Benzoxaborole Antimalarial Agents. Part 5. Lead Optimization of Novel Amide Pyrazinyloxy Benzoxaboroles and Identification of a Preclinical Candidate

Article abstract of DOI:10.1021/acs.jmedchem.7b00621

Carboxamide pyrazinyloxy benzoxaboroles were investigated with the goal to identify a molecule with satisfactory antimalarial activity, physicochemical properties, pharmacokinetic profile, in vivo efficacy, and safety profile. This optimization effort discovered 46, which met our target candidate profile. Compound 46 had excellent activity against cultured Plasmodium falciparum, and in vivo against P. falciparum and P. berghei in infected mice. It exhibited good PK properties in mice, rats, and dogs. It was highly active against the other 11 P. falciparum strains, which are mostly resistant to chloroquine and pyrimethamine. The rapid parasite in vitro reduction and in vivo parasite clearance profile of 46 were similar to those of artemisinin and chloroquine, two rapid-acting antimalarials. It was nongenotoxic in an Ames assay, an in vitro micronucleus assay, and an in vivo rat micronucleus assay when dosed orally up to 2000 mg/kg. The combined properties of this novel benzoxaborole support its progression to preclinical development.

Full text of DOI:10.1021/acs.jmedchem.7b00621

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Article
Benzoxaborole Antimalarial Agents. Part 5. Lead Optimization of Novel Amide
Pyrazinyloxy Benzoxaboroles and Identification of a Preclinical Candidate
Yong-Kang Zhang, Jacob J. Plattner, Eric E. Easom, Robert Toms Jacobs, Denghui
Guo, Yvonne R. Freund, Pamela Berry, Vic Ciaravino, John C. L. Erve, Philip J.
Rosenthal, Brice Campo, Francisco-Javier Gamo, Laura M. Sanz, and Jianxin Cao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00621 • Publication Date (Web): 21 Jun 2017
Downloaded from http://pubs.acs.org on June 22, 2017
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Benzoxaborole Antimalarial Agents. Part 5. Lead Optimization
of Novel Amide Pyrazinyloxy Benzoxaboroles and
Identification of a Preclinical Candidate
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YongꢀKang Zhang, Jacob J. Plattner, Eric E. Easom, Robert T. Jacobs, Denghui Guo,
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Yvonne R. Freund, Pamela Berry, Vic Ciaravino, John C. L. Erve, Philip J. Rosenthal, Brice
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Campo, FranciscoꢀJavier Gamo, Laura M. Sanz, Jianxin Cao
†
Anacor Pharmaceuticals, Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303, USA
§
Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, California 94143, USA
‡
Medicines for Malaria Venture, International Center Cointrin, Block G, 20 route de PréꢀBois, POB 1826, CH ꢀ
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215 Geneva
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Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760, Tres Cantos, Spain
┴
Shanghai ChemPartner, 998 Haꢀlei Road, Zhangjiang Highꢀtech Park, Pudong New Area, Shanghai 201203, China
KEYWORDS: Benzoxaborole; Antimalarial agent; Structure–activity relationship; Malaria;
Plasmodium falciparum; In vivo efficacy
ABSTRACT: Carboxamide pyrazinyloxy benzoxaboroles were investigated with the goal to
identify a molecule with satisfactory antimalarial activity, physicochemical properties,
pharmacokinetic profile, in vivo efficacy, and safety profile. This optimization effort discovered
46, which met our target candidate profile. Compound 46 had excellent activity against cultured
Plasmodium falciparum, and in vivo against P. falciparum and P. berghei in infected mice. It
exhibited good PK properties in mouse, rat and dog. It was highly active against other eleven P.
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falciparum strains, which are mostly resistant to chloroquine and pyrimethamine. The rapid
parasite in vitro reduction and in vivo parasite clearance profile of 46 were similar to those of
artemisinin and chloroquine, two rapidꢀacting antimalarials. It was nonꢀgenotoxic in an Ames
assay, an in vitro micronucleus assay and an in vivo rat micronucleus assay when dosed orally up
to 2000 mg/kg. The combined properties of this novel benzoxaborole support its progression to
preclinical development.
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INTRODUCTION
Malaria, a parasitic infection by the Plasmodium genera and spread through the bites of infected
mosquitoes, was responsible for an estimated 212 million clinical cases and 429,000 deaths
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worldwide in 2015, mostly among children under the age of five. This terrible disease, in
particular infection with Plasmodium falciparum, the most virulent human malaria parasite, traps
communities into poverty and disproportionately affects poor people who have limited access to
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health care. Following the discovery of artemisinin in the 1970’s , semiꢀsynthetic artemisininꢀ
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based combination therapies (ACTs) have become frontline medicines to treat malaria.
However, Plasmodium falciparum parasites in southeast Asia have shown evidence of resistance
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to artemisinins.
A deterioration of the susceptibility of P. falciparum to artemisinins and the
possibility that resistance will spread beyond the Greater Mekong subꢀregion are threats to global
strategies for the control, elimination and eradication of malaria. In addition, resistance to
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available artemisinin partner drugs is already widespread. Therefore, there is an urgent need to
enhance the pipeline of antimalarial medications that counter resistance and that are safe and
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easy for use in the most vulnerable populations.
In the process of creating richer antimalarial
development pipelines, many new chemical entities with different structures and sometimes also
with new mechanisms of action have entered preclinical and clinical development for the
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potential treatment of malaria in the last decade.
Maintaining such momentum as a sustained
effort is very important to the future of antimalarial therapeutics because attrition is expected
during drug development, and only a small portion of development compounds may reach the
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market. However, antimalarial drug discovery presents unique challenges. New antimalarials
should ideally meet multiple criteria, including excellent potency, ideally against multiple
plasmodial species; oral bioavailability, ideally with cure after a single dose; safety in children
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and pregnant women; and low cost of production.
We recently reported that 6ꢀ(2ꢀ(methoxycarbonyl)pyrazinylꢀ5ꢀoxy)ꢀ1,3ꢀdihydroꢀ1ꢀhydroxyꢀ
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,1ꢀbenzoxaborole (1 in Fig. 1) demonstrated potent in vitro activity against cultured parasites
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IC50 = 1.4 nM and 1.9 nM against P. falciparum W2 and 3D7 strains, respectively) and
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excellent in vivo efficacy against P. berghei in infected mice (ED90 = 7.0 mg/kg). The
limitation of this molecule was its poor oral pharmacokinetic (PK) profile, exemplified by a short
halfꢀlife (t = 1 h) and low bioavailability (F = 23%) in mice. This profile is readily attributable
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to hydrolysis of the methyl ester to the corresponding carboxylic acid. During this phase of the
project, it was also found that the primary carboxamide analog (2 in Fig. 1) had a muchꢀ
improved PK profile in mice (t1/2 = 2.1 h and F = 99%) and moderate in vivo efficacy (ED =
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8.7 mg/kg) despite being significantly less potent in vitro (IC = 210 nM against W2 and 350
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nM against 3D7). We also learned that incorporation of a 7ꢀMe substituent on the
benzoxaborole core can improve PK properties. Therefore, we focused our effort on the 7ꢀmethyl
benzoxaborole pyrazine carboxamide scaffold to optimize in vitro potency, stability of the
carboxamide group and PK profile in order to ultimately provide molecule(s) with better in vivo
efficacy. More specifically, we report here systematic modification of carboxamide region to
generate analogs 3ꢀ51 (Fig. 1), which were evaluated against P. falciparum; selected compounds
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were further profiled in a range of in vitro ADME, in vivo PK and in vivo mouse efficacy
models.
The
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6ꢀ(2ꢀ((3ꢀhydroxyꢀ3ꢀmethylazetidinꢀ1ꢀ
yl)carbonyl)pyrazinylꢀ5ꢀoxy)ꢀ1,3ꢀdihydroꢀ1ꢀhydroxyꢀ7ꢀmethylꢀ2,1ꢀbenzoxaborole (46), was
progressed to evaluate potency against resistant P. falciparum strains, in vivo parasite reduction
rate, and to preliminary genotoxicity studies. The results obtained on 46 support its progression
to preclinical development.
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CHEMISTRY
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The chemistry for the synthesis of compounds 1 and 2 was described previously. Preparation
of molecules 3ꢀ51 shared a common benzoxaborole intermediate 58, for which the synthetic
route is illustrated in Scheme 1. Formylation of 2ꢀmethylresorcinol (52) generated the aldehyde
53, which was selectively protected to give 54. The remaining hydroxyl group of 54 was
converted to a boronate ester in 56 via the triflate 55 and palladiumꢀmediated boronylation.
