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Bioorg Med Chem. Author manuscript; available in PMC 2017 March 15. Published in final edited form as: Bioorg Med Chem. 2016 March 15; 24(6): 1314–1321. doi:10.1016/j.bmc.2016.02.002.
Synthesis and Pharmacological Evaluation of Nucleoside Prodrugs Designed to Target Siderophore Biosynthesis in Mycobacterium tuberculosis
Author Manuscript
Surendra Dawadi†, Shuhei Kawamura†, Anja Rubenstein, Rory Remmel, and Courtney C. Aldrich* Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
Abstract
Author Manuscript
The nucleoside antibiotic, 5′-O-[N-(salicyl)sulfamoyl]adenosine (1), possesses potent whole-cell activity against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). This compound is also active in vivo, but suffers from poor drug disposition properties that result in poor bioavailability and rapid clearance. The synthesis and evaluation of a systematic series of lipophilic ester prodrugs containing linear and α-branched alkanoyl groups from two to twelve carbons at the 3′-position of a 2′-fluorinated analogue of 1 is reported with the goal to improve oral bioavailability. The prodrugs were stable in simulated gastric fluid (pH 1.2) and under physiological conditions (pH 7.4). The prodrugs were also remarkably stable in mouse, rat, and human serum (relative serum stability: human~rat>>mouse) displaying a parabolic trend in the SAR with hydrolysis rates increasing with chain length up to eight carbons (t1/2 = 1.6 h for octanoyl prodrug 7 in mouse serum) and then decreasing again with higher chain lengths. The permeability of the prodrugs was also assessed in a Caco-2 cell transwell model. All of the prodrugs were found to have reduced permeation in the apical-to-basolateral direction and enhanced permeation in the basolateral-to-apical direction relative to the parent compound 2, resulting in efflux ratios 5–28 times greater than 2. Additionally, Caco-2 cells were found to hydrolyze the prodrugs with SAR mirroring the serum stability results and a preference for hydrolysis on the apical side. Taken together, these results suggest that the described prodrug strategy will lead to lower than expected oral bioavailability of 2 and highlight the contribution of intestinal esterases for prodrug hydrolysis.
Author Manuscript
Graphical abstract
*
Corresponding author. Telephone: +1 612-625-7956. [email protected]. †These authors have contributed equally Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.xxxx.xx.xxx.
Dawadi et al.
Page 2
Author Manuscript
Keywords Prodrug; Nucleoside; Tuberculosis; Siderophore Biosynthesis Inhibitor
1. Introduction Author Manuscript Author Manuscript
Tuberculosis (TB), one of the oldest recorded diseases of humankind, is caused by the slowgrowing bacterium Mycobacterium tuberculosis (Mtb) as well as several closely related mycobacterial species. TB is a devastating disease clinically manifested as a persistent cough followed by hemoptysis, general malaise and fatigue, and severe weight loss as the disease progresses. TB is extremely difficult to treat relative to other bacterial infections due to a number of unique factors in the pathology and metabolism of Mtb.1–4 Thus, in the case of the simplest drug-sensitive TB, one must employ a four-drug regimen comprised of isoniazid, rifampicin, ethambutol, and pyrazinamide for the first two months followed by 4– 7 months of isoniazid and rifampicin. Drug-resistant strains are even more challenging to treat with corresponding lower cure rates. As a result, TB has now overtaken malaria and HIV as the leading cause of infectious disease mortality.5 The development of new antitubercular agents that are effective against drug-resistant strains and reduce the duration of treatment will be necessary to bring TB back under control.
Author Manuscript
The nucleoside antibiotic 5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS (1), Figure 1) originally described by Quadri, Tan and co-workers was rationally designed to inhibit siderophore biosynthesis in Mtb, an essential process under iron-deficient conditions found in the host.6–9 Sal-AMS (1) possesses nanomolar enzyme inhibition of MbtA, which catalyzes the first committed step of mycobactin biosynthesis, and potent on-target wholecell activity. Proof-of-concept in vivo efficacy was also demonstrated; however, 1 suffers from poor physicochemical properties that result in high clearance and low oral bioavailability.10 To further advance this new class of antibiotics, we previously explored a large number of modifications to 1.8,11–16 The 2′-Fluoro analogue 2 emerged as a lead compound with improved in vitro antitubercular activity (2-fold more potent than 1) while displaying enhanced in vitro (Caco-2 permeability of 4.2 × 10−6 cm/s or 3.5-fold greater than 1, indicative of a medium-permeable compound) and in vivo drug disposition properties (3-fold reduced clearance resulting in a commensurate 3-fold improved oral exposure relative to 1), but no improvement in bioavailability.15 A common strategy to improve oral bioavailability is to synthesize an ester prodrug that increases the lipophilicity and thereby enhances gastrointestinal absorption.17,18 The ester prodrug is then cleaved by serum or tissue esterases to release the parent drug. Herein we
Bioorg Med Chem. Author manuscript; available in PMC 2017 March 15.
