; MacDonald, S. J. F.; Young, R. J.; Pickett, S. D.The impact of aromatic ring count on compound developability: further insights by examiing carbo- and hetero-aromatic and -aliphatic ring types Drug Discovery Today 2011, 16, 164– 171, DOI: 10.1016/j.drudis.2010.11.014[CrossRef], [PubMed], [CAS]9b.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvVyqsr0%253D&md5=8e2d0e4e499d2f07d66ca2f385defb2dRitchie, Timothy J.; MacDonald, Simon J. F.; Young, Robert J.; Pickett, Stephen D.Drug Discovery Today (2011),A review. The impact of carboarom., heteroarom., carboaliph. and heteroaliph. ring counts and fused arom. ring count on several developability measures (soly., lipophilicity, protein binding, P 450 inhibition and hERG binding) is the topic for this review article. Recent results indicate that increasing ring counts have detrimental effects on developability in the order carboaroms. » heteroaroms. > carboaliphatics > heteroaliphatics, with heteroaliphatics exerting a beneficial effect in many cases. Increasing arom. ring count exerts effects on several developability parameters that are lipophilicity- and size-independent, and fused arom. systems have a beneficial effect relative to their nonfused counterparts. Increasing arom. ring count has a detrimental effect on human bioavailability parameters, and heteroarom. ring count (but not other ring counts) has increased over time in marketed oral drugs. - 10.El Ashry
, E. S. H.; Ramadan, E. S.; Abdel Hamid, H.; Hagar, M.Microwave-assisted synthesis of quinoline derivatives from isatin Synth. Commun. 2005, 35, 2243– 2250, DOI: 10.1080/00397910500184719[CrossRef], [CAS]10.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpvVShtb0%253D&md5=191be26d9a70311bbeaa8d54e5ee6333El Ashry, El Sayed; Ramadan, El Sayed; Abdel Hamid, Hamida; Hagar, MohamedSynthetic Communications (2005),Microwave irradn. (MWI) was used for a rapid and efficient synthesis of quinoline-4-carboxylic acids and 1,2,3,4-tetrahydroacridine-9-carboxylic acid from the reaction of isatins with acyclic and cyclic ketones in basic medium. 2-Hydroxyquinoline-4-carboxylic acid was also obtained by irradiating a mixt. of isatin and malonic acid in AcOH. The esters of 2-phenyl- and 2-hydroxy-quinoline-4-carboxylic acids their resp. hydrazides were also prepd. under MWI. - 11.Leeson
, P. D.; Springthorpe, B.The influence of drug-like concepts on decision-making in medicinal chemistry Nat. Rev. Drug Discovery 2007, 6, 881– 890, DOI: 10.1038/nrd2445[CrossRef], [PubMed], [CAS]11.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1els7rL&md5=5665a9e2dc266a1ee096e20625757ef7Leeson, Paul D.; Springthorpe, BrianNature Reviews Drug Discovery (2007),A review. Despite the wide acceptance of drug-like principles such as the 'rule of five', this anal. of mols. currently being synthesized in leading pharmaceutical companies reveals that their phys. properties differ significantly from those of recently discovered oral drugs. The marked increase in lipophilicity in particular could increase the likelihood of attrition in drug development. The application of guidelines linked to the concept of drug-likeness, such as the 'rule of five', has gained wide acceptance as an approach to reduce attrition in drug discovery and development. However, despite this acceptance, anal. of recent trends reveals that the phys. properties of mols. that are currently being synthesized in leading drug discovery companies differ significantly from those of recently discovered oral drugs and compds. in clin. development. The consequences of the marked increase in lipophilicity - the most important drug-like phys. property - include a greater likelihood of lack of selectivity and attrition in drug development. Tackling the threat of compd.-related toxicol. attrition needs to move to the mainstream of medicinal chem. decision-making. - 12.Dulla
, B.; Wan, B.; Franzblau, S. G.; Kapavarapu, R.; Reiser, O.; Iqbal, J.; Pal, M.Construction and functionalization of fused pyridine ring leading to novel compounds as potential antitubercular agents Bioorg. Med. Chem. Lett. 2012, 22, 4629– 4635, DOI: 10.1016/j.bmcl.2012.05.096[CrossRef], [PubMed], [CAS]12.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XovFWktL4%253D&md5=2f86e4b13258be4966f5da4a22f25e56Dulla, Balakrishna; Wan, Baojie; Franzblau, Scott G.; Kapavarapu, Ravikumar; Reiser, Oliver; Iqbal, Javed; Pal, ManojitBioorganic & Medicinal Chemistry Letters (2012),A series of fused and functionalized pyridine derivs. was designed, synthesized and tested for potential antitubercular properties. All these novel compds. were prepd. by using multistep methods involving the construction of the pyridine ring as a key synthetic step. Some of these compds. were found to be interesting when tested for their antitubercular properties in vitro and one of them, I, emerged as an attractive and potential antitubercular agent. In addn., mol. docking to the shikimate dehydrogenase protein of Mycobacterium tuberculosis was carried out for several of the target mols. - 13.Böhm
, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Müller, K.; Obst-Sander, U.; Stahl, M.Fluorine in medicinal chemistry ChemBioChem 2004, 5, 637– 643, DOI: 10.1002/cbic.200301023[CrossRef], [PubMed], [CAS]13.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2c3it1egug%253D%253D&md5=fa5ca2cfc278b83c80d5c96b18dfe312Bohm Hans-Joachim; Banner David; Bendels Stefanie; Kansy Manfred; Kuhn Bernd; Muller Klaus; Obst-Sander Ulrike; Stahl MartinChembiochem : a European journal of chemical biology (2004),Fluorinated compounds are synthesized in pharmaceutical research on a routine basis and many marketed compounds contain fluorine. The present review summarizes some of the most frequently employed strategies for using fluorine substituents in medicinal chemistry. Quite often, fluorine is introduced to improve the metabolic stability by blocking metabolically labile sites. However, fluorine can also be used to modulate the physicochemical properties, such as lipophilicity or basicity. It may exert a substantial effect on the conformation of a molecule. Increasingly, fluorine is used to enhance the binding affinity to the target protein. Recent 3D-structure determinations of protein complexes with bound fluorinated ligands have led to an improved understanding of the nonbonding protein-ligand interactions that involve fluorine. - 14.(a) Meister
, S.; Plouffe, D. M.; Kuhen, K. L.; Bonamy, G. M. C.; Wu, T.; Barnes, S. W.; Bopp, S. E.; Borboa, R.; Bright, A. T.; Che, J.; Cohen, S.; Dharia, N. V.; Gagaring, K.; Gettayacamin, M.; Gordon, P.; Groessl, T.; Kato, N.; Lee, M. C. S.; McNamara, C. W.; Fidock, D. A.; Nagle, A.; Nam, T.-g.; Richmond, W.; Roland, J.; Rottmann, M.; Zhou, B.; Froissard, P.; Glynne, R. J.; Mazier, D.; Sattabongkot, J.; Schultz, P. G.; Tuntland, T.; Walker, J. R.; Zhou, Y.; Chatterjee, A.; Diagana, T. T.; Winzeler, E. A.Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery Science 2011, 334, 1372– 1377, DOI: 10.1126/science.1211936[CrossRef], [PubMed], [CAS]14a.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFOjt7vN&md5=45b2993be36e36eabe1d9280c86c3db2Meister, Stephan; Plouffe, David M.; Kuhen, Kelli L.; Bonamy, Ghislain M. C.; Wu, Tao; Barnes, S. Whitney; Bopp, Selina E.; Borboa, Rachel; Bright, A. Taylor; Che, Jianwei; Cohen, Steve; Dharia, Neekesh V.; Gagaring, Kerstin; Gettayacamin, Montip; Gordon, Perry; Groessl, Todd; Kato, Nobutaka; Lee, Marcus C. S.; McNamara, Case W.; Fidock, David A.; Nagle, Advait; Nam, Tae-gyu; Richmond, Wendy; Roland, Jason; Rottmann, Matthias; Zhou, Bin; Froissard, Patrick; Glynne, Richard J.; Mazier, Dominique; Sattabongkot, Jetsumon; Schultz, Peter G.; Tuntland, Tove; Walker, John R.; Zhou, Yingyao; Chatterjee, Arnab; Diagana, Thierry T.; Winzeler, Elizabeth A.Science (Washington, DC, United States) (2011),Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 com. available compds. with previously demonstrated blood-stage activity (median inhibitory concn. < 1 micromolar) and identified chem. scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compds. active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 mg/kg) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chem. tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biol. of exo-erythrocytic forms.(b) Delves, M.; Plouffe, D.; Scheurer, C.; Meister, S.; Wittlin, S.; Winzeler, E. A.; Sinden, R. E.; Leroy, D.The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites PLoS Med. 2012, 9, e1001169, DOI: 10.1371/journal.pmed.1001169[CrossRef], [PubMed], [CAS]14b.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC383mtVeisw%253D%253D&md5=f2f23a53d58ad1ba583f575973b928b6Delves Michael; Plouffe David; Scheurer Christian; Meister Stephan; Wittlin Sergio; Winzeler Elizabeth A; Sinden Robert E; Leroy DidierPLoS medicine (2012),BACKGROUND: Malaria remains a disease of devastating global impact, killing more than 800,000 people every year-the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance. METHODS AND FINDINGS: Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony. CONCLUSIONS: These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle-wide analyses of drugs for other pathogens with complex life cycles. - 15.Duffy
, S.; Avery, V. M.Identification of inhibitors of Plasmodium falciparum gametocyte development Malar. J. 2013, 12, 408, DOI: 10.1186/1475-2875-12-408[CrossRef], [PubMed], [CAS]15.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmvFehs7k%253D&md5=5b38ae9bee99e2566f7765099ca7801cDuffy, Sandra; Avery, Vicky M.Malaria Journal (2013),Background: Plasmodium falciparum gametocytes, specifically mature stages, are the only stage in man transmissible to the mosquito vector responsible for malaria transmission. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria. The comprehensive profiling of in vitro activity of anti-malarial compds. against both early (I-III) and late (IV-V) stage P. falciparum gametocytes, along with the high throughput screening (HTS) outcomes from the MMV malaria box are described. Method: Two anti-gametocyte HTS assays based on confocal fluorescence microscopy, utilizing both a gametocyte specific protein (pfs16-Luc-GFP) and a viability marker (MitoTracker Red CM-H2XRos) (MTR), were used for the measurement of anti-gametocytocidal activity. This combination provided a direct observation of gametocyte no. per assay well, while defining the viability of each gametocyte imaged. Results: IC50 values were obtained for 36 current anti-malarial compds. for activities against asexual, early and late stage gametocytes. The MMV malaria box was screened and actives progressed for IC50 evaluation. Seven % of the "drug-like" and 21% of the "probe-like" compds. from the MMV malaria box demonstrated equiv. activity against both asexual and late stage gametocytes. Conclusions: The assays described were shown to selectively identify compds. with gametocytocidal activity and have been demonstrated suitable for HTS with the capability of screening in the order of 20,000 compds. per screening campaign, two to three times per seven-day week.