Reduction of the aldehyde 56 and simultaneous cyclization produced the 6ꢀOꢀbenzyl
benzoxaborole 57, which was deprotected by catalytic hydrogenation to give 58. Methods for the
synthesis of various substituted 2ꢀaminoethanol intermediates are illustrated in Schemes 2ꢀ5. The
condensation product 63 from 61 and 62 reacted with the Grignard reagent 60, which was
generated from 59, to provide the silylmethyl adduct 64. Oxidative desilylation of 64 gave 65,
followed by hydrolysis to yield the desired 2ꢀaminoethanol 66. Its enantiomer 68 was obtained
by following the same methods starting from 67. For the preparation of 74 in Scheme 3, the
triflate (70) of trifluoroethanol (69) was used to alkylate 71, providing the alkylation product 72
that was hydrolyzed to provide the amino acid 73, which was reduced to the aminoalcohol 74. In
Scheme 4, the ester group of the cyclization product 77 from 75 and 76 was reduced to the
alcohol 78. Methylation of 78 produced the ether 79, which was hydrolyzed to give the 2ꢀ
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aminoethanol 80 containing the ether side group. For the synthesis of the thioether analog 86, the
Bocꢀprotected ester product 83 from 81 and 82 was methylated to give 84 that was hydrolyzed
under an acidic condition to afford 85, followed by reduction resulting in 86. As shown in
Scheme 5, the tertiary alcohols 93ꢀ95 and 102ꢀ104 can be conveniently generated by Grignard
reaction from their corresponding amino acid methyl esters 90ꢀ92 and 99ꢀ101, which were
obtained from amino acids 87ꢀ89 and 96ꢀ98, respectively. Synthesis of substituted azetidines
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112ꢀ117 is shown in Scheme 6. Protected azetidinone 105 was reacted with nucleophilic agents,
RMgBr or Me SiCF , to give the alcohols 106ꢀ111 that were deprotected by catalytic
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hydrogenation to provide the hydroxylated azetidines 112ꢀ117. With both the key benzoxaborole
and amine fragments in hand, two general methods to generate the amides 3ꢀ51 have been used
as illustrated in Scheme 7. In general method A, the pyrazine amides were prepared from amines
118 and acid 119 prior to the substitution reaction between the phenolic benzoxaborole 58 and
the amide pyrazinyl chloride 120. In general method B, the amide formation was conducted on
the acid intermediate 123 derived from the ester 122, which was produced by a substitution
reaction between 58 and 121.
RESULTS AND DISCUSSION
Structure-Activity Relationships. The in vitro activities (IC s) of test compounds for
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growth inhibition of the 3D7 (chloroquine sensitive) and W2 (chloroquine resistant) strains of P.
falciparum were determined (see Table 1). Compound 3 with a Nꢀ(2’ꢀmethoxyethyl) group at the
amide had similar potency as 4 with a Nꢀ(2’ꢀhydroxyethyl) group. Compound 5 with an extra 2’ꢀ
methyl, designed to decrease oxidative metabolism of the hydroxyl group, was slightly less
potent than 4, whereas the 2’,2’ꢀdimethyl analog 6 was equipotent with 4. The corresponding
1’,1’ꢀdimethyl analog 7 was also equipotent with 4, but its cyclopropyl analog 8 had decreased
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activity. More analogs (9ꢀ29) with various 1’ꢀmonoalkyl substituents in the amide side chain, to
modulate lipophilicities and improve PK properties, had potencies ranging from 31 to 1990 nM
and showed that the (R)ꢀenantiomer was generally slightly more active than the (S)ꢀisomer.
Compounds with 1’,2’ꢀdialkyls (30ꢀ33) mostly gave potency around 200 nM. Similar activities
were seen with (34ꢀ39) that have 1’,2’,2’ꢀtrialkyls in the amide side chain. Targeting to improve
the metabolic stability of the amide bond, N,Nꢀdisubstituted amide analogs (40ꢀ44) were
synthesized, and their IC values ranged from 48 to 437 nM indicating that these substitutions
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were tolerated. Considering this N,Nꢀdisubstitution pattern and a previous observation that
compounds 37 and 38 containing a terminal tertiary alcohol had good activity promoted the idea
to synthesize the cyclic pyrrolidinyl molecule 45, which showed IC = 139 nM (W2 strain).
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Further structural simplification to remove the chiral center in 45 resulted in the methylazetidinyl
compound 46 with improved potency of IC 33 nM (W2 strain) and 43 nM (3D7 strain). Five
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additional azetidinyl analogs (47ꢀ51) with ethyl, nꢀpropyl, isopropyl, cyclopropyl or
trifluoromethyl replacing the 3’ꢀmethyl at azetidine in 46 were also very active (IC from 7 nM
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to 134 nM).
Metabolic Stability and Pharmacokinetic Study. As in vitro potency was maintained with
a relatively broad array of amide substitution patterns, a key driver for the project was to build
good pharmacokinetic properties into the series to enable provision of a single dose cure. This
feature is critical for treatment of malaria patients in disease endemic areas to ensure compliance.
Therefore, the pharmacokinetic properties of selected compounds following oral administration
to mice were determined (Table 2). The hydroxyethyl amide 9 had high plasma concentration,
good exposure, moderate halfꢀlife, and excellent bioavailability. It was hypothesized that
increasing length of exposure (as indicated by halfꢀlife) was important to achieve our goal of a
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single dose cure molecule. In order to achieve this goal, we further hypothesized that increasing
molecule lipophilicity would lead to a higher volume of distribution and a longer halfꢀlife. Of
course, it was important that changes in lipophilicity could not be large, as it was expected that
metabolic stability could be adversely affected by such changes. Therefore, compounds 9 with
methyl substitution on the amide side chain (cLogD = 0.66), 20 with trifluoroethyl (cLogD =
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1.54) and 14 with nꢀpropyl (cLogD = 1.53) were compared. Compound 14 gave a longer halfꢀlife
(4.5 h) than 9 (2.7 h) and 20 (3.0 h), while higher clearance was unsurprisingly observed with
more lipophilic compounds (CL = 243, 238 and 128 mL/h/kg with 14, 20 and 9, respectively).
To further improve the halfꢀlife of 14, minimizing the amide hydrolysis was strategized by using
N,Nꢀdisubstitution to generate steric hindrance around the amide bond. Encouragingly, the potent
3ꢀmethylꢀ3ꢀhydroxyazetidinyl amide 46 had a longer halfꢀlife (6.8 h) in mice following oral
administration which enabled us to combine high in vitro antiparasitic activity and good in vivo
pharmacokinetic properties in a single molecule. Five additional azetidinyl amide analogs were
designed and synthesized with variation of the alkyl to ethyl (47), nꢀpropyl (48), isopropyl (49),
cyclopropyl (50) or trifluoromethyl (51). In order to differentiate these six close analogs, their
metabolic stabilities were assessed in an in vitro rat and human hepatocyte assays, which
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incubated 7 x 10 cells/well and 20 µM benzoxaborole for 44 hours. In the experiment with rat
heptocytes, and as expected, an inverse relationship between metabolic stability and lipophilicity
was observed. The rank order of stability was 46 (Me) > 47 (Et) > 50 (cꢀPr) > 49 (iꢀPr) > 48 (nꢀ
Pr) » 51 (CF ). In human hepatocytes, the compounds were generally more stable than in rat
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hepatocytes, and the rank order was similar, e.g. 46 (Me) ≈ 47 (Et) ≈ 50 (cꢀPr) > 49 (iꢀPr) ≈ 48
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dog pharmacokinetic studies. Data from these studies is presented in Table 2. Briefly, compound
46 exhibited excellent oral exposure, low clearance and long halfꢀlife in both species.
In Vivo Efficacy in P. berghei and P. falciparum Mouse Models. Compounds were selected
for in vivo testing based on their chemical diversity, in vitro potency, physicochemical properties
and pharmacokinetic properties. Twelve compounds (4, 9, 12, 14, 16, 20, 46, 47, 48, 49, 50 and
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1) were tested in a P. berghei mouse model. In this model, compounds were dosed orally
once a day for four consecutive days, beginning 1 hour after infection by intraperitoneal
administration of P. bergheiꢀinfected erythrocytes. Compound 4 with the hydroxyethyl side
chain to the amide nitrogen had a low efficacy of ED = 56.1 mg/kg. Due to the improved
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pharmacokinetic profiles exhibited by hydroxyethyl amides containing substituents, in vivo
efficacy was also improved for compounds 9 (ED = 44.3 mg/kg), 14 (ED = 11.7 mg/kg) and
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this trend (ED = 26.7 mg/kg). The azetidinyl amide analogs (46ꢀ51) further improved efficacies
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with the ED values ranging from 1.9 to 10.3 mg/kg, and 46 had the best efficacy among these
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molecules, which was consistent with the result of in vitro metabolic stability evaluation.