Dawadi et al.
Page 3
Author Manuscript
report the synthesis, chemical and enzymatic stability, as well as the in vitro membrane permeability of a systematic series of eight prodrugs (3–10) of the 2′-fluoro analogue 2 through esterification at the 3′-OH group (Figure 1). Prodrugs 3–8 contain a linear alkyl chain from two to twelve carbons while 9 and 10 are branched at the α-position to explore the importance of steric hindrance of the promoiety. The pivaloyl ester, which contains a tertiary carbon at the α-position, was not prepared due to the chronic toxicity of pivalate.19 The phenolic hydroxyl group was left unmasked because the formation of an intramolecular hydrogen bond with the charged acylsulfamide moiety was expected to shield the polarity through charge delocalization.14
2. Results and Discussion 2.1. Chemistry
Author Manuscript Author Manuscript
Synthesis of the key intermediate 15 for preparation of the prodrugs began with commercially available 2′-deoxy-2′-fluoroadenosine 11 (Scheme 1). Regioselective 5′azidation of 11 with NaN3 using the classic Appel conditions (CBr4, PPh3, DMF) afforded 12 in 78% yield. The 3′-OH in 12 was then protected as the TBS ether 13 in 80% yield. Catalytic hydrogenation of 13 employing wet Pd/C led to reduction of the azide to the corresponding amine in quantitative yield. The crude aminonucleoside intermediate was refluxed with sulfamide (NH2SO2NH2) in 1,4-dioxane to provide 14 in 85% yield over two steps. Deprotection of the TBS group with HCl furnished the desired sulfamide 15 in quantitative yield. The conversion of azide 12 to sulfamide 15 was accomplished in four steps with a 68% overall yield by this optimized synthetic route. We also demonstrated that azide 12 could be directly converted to sulfamide 15 in 58% overall yield in two steps circumventing the TBS protection-deprotection sequence utilizing an analogous series of reactions for the conversion of 13 to 14. However, the former route was preferred due to its higher overall yield and avoidance of chromatographic purification of the polar nucleoside 15.
Author Manuscript
With an efficient route to the common intermediate 15 in place, we then developed a reliable procedure for introduction of the promoieties onto the 3′-OH of the nucleoside. Although selective acylation of the 3′-OH over the sulfamide could be accomplished at −5 °C; in practice the extended reaction times made this experimentally impractical. Thus, the reactions were conducted at 0 °C leading to dual acylation. The acyl group on the sulfamide moiety was subsequently cleaved in situ by treatment with formic acid in methanol. By this method, the 3′-O-acylated sulfamides 16–23 were obtained in 61–88% yield. The salicyl group was introduced by Cs2CO3 mediated coupling with N-hydroxysuccinimdyl ester 24 followed by hydrogenolysis of the benzyl protected phenol and purification by column chromatography (coelution with 2% Et3N) afforded prodrugs 3–10 as triethylammonium salts in 37–57% yield over these final two steps. 2.2. Aqueous and plasma stability The ideal prodrug for our application should be stable in aqueous solutions (for 1–2 hours to enable oral absorption), but rapidly hydrolyzed to the parent drug in plasma. The aqueous stability of the prodrugs 3–10 was thus studied in simulated gastric fluid (SGF, pH 1.2) and
Bioorg Med Chem. Author manuscript; available in PMC 2017 March 15.
Dawadi et al.
Page 4
Author Manuscript
HEPES buffer (pH 7.4), which mimic gastric and physiological pH, respectively. The prodrugs 3–10 (100 µM) were incubated at 37 °C in the indicated buffers containing 2% DMSO and the amount of both hydrolyzed product (i.e. parent drug 2) and the prodrug remaining in the solution were monitored by HPLC. Most of the prodrugs were extremely stable and showed little degradation or hydrolysis at 2 hours in both buffers (Table 1). Surprisingly, in the case of most lipophilic dodecanoyl prodrug 8 at pH 1.2, only 76% of the prodrug remained in solution after 2 hours at 37 °C. Chemical hydrolysis to release the parent drug 2 was not observed. Instead we noticed that the prodrug began to slowly precipitate over time, due to the low solubility at pH 1.2. This was also observed with octanoyl prodrug 7 to a minor extent (93% remaining in solution at 2 hours). Only the acetate prodrug 3 was slightly hydrolyzed to parent drug 2 (8% hydrolyzed at pH 7.4 at 2 h). These results confirm that the prodrugs are stable under aqueous conditions.