Selected compounds were also tested in a P. falciparum mouse model, which evaluates the
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therapeutic efficacy of a compound against strain Pf3D7
growing in the peripheral blood of
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NODscidIL2Rγ mice engrafted with human erythrocytes (Table 4). Compounds were dosed
orally once a day for four consecutive days. Compound 14 demonstrated excellent efficacy (ED90
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azetidinyl amide analogs (46, 47, 49, 50 and 51), an initial test indicated that all of these five
molecules were very efficacious (ED < 5 mg/kg). Based on our understanding of metabolic
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for determination of their ED values. In these experiments, potency and exposure of 46 (ED
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similar (Table 4). Furthermore, as shown in Figure 2, the result of the P. falciparumꢀinfected
mouse model experiment demonstrated that the in vivo parasite clearance profile of 46 was rapid
and similar to artesunate and chloroquine, two wellꢀknown fast parasiteꢀkilling antimalarial
medicines.
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Since compound 46 demonstrated the best overall profile of the in vitro potency, in vitro
metabolic stability, PK properties and in vivo efficacies amongst these amides 3ꢀ51, it was
further studied for activity against other P. falciparum strains, in vitro parasite reduction rate
(PRR), and genotoxicity properties.
Since the malaria patient population includes many children and women within childbearing
years, safety of antimalarial treatment has the profound priority. This research program adapted a
strategy at research stage to screen representative compounds in micronucleus genetic toxicity
assays. Compound 4 with a terminal hydroxyl group on the ethyl was in vitro micronucleus
negative. It was further tested in a micronucleus in vivo rat model, and the result was also
micronucleus negative.
Activities of Compound 46 against Multiple P. falciparum Parasite Strains. As
summarized in Table 5, compound 46 was tested against an additional eleven P. falciparum
strains and demonstrated excellent potency with IC ’s in the range of 36ꢀ80 nM. Many of these
50
parasite strains are chloroquine and pyrimethamine resistant, which indicated that compound 46
would not be expected to be cross resistant against these multiple drug resistant strains and it
may have possible novel mechanism of action or resistance distinct from those of chloroquine
and pyrimethamine.
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In Vitro P. falciparum Parasite Killing Rate of Compound 46. A parasite reduction ratio
PRR) P. falciparum in vitro assay was used to compare parasite killing rates at different
(
concentrations. Three concentrations used were 10×, 30× and 100× IC . The assay allowed us to
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determine the molecule’s important antiparasitic parameters such as lag phase (the time needed
to show maximal killing), PRR (parasite reduction ratio or number of parasites the compound
could kill in a parasite life cycle) and PCT99.9% (parasite clearance time to kill 99.9% of the initial
parasite population). The timeꢀkill curves for compound 46 in Figure 3 suggested that the
concentration required for 46 to achieve a maximal rate of parasiteꢀkilling was 10× to 30× IC .
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The data were analyzed to fit to the linear portion of the curve to determine the PRR parameters
as summarized in Table 6, together with standard antimalarial agents for comparison. The result
indicated that the antiparasitic rate of action of compound 46 was fast and similar to the rates for
artemisinin and chloroquine.
Ames and Genetic Toxicity Test Results of Compound 46. The bacterial reverse mutation
assay (Ames test) was conducted to evaluate the potential of compound 46 to induce mutation in
Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 as well as the E. coli strain
WP2 uvrA in the presence and absence of an exogenous metabolic activation system (S9). The
results are presented in Table 7 demonstrating that molecule 46 was not mutagenic in the Ames
assay under the conditions tested. The molecule was also tested in the in vitro micronucleus
assay using human lymphocytes in the presence and absence of an Aroclor 1254 induced rat liver
S9 activation system for evaluating its clastogenic potential. A dose range cytotoxicity test was
performed, establishing the dose range for the definitive micronucleus assay, which was ≤
355.15 ꢁg/ mL of 46. In the in vitro micronucleus assay, human lymphocytes were treated for 3
and 28 hours in the nonꢀactivated test system and for 3 hours in the S9 activated test system. As
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shown in Table 8, in comparison with the 1% DMSO control, there were no statistically
significant increases in the frequency of micronucleated cells observed in the groups treated with
compound 46 under any conditions, indicating that compound 46 was not clastogenic at 3 h, with
and without S9 activation, and at 28 h without S9 activation. This encouraging result led us to
test compound 46 in the in vivo rat micronucleus model for further genotoxicity assessment by
analyzing micronuclei in polychromatic erythrocyte cells (PCEs) in rat bone marrow. Three
single oral doses were tested at 500, 1000 and 2000 mg/kg. The toxicokinetic plasma samples
were collected and analyzed. The exposure of 46 at the highest dose of 2000 mg/kg was 742
µg×h/mL, confirming that there was adequate exposure of 46 in the in vivo micronucleus assay.
There were no statistically significant increases in the incidence of micronucleated PCEs
(MnPCEs) in the 46ꢀtreated groups relative to the negative control group, and therefore, the
compound did not have a micronucleus risk at all three test doses in this study (Table 9).
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CONCLUSION
In summary, this lead optimization program has discovered a new potent antimalarial agent, 6ꢀ
(2ꢀ((3ꢀhydroxyꢀ3ꢀmethylazetidinꢀ1ꢀyl)carbonyl)pyrazinylꢀ5ꢀoxy)ꢀ1,3ꢀdihydroꢀ1ꢀhydroxyꢀ7ꢀ
methylꢀ2,1ꢀbenzoxaborole (46). This molecule had good pharmacokinetic properties and
demonstrated excellent in vivo efficacies against P. falciparum (ED = 0.85 mg/kg) and against
90
P. berghei (ED = 1.92 mg/kg) in infected mice. It was highly active against various multidrug
9
0
resistant P. falciparum strains, indicating no crossꢀresistance and a possible novel mechanism of
action. In addition, the rapid parasite in vitro reduction and in vivo clearance profile of 46 was
similar to those of artemisinin and chloroquine. Exploratory safety studies demonstrated it was
Ames negative and micronucleus negative in both in vitro and in vivo models. Based on the
current data, this novel benzoxaborole has been selected as a preclinical development candidate.
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EXPERIMENTAL SECTION
. General Methods. Starting materials and solvents purchased from chemical companies were
used without further purification except where noted. High performance liquid chromatography
1
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(HPLC) was used to determine the purity of the compounds synthesized. The data confirmed that
1
the target compounds had ≥95% purity. Proton ( H) NMR spectra were recorded at room
temperature on Bruker 300 or 400 instruments (Bruker Corporation, Billerica, Massachusetts,
USA) using DMSOꢀd as solvent. Chemical shifts are given in parts per million (ppm).
6
Electrospray ionization–mass spectrometry (ESI–MS) and LC–MS were carried out on an
API2000 (AB Sciex, Framingham, Massachusetts, USA) or a Finnigan LCQ mass spectrometer
(Thermo Fisher Scientific, Waltham, Massachusetts, USA). HPLC was conducted on an Agilent
1200 system (Agilent Technologies, Santa Clara, California, USA) using a BDS Hypersil Cꢀ18
column (150×4.6 mm, 5 micron, 120 nm pore size, Thermo Fisher Scientific, Waltham,
Massachusetts, USA). The mobile phase used was composed of buffer A (H O containing 0.1%
2
phosphoric acid) and buffer B (CH CN). The column was eluted with a gradient of 95% buffer A
3
and 5% buffer B to 40% buffer A and 60% buffer B over 10 min, followed by 40% buffer A and
60% buffer B for 1 min, followed by a gradient over 1 min to 95% buffer A and 5% buffer B that
was maintained for three more minutes at a flow rate of 1.0mL/min with a column temperature
o
of 30 C and UV detection at 220 nm and 254 nm. Silica gel 60 nm (200–425 mesh, Thermo
Fisher Scientific, Waltham, Massachusetts, USA) was used for flash column chromatography.
2. Care and use of experimental animals. Pharmacokinetic studies with experimental animals
were designed and performed with reference to the guidelines of the Institutional Animal Care
and Use Committee (Anacor Pharmaceuticals, Inc., ANꢀ7ꢀ2007). The in vivo efficacy work with
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experimental animals was performed in accordance with the institutional guidelines as defined
by the Institutional Animal Care and Use Committee (UCSF, AN109984).
3. Methods for testing in vitro antimalarial activity. Experimental procedures used for
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determining in vitro antimalarial activities of the newly synthesized benzoxaboroles against a
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variety of P. falciparum parasite strains can be found in a previous report.
4
. Method for pharmacokinetics measurement. Female CDꢀ1 mice, male SD rats, and beagle
dogs received the benzoxaborole test material either by intravenous injection or by oral gavage in
formulated solutions. The dose of 4, 9, 14, 20 and 46 was set at 5 mg/kg for both iv and oral, and
the formulation used polyethylene glycol 300, propylene glycol and water (ratio 55/25/20, pH =
6). Blood samples were collected and analyzed for drug content using HPLC coupled to tandem
mass spectrometry.
5. Method for testing in vivo efficacy using a P. berghei mouse model. Molecules 4, 9, 12, 14,
16, 20, 46, 47, 48, 49, 50 and 51 were tested in a P. berghei mouse malaria model by following
2
0
the method reported previously.
6. Method for testing in vivo efficacy using a P. falciparum mouse model. Efficacies of
compounds 14, 46, 47, 49, 50 and 51 in a P. falciparum mouse malaria model were evaluated by
2
1
using the method described previously.
7. Method of in vitro parasite reduction ratio (PRR) assay. The parasite reduction ratio
2
2
parameters of molecule 46 were determined by using the procedures reported previously.
8. Method for testing 46 in bacterial reverse mutation assay (Ames assay). The study
evaluated the potential of molecule 46 to induce reverse mutation in Salmonella typhimurium
strains TA98, TA100, TA1535 and TA1537 and the E. coli strain WP2 uvrA in the presence and
absence of an exogenous metabolic activation system (rat liver S9). The compound was
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dissolved in DMSO and tested using the plate incorporation method at eight dose levels (1.5, 5,
5, 50, 150, 500, 1500, 5000 µg/plate). All positive and vehicle control values were within
acceptable ranges and all criteria for a valid assay were met.
. Methods of in vitro and in vivo micronucleus assays. The compound was tested in the in
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vitro micronucleus assay in human lymphocytes in the presence or absence of a rat liver S9
metabolic activation system to evaluate its clastogenic/aneugenic potential. A dose rangeꢀfinding
assay was performed to establish the dose range for the definitive micronucleus assay. In the
dose rangeꢀfinding assay, human lymphocytes were treated for 3 and 28 hours in the absence of
S9 and for 3 hours in the presence of S9. All cells were harvested 28 hours after initiation of test
article treatment. The test article was dissolved in DMSO at concentrations up to 355.15 mg/mL
(1 mM). No precipitate was observed in the treatment medium at any concentration at the
beginning and the end of the treatment period. Single cultures of human lymphocytes were
incubated with the compound at 0.5, 1, 2.5, 5, 10, 25, 50, 100, 200, and 355 ꢁg/mL in all three
exposure series. Selection of concentrations for the definitive micronucleus assay was based on
the cytotoxicity of the test article relative to the concurrent solvent control. At least 1000
binucleated cells for each culture and 2000 for each concentration were scored. The validity of
the assay was confirmed.
The in vivo mammalian erythrocyte micronucleus assay in rats was conducted according to
2
4,25
established procedures.
The test article was evaluated for its clastogenic activity and/or
disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte cells in
rat bone marrow. Carboxymethylcellulose (CMC, 1%, medium viscosity) in deionized water was
used as the vehicle. Test and control article formulations were administered at a dose volume of
1
0 mL/kg via oral gavage. In the dose range finding assay, doses tested were 500, 1000 and 2000
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mg/kg in three male rats/group. No mortality occurred at any dose. All animals appeared normal
throughout the observation period. Therefore, the maximum tolerable dose for the definitive
micronucleus assay was set at 2000 mg/kg, which is the maximum regulatory required dose. In
the definitive assay, the study was designed to include eight groups (five rats/group): two vehicle
groups for euthanasia 24 and 48 h after treatment, one 500 mg/kg/day group and one 1000
mg/kg/day for euthanasia at 24 h, two 2000 mg/kg/day groups for euthanasia at 24 and 48 h, one
positive control (cyclophosphamide monohydrate, CP) group and one vinblastine positive
control group for CREST analysis. Femoral bone marrow was collected at approximately 24 or
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48 h after the dose. Animals were euthanized by carbon dioxide inhalation. Immediately
following euthanasia, the femurs were exposed, and cut just above the knee. The bone marrow
was transferred to a centrifuge tube containing approximately 2 mL fetal bovine serum and the
cells were pelleted by centrifugation. The supernatant was drawn off leaving a small amount of
fetal bovine serum with the pellet. Four preꢀcolumn slides for micronucleus evaluation and two
postꢀcolumn slides for CREST analysis were prepared from each animal. The slides were airꢀ
dried and fixed by dipping in methanol. One set of two preꢀcolumn slides was stained with
acridine orange for microscopic evaluation. The other set of slides was kept as backup. Postꢀ
column slides for potential CREST staining were prepared by processing the remaining bone
marrow suspensions through a cellulose column containing 0.6 g of a 50:50 mixture of cellulose
type 50 and alphaꢀcellulose (one column per animal) and using 6 mL fetal bovine serum as the
eluate. Eluted cells were pelleted by centrifugation, and the supernatant was drawn off leaving a
small amount of fetal bovine serum with the pellet. The cell pellet was reꢀsuspended for slide
preparation. Two postꢀcolumn slides per animal were prepared at relatively high cell density and
stored at ꢀ10 to ꢀ30 ºC for CREST analysis. Each pre and postꢀcolumn slide was identified by the
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harvest date, study number and animal number. Slides were coded using a random number table
by an individual not involved with the scoring process. Post column slides were reserved only for
CREST analysis. Initially, one set of postꢀcolumn slides was stained with acridine orange for
micronucleus evaluation and the other set was kept as backup.
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10. Methods for the syntheses of final amide benzoxaborole compounds 3-51. General
Method A (Substitution Method): This method involved a substitution reaction between the
benzoxaborole intermediate 58 and an amide intermediate 120 in the final step. Representative
experimental procedures are described below. 7ꢀMethylbenzo[c][1,2]oxaboroleꢀ1,6(3H)ꢀdiol
(
58), 5ꢀchloropyrazineꢀ2ꢀcarboxamide (120, 1.1 eq), Cs CO (2.5 eq) and DMF were added to a
2 3
o
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ꢀnecked flask. The reaction mixture was stirred at 40ꢀ50 C until completion as monitored by
HPLC. Water was added, and then HCl (0.2 N) was added dropwise to the mixture. It was stirred
for 20 min while white solid was precipitated. It was filtered and the solid cake was dissolved in
NaOH (1 N) and extracted by dichloromethane. The separated inorganic layer was acidified by
HCl (1 N) to pH = 3, and the white solid precipitate was collected by filtration. The solid cake
was stirred in water for 30 min and filtered. And then the solid was stirred in acetone and
filtered. The solid was dried under high vacuum to give the desired product. If the compound
didn’t meet the HPLC purity requirement (≥95%), it was further purified by column
chromatography so that the material reached ≥95% purity by HPLC. General Method B
(Amidation Method): This method included an amidation reaction from the benzoxaborole acid
intermediate 123 and an amine intermediate in the final step. Representative experimental
procedures are described below. To a solution of 5ꢀchloropyrazineꢀ2ꢀcarboxylic acid (7.9 g, 50
o
mmol) in MeOH (50 mL) was added SOCl (3.0 g, 25 mmol) dropwise at 0 C. The reaction
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mixture was stirred at 70 C for 2 h, and then concentrated under the reduced pressure to give
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methyl 5ꢀchloropyrazineꢀ2ꢀcarboxylate (4.5 g) as a white solid which was used in next step
without further purification. MS (ESI+): m/z = 173 (M+1). A solution of this intermediate (2.9 g,
1
6.86 mmol), 7ꢀmethylbenzo[c][1,2]oxaboroleꢀ1,6(3H)ꢀdiol (1.94 g, 11.80 mmol) and Cs CO
2 3
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(
7.74 g, 23.60 mmol) in DMF (20 mL) was stirred at rt for 4 h. The mixture was poured into
water (500 mL) and the solid was collected. The solid was dried under the reduced pressure to
give methyl 5ꢀ(1ꢀhydroxyꢀ7ꢀmethylꢀ1,3ꢀdihydrobenzo[c][1,2]oxaborolꢀ6ꢀyloxy)pyrazineꢀ2ꢀ
carboxylate (2.6 g, yield 73%) as a lightꢀbrown solid. MS (ESI+): m/z = 301 (M+1). To a
solution of this ester intermediate (2.6 g, 8.7 mmol) in MeOH (30 mL) was added 1N LiOH (34
mL). The reaction mixture was stirred at rt for 3 h, and then 1N HCl was added to pH = 5. The
solid was collected and dried under the reduced pressure to give the acid 123 (2.3 g, yield 93%)
1
as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 13.40 (br. s, 1H), 9.09 (s, 1H), 8.73ꢀ8.72 (m,
6
1
H), 8.68ꢀ8.67 (m, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 5.01 (s, 2H), 2.24 (s,
H) ppm; HPLC purity: 100% at 214 nm and 100% at 254 nm; MS (ESI+): m/z = 287 (M+1). To
3
a solution of 123 (100 mg, 0.35 mmol) and DIPEA (135 mg, 1.05 mmol) in DMF (3 mL) was
added HATU (200 mg, 0.53 mmol). The reaction mixture was stirred at rt for 30 min, and then
the corresponding amine (0.35mmol) was added. The reaction mixture was stirred at rt overnight.