Author Manuscript Author Manuscript
Next, the stability of the prodrugs in mouse, rat, and human plasma at 37 °C was investigated. The prodrugs were hydrolyzed very slowly in rat and human plasma. On the other hand, most of the prodrugs were hydrolyzed more rapidly by mouse plasma and followed first order kinetics allowing determination of their half-lives. For prodrugs that showed less than 50% hydrolysis at 6 hours, the percentage of prodrug remaining at this terminal time point was measured. The half-lives and percent remaining at 6 hours are listed in Table 1. In mouse plasma, the fastest rate of hydrolysis was observed with octanoyl prodrug 7 with a half-life (t1/2) of 0.7 hours. The hydrolysis rate was slightly slower for prodrugs with larger alkyl groups such as dodecanoyl 8 (t1/2 = 1.6 h) and much slower for branched alkyl chains such as 9 and 10. It was also slower for prodrugs 3, 4, 5, and 6 containing shorter ester promoieties with a clear trend paralleling chain length. In rat and human plasma, the hydrolysis was slow for all prodrugs with a trend of slower hydrolysis for longer and branched promoiet ies. Nonetheless, the prodrugs studied were far more susceptible to enzymatic hydrolysis than chemical hydrolysis in aqueous buffers. We also confirmed that the parent compound 2 was 100% stable at the last incubation time in both the aqueous and plasma stability experiments. 2.3 Caco-2 permeability
Author Manuscript
A representative set of prodrugs (acetyl 3, butanoyl 5, octanoyl 7, isobutyryl 9, and 2ethylbutyryl 10) along with the parent drug 2 were then evaluated in the bidirectional Caco-2 cell transwell model to measure their permeability and potential for improved oral absorption. Because ester prodrugs can be hydrolyzed by esterases produced by Caco-2 cells, the concentration of both the parent compound and the prodrug were monitored. The permeability coefficients Papp of each prodrug for transport from the apical-to-basolateral (AP-BL) and the converse direction (BL-AP) were determined under initial velocity conditions (
Bioorg Med Chem. Author manuscript; available in PMC 2017 March 15. Published in final edited form as: Bioorg Med Chem. 2016 March 15; 24(6): 1314–1321. doi:10.1016/j.bmc.2016.02.002.
Synthesis and Pharmacological Evaluation of Nucleoside Prodrugs Designed to Target Siderophore Biosynthesis in Mycobacterium tuberculosis
Author Manuscript
Surendra Dawadi†, Shuhei Kawamura†, Anja Rubenstein, Rory Remmel, and Courtney C. Aldrich* Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
Abstract
Author Manuscript
The nucleoside antibiotic, 5′-O-[N-(salicyl)sulfamoyl]adenosine (1), possesses potent whole-cell activity against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). This compound is also active in vivo, but suffers from poor drug disposition properties that result in poor bioavailability and rapid clearance. The synthesis and evaluation of a systematic series of lipophilic ester prodrugs containing linear and α-branched alkanoyl groups from two to twelve carbons at the 3′-position of a 2′-fluorinated analogue of 1 is reported with the goal to improve oral bioavailability. The prodrugs were stable in simulated gastric fluid (pH 1.2) and under physiological conditions (pH 7.4). The prodrugs were also remarkably stable in mouse, rat, and human serum (relative serum stability: human~rat>>mouse) displaying a parabolic trend in the SAR with hydrolysis rates increasing with chain length up to eight carbons (t1/2 = 1.6 h for octanoyl prodrug 7 in mouse serum) and then decreasing again with higher chain lengths. The permeability of the prodrugs was also assessed in a Caco-2 cell transwell model. All of the prodrugs were found to have reduced permeation in the apical-to-basolateral direction and enhanced permeation in the basolateral-to-apical direction relative to the parent compound 2, resulting in efflux ratios 5–28 times greater than 2. Additionally, Caco-2 cells were found to hydrolyze the prodrugs with SAR mirroring the serum stability results and a preference for hydrolysis on the apical side. Taken together, these results suggest that the described prodrug strategy will lead to lower than expected oral bioavailability of 2 and highlight the contribution of intestinal esterases for prodrug hydrolysis.
Author Manuscript
Graphical abstract
*
Corresponding author. Telephone: +1 612-625-7956. [email protected]. †These authors have contributed equally Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.xxxx.xx.xxx.