The crude product obtained from a normal workꢀup was purified by prepꢀHPLC to give the final
product as a solid.
5-(1-Hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(2-methoxyethyl)
pyrazine-2-carboxamide (3). To a solution of 5ꢀchloropyrazineꢀ2ꢀcarboxylic acid (2.0g, 12.62
mmol) in DCM (42 mL) was added SOCl (42 mL) at room temperature. The reaction was
2
o
stirred at 50 C overnight and then concentrated directly. To a solution of the residue in pyridine
(42mL) was added 2ꢀmethoxyethylamine (1.69 g, 22.52 mmol, 1.78 eq ) at room temperature.
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The reaction was stirred at 50 C for 3 h. The mixture was poured into water (100 mL), and then
extracted with EA (3 × 50 mL). The combined organic phase was washed with brine, dried over
MgSO , filtered and concentrated in vacuum to give the residue of 5ꢀchloroꢀNꢀ(2ꢀ
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methoxyethyl)pyrazineꢀ2ꢀcarboxamide, which was used in the next step without further
purification. The final step was conducted by following the procedure described in general
1
method A. It was obtained in 13% yield of the final step as a white solid. H NMR (300 MHz,
DMSOꢀd ): δ 9.07 (s, 1H), 8.66 (s, 1H), 8.62ꢀ8.59 (m, 1H), 8.60 (s, 1H), 7.29 (d, J = 8.1 Hz,
6
1H), 7.25 (d, J = 8.1 Hz, 1H), 4.99 (s, 2H), 3.55ꢀ3.40 (m, 4H), 3.26 (s, 3H), 2.22 (s, 3H) ppm.
HPLC purity: 98.2% at 220 nm and 96.9% at 254 nm; Mass (ESI+): m/z = 344 (M+1).
5
-(1-Hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(2-hydroxyethyl)
pyrazine-2-carboxamide (4). To a solution of 5ꢀchloropyrazineꢀ2ꢀcarboxylic acid (500 mg, 3.16
mmol) and TEA (352 mg, 3.47 mmol, 1.1 eq) in DCM (15.8 mL) was added isobutyl
o
chloroformate (944 mg, 6.96 mmol, 1.1 eq) at 0 C. The reaction mixture was stirred for 20 min.
o
Then 2ꢀaminoethanol (231 mg, 3.79 mmol, 1.2 eq) was added at 0 C and the reaction mixture
was stirred at rt for 1 h. After completion, the mixture was poured into water (100 mL) and
extracted with DCM (2 × 20 mL). The combined organic phase was washed with brine, dried
over MgSO , filtered and concentrated in vacuum. The residue was purified by column
4
chromatography to give 5ꢀchloroꢀNꢀ(2ꢀhydroxyethyl)pyrazineꢀ2ꢀcarboxamide (480 mg, 75%
yield) as a white solid. The next step was conducted by following general method A. The final
1
product was obtained in 51% yield of the final step as an offꢀwhite solid. H NMR (300 MHz,
DMSOꢀd ): δ 9.05 (s, 1H), 8.65 (s, 1H), 8.59 (s, 1H), 8.58ꢀ8.55 (m, 1H), 7.28 (d, J = 8.1 Hz,
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H), 7.24 (d, J = 8.1 Hz, 1H), 4.98 (s, 2H), 4.76 (t, J = 5.4 Hz, 1H), 3.54ꢀ3.48 (m, 2H), 3.39ꢀ3.30
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m, 2H), 2.21 (s, 3H) ppm; HPLC purity: 99.6% at 220 nm and 99.3% at 254 nm; Mass (ESI+):
m/z = 330 (M+1).
-(1-Hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(2-hydroxypropyl)
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pyrazine-2-carboxamide (5). This compound was synthesized by using 2ꢀhydroxypropylamine
as a starting material and following the procedures used for the analog 3. It was obtained in 29%
1
yield of the final step as a solid. H NMR (300 MHz, DMSOꢀd ): δ 9.06 (s, 1H), 8.66 (d, J = 1.2
6
Hz, 1H), 8.60 (d, J = 1.2 Hz, 1H), 8.48 (t, J = 6.0 Hz, 1H), 7.29 (d, J = 8 Hz, 1H), 7.25 (d, J = 8
Hz, 1H), 4.98 (s, 2H), 4.82 (d, J = 4.8 Hz, 1H), 3.83ꢀ3.76 (m, 1H), 3.28ꢀ3.19 (m, 2H), 2.22 (s,
3H), 1.06 (d, J = 6.0 Hz, 3H) ppm. HPLC purity: 99.1% at 220 nm and 98.9% at 254 nm; Mass
(ESI+): m/z = 344 (M+1).
N-(2-Hydroxy-2-methylpropyl)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxa-
borole-6-yloxy)pyrazine-2-carboxamide (6). This compound was synthesized by using 2ꢀ
hydroxyꢀ2ꢀmethylpropylamine as a starting material and following the procedures used for the
1
analog 7. It was obtained in 68% yield of the final step as a white solid. H NMR (300 MHz,
DMSOꢀd ): δ 9.10 (s, 1H), 8.68 (s, 1H), 8.64 (s, 1H), 8.25ꢀ8.40 (m, 1H), 7.30 (d, J = 8.0 Hz,
6
1H), 7.25 (d, J = 8.0 Hz, 1H), 4.99 (s, 2H), 4.73 (s, 1H), 3.28 (d, J = 6.3 Hz, 2H), 2.22 (s, 3H),
1
.11 (s, 6H) ppm. HPLC purity: 99.5% at 220 nm and 99.2% at 254 nm; Mass (ESI+): m/z = 358
M+1).
N-(1-Hydroxy-2-methylpropan-2-yl)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]
(
oxaborole-6-yloxy)pyrazine-2-carboxamide (7). This compound was synthesized by using 1ꢀ
hydroxyꢀ2ꢀmethylpropanꢀ2ꢀyl amine as a starting material and following the procedures used for
1
the analog 3. It was obtained in 38% yield of the final step as a solid. H NMR (500 MHz,
DMSOꢀd ): δ 9.04 (s, 1H), 8.65 (s, 1H), 8.57 (s, 1H), 7.94 (s, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.24
6
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d, J = 8.0 Hz, 1H), 5.13 (t, J = 5.0 Hz, 1H), 4.98 (s, 2H), 3.45 (d, J = 5.0 Hz, 2H), 2.21 (s, 3H),
1
.35 (s, 6H) ppm. HPLC purity: 97.8% at 220 nm and 96.8% at 254 nm; Mass (ESI+): m/z = 358
M+1).
-(1-Hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-
(
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(hydroxymethyl)cyclopropyl)pyrazine-2-carboxamide (8). This compound was synthesized
by using 1ꢀ(hydroxymethyl)cyclopropylamine as a starting material and following the procedures
1
used for the analog 3. It was obtained in 24% yield of the final step as a solid. H NMR (300
MHz, DMSOꢀd ): δ 9.07 (s, 1H), 8.67 (s, 1H), 8.64 (s, 1H), 8.57 (s, 1H), 7.30 (d, J = 8.0 Hz,
6
1
H), 7.25 (d, J = 8.0 Hz, 1H), 4.99 (s, 2H), 4.71 (t, J = 5.7 Hz, 1H), 3.52 (d, J = 5.7 Hz, 2H),
2
.21 (s, 3H), 0.78 (s, 4H) ppm. HPLC purity: 96.5% at 220 nm and 99.2% at 254 nm; Mass
(ESI+): m/z = 356 (M+1).
-(1-Hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-hydroxypropan-
-yl)pyrazine-2-carboxamide (9). This compound was synthesized by using 2ꢀaminopropanꢀ1ꢀ
5
2
ol as a starting material and following the procedures used for the analog 4. It was obtained in
1
5
7% yield of the final step as a white solid. H NMR (500 MHz, DMSOꢀd ): δ 9.04 (s, 1H), 8.66
6
(s, 1H), 8.59 (s, 1H), 8.27 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 8.3 Hz, 1H),
.98 (s, 2H), 4.80 (t, J = 5.5 Hz, 1H), 4.10ꢀ4.00 (m, 1H), 3.50ꢀ3.40 (m, 2H), 2.22 (s, 3H), 1.15 (d,
4
J = 7.0 Hz, 3H) ppm. HPLC purity: 96.5% at 220 nm and 95.1% at 254 nm; Mass (ESI+): m/z =
344 (M+1).
(R)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-hydroxy
propan-2-yl)pyrazine-2-carboxamide (10). This compound was synthesized by using (R)ꢀ2ꢀ
aminopropanꢀ1ꢀol as a starting material and following the procedures used for the analog 4. It
1
was obtained in 40% yield of the final step as a white solid. H NMR (300 MHz, DMSOꢀd ): δ
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.08 (s, 1H), 8.66 (s, 1H), 8.60 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.25
(d, J = 8.0 Hz, 1H), 4.99 (s, 2H), 4.83 (t, J = 5.5 Hz, 1H), 4.10ꢀ4.00 (m, 1H), 3.50ꢀ3.40 (m, 2H),
2.22 (s, 3H), 1.15 (d, J = 6.6 Hz, 3H) ppm. HPLC purity: 99.3% at 220 nm and 99.7% at 254 nm;
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Chiral HPLC purity: 100% at 240 nm; Mass (ESI+): m/z = 344 (M+1).
(S)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-hydroxy
propan-2-yl)pyrazine-2-carboxamide (11). This compound was synthesized by using (S)ꢀ2ꢀ
aminopropanꢀ1ꢀol as a starting material and following the procedures used for the analog 4. It
1
was obtained in 10% yield of the final step as a solid. H NMR (300 MHz, DMSOꢀd ): δ 9.08 (s,
6
1H), 8.66 (s, 1H), 8.60 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 8.0
Hz, 1H), 4.99 (s, 2H), 4.83 (t, J = 5.7 Hz, 1H), 4.10ꢀ4.00 (m, 1H), 3.50ꢀ3.40 (m, 2H), 2.22 (s,
3H), 1.15 (d, J = 6.6 Hz, 3H) ppm. HPLC purity: 99.3% at 220 nm and 99.6% at 254 nm; Chiral
HPLC purity: 99.8% at 240 nm; Mass (ESI+): m/z = 344 (M+1).
(R)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-hydroxy
butan-2-yl)pyrazine-2-carboxamide (12). This compound was synthesized by using (R)ꢀ2ꢀ
aminobutanꢀ1ꢀol as a starting material and following the procedures used for the analog 4. It was
1
obtained in 59% yield of the final step as a white solid. H NMR (300 MHz, DMSOꢀd ): δ 9.08
6
(s, 1H), 8.66 (s, 1H), 8.61 (s, 1H), 8.23 (d, J = 9.0 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.25 (d, J =
8.4 Hz, 1H), 4.99 (s, 2H), 4.77 (t, J = 5.6 Hz, 1H), 3.95ꢀ3.80 (m, 1H), 3.50ꢀ3.35 (m, 2H), 2.22 (s,
3H), 1.75ꢀ1.45 (m, 2H), 0.86 (t, J = 7.5 Hz, 3H) ppm. HPLC purity: 100% at 220 nm and 100%
at 254 nm; Chiral HPLC purity: 100% at 240 nm; Mass (ESI+): m/z = 358 (M+1).
(S)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-hydroxy
butan-2-yl)pyrazine-2-carboxamide (13). This compound was synthesized by using (S)ꢀ2ꢀ
aminobutanꢀ1ꢀol as a starting material and following the procedures used for the analog 4. It was
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obtained in 59% yield of the final step as a white solid. H NMR (300 MHz, DMSOꢀd ): δ 9.08
6
(
s, 1H), 8.66 (s, 1H), 8.61 (s, 1H), 8.23 (d, J = 8.7 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.25 (d, J =
8
.4 Hz, 1H), 4.99 (s, 2H), 4.78 (t, J = 5.0 Hz, 1H), 3.95ꢀ3.80 (m, 1H), 3.60ꢀ3.40 (m, 2H), 2.22 (s,
H), 1.75ꢀ1.45 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H) ppm. HPLC purity: 100% at 220 nm and 100%
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at 254 nm; Chiral HPLC purity: 100% at 240 nm; Mass (ESI+): m/z = 358 (M+1).
R)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-hydroxy
(
pentan-2-yl)pyrazine-2-carboxamide (14). To a solution of (R)ꢀ2ꢀaminopentanoic acid (10 g,
o
85.4 mmol) in THF (200 mL) was added NaBH (10g, 85.4 mmol, 2.4 eq) at 0 C under N . A
4
2
solution of I (21.7 g, 85.4 mmol, 1 eq) in THF (85 mL) was added dropwise into the mixture for
2
4
0 min until no gas was generated from the mixture. The reaction mixture was stirred overnight
o
at 70 C. After completion, methanol was added dropwise until the mixture was clear. It was
added with 20% KOH (150 mL) and stirred for 4 h. The mixture was diluted with H O (100 mL)
2
and extracted with DCM (3 × 150 mL). The organic phase was washed with brine and dried over
anhydrous Na SO After filtration, the filtrate was concentrated to give (R)ꢀ2ꢀaminopentanꢀ1ꢀol
2
4.
(
8.1 g, 92% yield). A solution of (R)ꢀ2ꢀaminopentanꢀ1ꢀol (6 g, 58.1 mmol) in 4 N HCl MeOH
(150 mL) was stirred at rt for 40 min. The residue after rotary evaporation was mixed with 40%
EA/PE (150 mL). The precipitated solid was collected by filtration to give (R)ꢀ2ꢀaminopentanꢀ1ꢀ
ol HCl salt (4.6 g, 56.6% yield). To a solution of 5ꢀchloropyrazineꢀ2ꢀcarboxylic acid (1.13 g,
7.16 mmol) in NMP (48 mL) was added dropwise a solution of (R)ꢀ2ꢀaminopentanꢀ1ꢀol
hydrochloride (1 g, 7.16 mmol, 1 eq) and 2ꢀchloroꢀ1ꢀmethylpyridinium iodide (CMPI, 4.6 g, 18
mmol, 2.5 eq) in NMP (5 mL) with stirring under N . The reaction was stirred at rt for 2 h. After
2
completion, the reaction was diluted with H O (150 mL), adjusted to pH 3ꢀ4 with 1N HCl, and
2
extracted with EA (2 × 150 mL). The organic phases were washed with brine (3 × 250 mL) and
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dried over anhydrous Na SO The residue after rotary evaporation was purified by column
2
4.
chromatography to give (R)ꢀ5ꢀchloroꢀNꢀ(1ꢀhydroxypentanꢀ2ꢀyl)pyrazineꢀ2ꢀcarboxamide (1.73g,
99% yield). The final step was conducted by following general method A. The product was
10
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obtained in 63% yield of the final step as a white solid. H NMR (500 MHz, DMSOꢀd ): δ 9.09
6
(s, 1H), 8.66 (d, J = 1.0 Hz, 1H), 8.61 (d, J = 1.0 Hz, 1H), 8.24 (d, J = 9.0 Hz, 1H), 7.30 (d, J =
8
.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 4.99 (s, 2H), 4.78 (t, J = 5.5 Hz, 1H), 4.01ꢀ3.94 (m, 1H),
3
.51ꢀ3.45 (m, 1H), 3.45ꢀ3.40 (m, 1H), 2.22 (s, 3H), 1.61ꢀ1.45 (m, 2H), 1.40ꢀ1.20 (m, 2H), 0.87
(t, J = 6.9 Hz, 3H) ppm. HPLC purity: 99.2% at 220 nm and 99.3% at 254 nm; Chiral HPLC
purity: 100% at 240 nm; Mass (ESI+): m/z = 372 (M+1).
(
S)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-
hydroxypentan-2-yl)pyrazine-2-carboxamide (15). This compound was synthesized by using
(
S)ꢀ2ꢀaminopentanoic acid as a starting material and following the procedures used for the
1
isomer 14. It was obtained in 31% yield of the final step as a white solid. H NMR (300 MHz,
DMSOꢀd ): δ 9.08 (s, 1H), 8.66 (s, 1H), 8.60 (s, 1H), 8.22 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 8.0
6
Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 4.99 (s, 2H), 4.77 (t, J = 5.5 Hz, 1H), 4.05ꢀ3.90 (m, 1H), 3.55ꢀ
3.35 (m, 2H), 2.22 (s, 3H), 1.68ꢀ1.40 (m, 2H), 1.40ꢀ1.20 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H) ppm.
HPLC purity: 99.7% at 220 nm and 99.5% at 254 nm; Mass (ESI+): m/z = 372 (M+1).