Dawadi et al.
Page 2
Author Manuscript
Keywords Prodrug; Nucleoside; Tuberculosis; Siderophore Biosynthesis Inhibitor
1. Introduction Author Manuscript Author Manuscript
Tuberculosis (TB), one of the oldest recorded diseases of humankind, is caused by the slowgrowing bacterium Mycobacterium tuberculosis (Mtb) as well as several closely related mycobacterial species. TB is a devastating disease clinically manifested as a persistent cough followed by hemoptysis, general malaise and fatigue, and severe weight loss as the disease progresses. TB is extremely difficult to treat relative to other bacterial infections due to a number of unique factors in the pathology and metabolism of Mtb.1–4 Thus, in the case of the simplest drug-sensitive TB, one must employ a four-drug regimen comprised of isoniazid, rifampicin, ethambutol, and pyrazinamide for the first two months followed by 4– 7 months of isoniazid and rifampicin. Drug-resistant strains are even more challenging to treat with corresponding lower cure rates. As a result, TB has now overtaken malaria and HIV as the leading cause of infectious disease mortality.5 The development of new antitubercular agents that are effective against drug-resistant strains and reduce the duration of treatment will be necessary to bring TB back under control.
Author Manuscript
The nucleoside antibiotic 5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS (1), Figure 1) originally described by Quadri, Tan and co-workers was rationally designed to inhibit siderophore biosynthesis in Mtb, an essential process under iron-deficient conditions found in the host.6–9 Sal-AMS (1) possesses nanomolar enzyme inhibition of MbtA, which catalyzes the first committed step of mycobactin biosynthesis, and potent on-target wholecell activity. Proof-of-concept in vivo efficacy was also demonstrated; however, 1 suffers from poor physicochemical properties that result in high clearance and low oral bioavailability.10 To further advance this new class of antibiotics, we previously explored a large number of modifications to 1.8,11–16 The 2′-Fluoro analogue 2 emerged as a lead compound with improved in vitro antitubercular activity (2-fold more potent than 1) while displaying enhanced in vitro (Caco-2 permeability of 4.2 × 10−6 cm/s or 3.5-fold greater than 1, indicative of a medium-permeable compound) and in vivo drug disposition properties (3-fold reduced clearance resulting in a commensurate 3-fold improved oral exposure relative to 1), but no improvement in bioavailability.15 A common strategy to improve oral bioavailability is to synthesize an ester prodrug that increases the lipophilicity and thereby enhances gastrointestinal absorption.17,18 The ester prodrug is then cleaved by serum or tissue esterases to release the parent drug. Herein we
Bioorg Med Chem. Author manuscript; available in PMC 2017 March 15.
Dawadi et al.
Page 3
Author Manuscript
report the synthesis, chemical and enzymatic stability, as well as the in vitro membrane permeability of a systematic series of eight prodrugs (3–10) of the 2′-fluoro analogue 2 through esterification at the 3′-OH group (Figure 1). Prodrugs 3–8 contain a linear alkyl chain from two to twelve carbons while 9 and 10 are branched at the α-position to explore the importance of steric hindrance of the promoiety. The pivaloyl ester, which contains a tertiary carbon at the α-position, was not prepared due to the chronic toxicity of pivalate.19 The phenolic hydroxyl group was left unmasked because the formation of an intramolecular hydrogen bond with the charged acylsulfamide moiety was expected to shield the polarity through charge delocalization.14
2. Results and Discussion 2.1. Chemistry
Author Manuscript Author Manuscript
Synthesis of the key intermediate 15 for preparation of the prodrugs began with commercially available 2′-deoxy-2′-fluoroadenosine 11 (Scheme 1). Regioselective 5′azidation of 11 with NaN3 using the classic Appel conditions (CBr4, PPh3, DMF) afforded 12 in 78% yield. The 3′-OH in 12 was then protected as the TBS ether 13 in 80% yield. Catalytic hydrogenation of 13 employing wet Pd/C led to reduction of the azide to the corresponding amine in quantitative yield. The crude aminonucleoside intermediate was refluxed with sulfamide (NH2SO2NH2) in 1,4-dioxane to provide 14 in 85% yield over two steps. Deprotection of the TBS group with HCl furnished the desired sulfamide 15 in quantitative yield. The conversion of azide 12 to sulfamide 15 was accomplished in four steps with a 68% overall yield by this optimized synthetic route. We also demonstrated that azide 12 could be directly converted to sulfamide 15 in 58% overall yield in two steps circumventing the TBS protection-deprotection sequence utilizing an analogous series of reactions for the conversion of 13 to 14. However, the former route was preferred due to its higher overall yield and avoidance of chromatographic purification of the polar nucleoside 15.