(
R)-N-(1-hydroxy-3-methylbutan-2-yl)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]
oxaborol-6-yloxy)pyrazine-2-carboxamide (16). This compound was synthesized by using Dꢀ
valine as a starting material and following the procedures used for the analog 14. It was obtained
1
in 20% yield of the final step as a white solid. H NMR (500 MHz, DMSOꢀd ): δ 9.08 (s, 1H),
6
8
.67 (s, 1H), 8.62 (s, 1H), 8.13 (d, J = 9.5 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 8.0 Hz,
H), 4.99 (s, 2H), 4.72 (t, J = 5.5 Hz, 1H), 3.82ꢀ3.70 (m, 1H), 3.65ꢀ3.49 (m, 2H), 2.22 (s, 3H),
1
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.98ꢀ1.92 (m, 1H), 0.92 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.5 Hz, 3H) ppm. HPLC purity: 100% at
2
20 nm and 100% at 254 nm; Chiral HPLC purity: 100% at 240 nm; Mass (ESI+): m/z = 372
(M+1).
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(S)-N-(1-hydroxy-3-methylbutan-2-yl)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]
oxaborol-6-yloxy)pyrazine-2-carboxamide (17). This compound was synthesized by using Lꢀ
valine as a starting material and following the procedures used for the analog 14. It was obtained
1
in 36% yield of the final step as a white solid. H NMR (300 MHz, DMSOꢀd ): δ 9.08 (s, 1H),
6
8.67 (s, 1H), 8.62 (s, 1H), 8.13 (d, J = 9.5 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 8.0 Hz,
1
H), 4.99 (s, 2H), 4.72 (t, J = 5.5 Hz, 1H), 3.85ꢀ3.70 (m, 1H), 3.65ꢀ3.45 (m, 2H), 2.22 (s, 3H),
.02ꢀ1.88 (m, 1H), 0.92 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H) ppm. HPLC purity: 99.8%
2
at 220nm and 99.6% at 254nm; Chiral HPLC purity: 100% at 240 nm; Mass (ESI+): m/z = 372
M+1).
R)-N-(1-cyclopropyl-2-hydroxyethyl)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]
oxaborol-6-yloxy)pyrazine-2-carboxamide (18). To a solution of cyclopropanecarbaldehyde
(
(
(7.0 g, 99.12 mmol, 1.2 eq) in DCM (138 mL) were added Ti(OEt) (22.6 g, 99.12mmol, 1.2 eq)
4
o
and (S)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide (10 g, 82.6 mmol) at rt. The mixture was stirred at 50 C
overnight and then was poured into saturated NaHCO (138 mL) and diatomite (30g) with good
3
stirring for 0.5 h. The mixture was filtered and separated. The organic phase was washed with
brine, dried over MgSO , filtered and concentrated in vacuum to give (S,E)ꢀNꢀ
4
(cyclopropylmethylene)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide (12.5g, 87% yield) as oil. To a mixture
of Mg (2.86 g, 119.16 mmol, 1.65 eq) in dry THF (75 mL) were added I (40 mg) and
2
(chloromethyl)(isopropoxy)dimethylsilane (6.0 g, 36.11 mmol, 0.5 eq) under N . The mixture
2
was refluxed for 1 h, and then additional (chloromethyl)(isopropoxy)dimethylsilane (12 g, 72.22
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mmol, 1.0 eq) was added dropwise. The reaction mixture was refluxed for 2 h. A solution of
(S,E)ꢀNꢀ(cyclopropylmethylene)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide (12.5 g, 72.22 mmol, 1.0 eq) in
o
dry THF (38 mL) was added to the reaction mixture at ꢀ20 C under N . The mixture was stirred
2
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at rt for 1 h, poured into water (300 mL) and extracted with EA (3 × 100 mL). The combined
organic phase was washed with brine, dried over MgSO , filtered and concentrated in vacuum to
4
give
(S)ꢀNꢀ((R)ꢀ1ꢀcyclopropylꢀ2ꢀ(isopropoxydimethylsilyl)ethyl)ꢀ2ꢀmethylpropaneꢀ2ꢀ
sulfinamide (22.5 g, 100% yield) as oil. To a solution of this intermediate (22.3 g, 73.07 mmol)
in a mixed solution [MeOH (53.5 mL) / THF (53.5 mL)] were added KHCO (7.32 g, 73.07
3
o
mmol, 1 eq), KF (8.5 g, 146.14 mmol, 2 eq) and H O (22.3 mL) under N at 0 C. The reaction
2
2
2
o
mixture was stirred at 45 C for 2 h, poured into water (300 mL) and extracted with EA (3 ×
100mL). The combined organic phase was washed with brine, dried over MgSO , filtered and
4
concentrated in vacuum. The residue was purified by column chromatography to give (S)ꢀNꢀ((R)ꢀ
1ꢀcyclopropylꢀ2ꢀhydroxyethyl)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide (10 g, 67% yield) as a white
solid. To a solution of this intermediate (2 g, 9.75 mmol) in MeOH (50 mL) was added 1 N HCl
o
(
24 mL) at 0 C. The reaction was stirred at room temperature for 2 h and concentrated in
vacuum to give (R)ꢀ2ꢀaminoꢀ2ꢀcyclopropylethanꢀ1ꢀol hydrochloride (1.3 g, 100% yield) as a
white solid that was used in the next step without further purification. The subsequent reactions
were conducted by following the corresponding procedures used for the analog 14. Compound
1
18 was obtained in 12% yield of the final step as a white solid. H NMR (300 MHz, DMSOꢀd ):
6
δ 9.07 (s, 1H), 8.64 (s, 1H), 8.60 (s, 1H), 8.37 (d, J = 9.0 Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H), 7.24
(d, J = 8.1 Hz, 1H), 4.97 (s, 2H), 4.78 (t, J = 5.5 Hz, 1H), 3.65ꢀ3.50 (m, 2H), 3.40ꢀ3.30 (m, 1H),
2.20 (s, 3H), 1.10ꢀ1.00 (m, 1H), 0.50ꢀ0.20 (m, 4H) ppm. HPLC purity: 99.6% at 220 nm and
99.7% at 254 nm; Chiral HPLC purity: 100% at 240 nm; Mass (ESI+): m/z = 370 (M+1).
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(
S)-N-(1-cyclopropyl-2-hydroxyethyl)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]
oxaborol-6-yloxy)pyrazine-2-carboxamide (19). This compound was prepared by following
the methodology similar to that described above for 18 using (R)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide
0
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to replace (S)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide in the first step. Compound 19 was obtained in 6%
1
yield of the final step as a white solid. H NMR (300 MHz, DMSOꢀd ): δ 9.07 (s, 1H), 8.66 (s,
6
1
H), 8.61 (s, 1H), 8.37 (d, J = 9.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H),
.99 (s, 2H), 4.79 (t, J = 5.5 Hz, 1H), 3.65ꢀ3.50 (m, 2H), 3.50ꢀ3.32 (m, 1H), 2.22 (s, 3H), 1.10ꢀ
4
1.00 (m, 1H), 0.50ꢀ0.20 (m, 4H) ppm. HPLC purity: 96.6% at 220 nm and 95.9% at 254 nm;
Mass (ESI+): m/z = 370 (M+1).
(R)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(4,4,4-trifluoro-
1
-hydroxybutan-2-yl)pyrazine-2-carboxamide (20). To a solution of 5ꢀchloropyrazineꢀ2ꢀ
carboxylic acid (372 mg, 2.35 mmol) and HATU (1.07 g, 2.82 mmol) in DMF (10 mL) was
added DIPEA (606 mg, 4.97 mmol). The mixture was stirred at rt for 30 min, and then 2ꢀaminoꢀ
4
,4,4ꢀtrifluorobutanꢀ1ꢀol hydrochloride (506 mg, 2.82 mmol) was added. The resulting mixture
was stirred at rt for 1 h. Water (30 mL) was added and the solution was extracted with EA (3 ×
50mL). The combined organic layer was washed with brine, dried over Na SO , filtered and
2
4
concentrated under reduced pressure. The residue was purified by Combiflash (PE: EA=1:1) to
give 5ꢀchloroꢀNꢀ(4,4,4ꢀtrifluoroꢀ1ꢀhydroxybutanꢀ2ꢀyl)pyrazineꢀ2ꢀcarboxamide (435 mg, 65%) as
a white solid. MS (ESI+): m/z = 284.0 (M+1). For the next step, the racemic compound was
prepared by general method A in 57% yield of the final step. Chiral HPLC method was used for
the separation of the racemic mixture and the chiral HPLC condition is shown as following:
*
instrument SFCꢀ80 (Thar, Waters), column CHIRALPAK AD 20 250 mm, 5 µm, column
o
temperature 35 C, mobile phase CO /Methanol = 70/30, flow rate 80g/min, back pressure 100
2
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bar, detection wavelength 214 nm or 230 nm, cycle time 6.0 min, sample solution 300 mg in 60
mL MeOH, and injection volume 4.5 mL (loading 23 mg per injection). The separation gave the
1
enantiomer 20 in 94.4% yield. H NMR (400 MHz, DMSOꢀd ): δ 9.10 (s, 1H), 8.74 (d, J = 8.8
6
10
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13
14
15
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18
19
20
21
22
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Hz, 1H), 8.67 (d, J = 1.2 Hz, 1H), 8.63 (d, J = 1.2 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.26 (d, J =
8.2 Hz, 1H), 5.12 (t, J = 6.0 Hz, 1H), 4.99 (s, 2H), 4.36ꢀ4.32 (m, 1H), 3.50ꢀ3.46 (m, 1H), 3.45ꢀ
3
.39 (m, 1H), 2.72ꢀ2.57 (m, 2H), 2.22 (s, 3H) ppm; HPLC purity: 100% at 220 nm and 100% at
54 nm; Chiral HPLC purity: 100% at 230 nm; MS (ESI+): m/z = 412 (M+1).
2
(S)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(4,4,4-trifluoro-
-hydroxybutan-2-yl)pyrazine-2-carboxamide (21). The racemic compound was prepared by
1
the methodology described above for 20. Chiral HPLC separation gave the enantiomer 21 in
1
18.4% yield. H NMR (400 MHz, DMSOꢀd ): δ 9.10 (s, 1H), 8.73 (d, J = 9.6 Hz, 1H), 8.67 (d, J
6
=
1.2 Hz, 1H), 8.63 (d, J = 1.2 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.26 (d, J = 8.2 Hz, 1H), 5.11
t, J = 6.0 Hz, 1H), 4.99 (s, 2H), 4.39ꢀ4.30 (m, 1H), 3.49ꢀ3.45 (m, 1H), 3.43ꢀ3.38 (m, 1H), 2.72ꢀ
.59 (m, 2H), 2.22 (s, 3H) ppm; HPLC purity: 100% at 220 nm and 100% at 254 nm; Chiral
(
2
HPLC purity: 98.8% at 230 nm; MS (ESI+): m/z = 412 (M+1).
(R)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(5,5,5-trifluoro-
o
1
-hydroxypentan-2-yl)pyrazine-2-carboxamide (22). At ꢀ25 C, 2,6ꢀlutidine (1.41 g, 13.2
mmol) in DCM (50 mL) was mixed with Tf O (3.47 g, 12.3 mmol), and the mixture was stirred
2
for 5 min. To the mixture was added 3,3,3ꢀtrifluoropropanꢀ1ꢀol (1.0 g, 8.8 mmol). After 2 h, it
was warmed to rt and stirred for 1h. The solvent was removed and the residue was purified by
silica gel column chromatography using PE:EA = 2:1 to give 3,3,3ꢀtrifluoropropyl
1
trifluoromethanesulfonate (460 mg, yield 21%) as a yellow oil. H NMR (400 MHz, CDCl ): δ
3
4.73ꢀ4.70 (m, 2H), 2.72ꢀ2.65 (m, 2H) ppm.
A
solution of tertꢀbutyl 2ꢀ
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(
diphenylmethyleneamino)acetate (424 mg, 1.44 mmol) in THF (20 mL) was cooled to ꢀ78°C
and treated dropwise with LDA (2M in THF, 1.08 mL, 2.16 mmol), and then added 3,3,3ꢀ
trifluoropropyl trifluoromethanesulfonate (460 mg, 1.87 mmol) in THF (2 mL) dropwise. The
reaction mixture was gradually warmed to rt and stirred for 4 h. The reaction was quenched with
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saturated NH Cl (50 mL) at 0°C and then extracted with ethyl acetate (2 × 100 mL). The organic
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layer was dried over Na SO and concentrated under reduced pressure. The residue was purified
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by silica gel column chromatography using PE:EA = 40:1 to give tertꢀbutyl 2ꢀ
1
(diphenylmethyleneamino)ꢀ5,5,5ꢀtrifluoropentanoate (400 mg, yield 71%) as a colorless oil. H
NMR (400 MHz, CDCl ): δ 7.66ꢀ7.16 (m, 10H), 3.98 (t, 1H), 2.18ꢀ2.07 (m, 4H), 1.43 (s, 9H)
3
ppm. A solution of this intermediate (400 mg, 1.02 mmol) in 50% HCl (5 mL) was refluxed
overnight. Water was removed to give 2ꢀaminoꢀ5,5,5ꢀtrifluoropentanoic acid hydrochloride (210
mg, yield 99%) as a white solid. MS (ESI+): m/z =172 (M+1). To a solution of 2ꢀaminoꢀ5,5,5ꢀ
trifluoropentanoic acid hydrochloride (210 mg, 1.0 mmol) in THF (10 mL) was added LAH (76
o
mg, 2.0 mmol). The mixture was stirred at 70 C overnight. Several drops of water were added
and the mixture was filtered. The filtrate was concentrated under reduced pressure. 4N HCl 1,4ꢀ
dioxane solution (2 mL) was added at rt and stirred for 30min. Water (10 mL) was added and
then the mixture was extracted with EtOAc (2 × 10 mL). The aqueous layer was freezeꢀdried to
give 2ꢀaminoꢀ5,5,5ꢀtrifluoropentanꢀ1ꢀol hydrochloride (150 mg, yield 77%) as a white solid. MS
(ESI+): m/z = 158 (M+1). The subsequent reactions and chiral separation were performed by
following the procedures described above for 20. Compound 22 was obtained in yield 30% of the
1
final step as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 9.09 (s, 1H), 8.67 (s, 1H), 8.62 (s,
6
1
H), 8.49 (d, J = 9.2 Hz, 2H), 7.30 (d, J = 8.2 Hz, 1H), 7.26 (d, J = 8.2 Hz, 1H), 4.99 (s, 2H),
.93 (t, J = 6.0 Hz, 1H), 4.05ꢀ4.01 (m, 1H), 3.51ꢀ3.45 (m, 2H), 2.30ꢀ2.20 (m, 2H), 2.22 (s, 3H),
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.88ꢀ1.74 (m, 2H) ppm; HPLC purity: 100% at 214 nm and 100% at 254 nm; Chiral HPLC
purity: 100% at 230 nm.; MS (ESI+): m/z = 426 (M+1).
S)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(5,5,5-trifluoro-
(
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-hydroxypentan-2-yl)pyrazine-2-carboxamide (23). This (S)ꢀisomer was obtained by a chiral
1
HPLC separation from its racemic mixture as described above for 22. H NMR (400 MHz,
DMSOꢀd ): δ 9.10 (s, 1H), 8.67 (s, 1H), 8.62 (s, 1H), 8.49 (d, J = 9.2 Hz, 2H), 7.30 (d, J = 8.3
6
Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 4.99 (s, 2H), 4.94 (t, J = 6.0 Hz, 1H), 4.05ꢀ4.01 (m, 1H), 3.51ꢀ
3.45 (m, 2H), 2.30ꢀ2.20 (m, 2H), 2.22 (s, 3H), 1.88ꢀ1.74 (m, 2H) ppm; HPLC purity: 100% at
2
14 nm and 100% at 254 nm; Chiral HPLC purity: 98.1% at 230 nm; MS (ESI+): m/z = 426
M+1).
R)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-(1-hydroxy
(
(
hexan-2-yl)pyrazine-2-carboxamide (24). This compound was synthesized by following the
procedures used for the analog 14 with (R)ꢀ2ꢀaminohexanoic acid as the starting material.
1
Compound 24 was obtained in 23% yield of the final step as a white solid. H NMR (300 MHz,
DMSOꢀd ): δ 9.08 (s, 1H), 8.66 (s, 1H), 8.60 (s, 1H), 8.23 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 8.4
6
Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 4.99 (s, 2H), 4.77 (t, J = 5.5 Hz, 1H), 3.98ꢀ3.85 (m, 1H), 3.55ꢀ
3
.35 (m, 2H), 2.22 (s, 3H), 1.70ꢀ1.42 (m, 2H), 1.35ꢀ1.15 (m, 4H), 0.85 (m, 3H) ppm; HPLC
purity: 99.2% at 220 nm and 98.9% at 254 nm; Chiral HPLC purity: 100% at 240 nm; Mass
(ESI+): m/z = 386 (M+1).
(R)-N-(2-hydroxy-1-phenylethyl)-5-(1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxa
borol-6-yloxy)pyrazine-2-carboxamide (25). This compound was synthesized by using (R)ꢀ2ꢀ
aminoꢀ2ꢀphenylethanꢀ1ꢀol as the starting material and following the procedures used for the
1
analog 14. Compound 25 was obtained in 33% yield of the final step as a white solid. H NMR
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