Author Manuscript
With an efficient route to the common intermediate 15 in place, we then developed a reliable procedure for introduction of the promoieties onto the 3′-OH of the nucleoside. Although selective acylation of the 3′-OH over the sulfamide could be accomplished at −5 °C; in practice the extended reaction times made this experimentally impractical. Thus, the reactions were conducted at 0 °C leading to dual acylation. The acyl group on the sulfamide moiety was subsequently cleaved in situ by treatment with formic acid in methanol. By this method, the 3′-O-acylated sulfamides 16–23 were obtained in 61–88% yield. The salicyl group was introduced by Cs2CO3 mediated coupling with N-hydroxysuccinimdyl ester 24 followed by hydrogenolysis of the benzyl protected phenol and purification by column chromatography (coelution with 2% Et3N) afforded prodrugs 3–10 as triethylammonium salts in 37–57% yield over these final two steps. 2.2. Aqueous and plasma stability The ideal prodrug for our application should be stable in aqueous solutions (for 1–2 hours to enable oral absorption), but rapidly hydrolyzed to the parent drug in plasma. The aqueous stability of the prodrugs 3–10 was thus studied in simulated gastric fluid (SGF, pH 1.2) and
Bioorg Med Chem. Author manuscript; available in PMC 2017 March 15.
Dawadi et al.
Page 4
Author Manuscript
HEPES buffer (pH 7.4), which mimic gastric and physiological pH, respectively. The prodrugs 3–10 (100 µM) were incubated at 37 °C in the indicated buffers containing 2% DMSO and the amount of both hydrolyzed product (i.e. parent drug 2) and the prodrug remaining in the solution were monitored by HPLC. Most of the prodrugs were extremely stable and showed little degradation or hydrolysis at 2 hours in both buffers (Table 1). Surprisingly, in the case of most lipophilic dodecanoyl prodrug 8 at pH 1.2, only 76% of the prodrug remained in solution after 2 hours at 37 °C. Chemical hydrolysis to release the parent drug 2 was not observed. Instead we noticed that the prodrug began to slowly precipitate over time, due to the low solubility at pH 1.2. This was also observed with octanoyl prodrug 7 to a minor extent (93% remaining in solution at 2 hours). Only the acetate prodrug 3 was slightly hydrolyzed to parent drug 2 (8% hydrolyzed at pH 7.4 at 2 h). These results confirm that the prodrugs are stable under aqueous conditions.
Author Manuscript Author Manuscript
Next, the stability of the prodrugs in mouse, rat, and human plasma at 37 °C was investigated. The prodrugs were hydrolyzed very slowly in rat and human plasma. On the other hand, most of the prodrugs were hydrolyzed more rapidly by mouse plasma and followed first order kinetics allowing determination of their half-lives. For prodrugs that showed less than 50% hydrolysis at 6 hours, the percentage of prodrug remaining at this terminal time point was measured. The half-lives and percent remaining at 6 hours are listed in Table 1. In mouse plasma, the fastest rate of hydrolysis was observed with octanoyl prodrug 7 with a half-life (t1/2) of 0.7 hours. The hydrolysis rate was slightly slower for prodrugs with larger alkyl groups such as dodecanoyl 8 (t1/2 = 1.6 h) and much slower for branched alkyl chains such as 9 and 10. It was also slower for prodrugs 3, 4, 5, and 6 containing shorter ester promoieties with a clear trend paralleling chain length. In rat and human plasma, the hydrolysis was slow for all prodrugs with a trend of slower hydrolysis for longer and branched promoiet ies. Nonetheless, the prodrugs studied were far more susceptible to enzymatic hydrolysis than chemical hydrolysis in aqueous buffers. We also confirmed that the parent compound 2 was 100% stable at the last incubation time in both the aqueous and plasma stability experiments. 2.3 Caco-2 permeability
Author Manuscript
A representative set of prodrugs (acetyl 3, butanoyl 5, octanoyl 7, isobutyryl 9, and 2ethylbutyryl 10) along with the parent drug 2 were then evaluated in the bidirectional Caco-2 cell transwell model to measure their permeability and potential for improved oral absorption. Because ester prodrugs can be hydrolyzed by esterases produced by Caco-2 cells, the concentration of both the parent compound and the prodrug were monitored. The permeability coefficients Papp of each prodrug for transport from the apical-to-basolateral (AP-BL) and the converse direction (BL-AP) were determined under initial velocity conditions (
