5′-O-[(N-Acyl)sulfamoyl]adenosines as Antitubercular Agents that Inhibit MbtA: An Adenylation Enzyme Required for Siderophore Biosynthesis of the Mycobactins

Abstract

A study of the structure–activity relationships (SAR) of 5′-O-[N-(Salicyl)sulfamoyl]adenosine (6), a potent inhibitor of the bifunctional enzyme salicyl-AMP ligase (MbtA, encoded by the gene Rv2384) in Mycobacterium tuberculosis, is described, targeting the salicyl moiety. A systematic series of analogues was prepared exploring the importance of substitution at the C-2 position revealing that a hydroxy group is required for optimal activity. Examination of a series of substituted salicyl derivatives indicated that substitution at C-4 was tolerated. Consequently, a series of analogues at this position provided 4-fluoro derivative, which displayed an impressive MIC₉₉ of 0.098 μM against whole-cell M. tuberculosis under iron-limiting conditions. Examination of other heterocyclic, cycloalkyl, alkyl, and aminoacyl replacements of the salicyl moiety demonstrated that these nonconserative modifications were poorly tolerated, a result consistent with the fairly strict substrate specificities of related non-ribosomal peptide synthetase (NRPS) adenylation enzymes.

Introduction

Tuberculosis (TB^a) is the leading cause of infectious disease mortality by a bacterial pathogen.¹ TB is extremely difficult to treat since the etiological agent, Mycobacterium tuberculosis, is slow growing, capable of switching its metabolism to a latent or non-replicating state, and possesses a nearly impenetrable cell-wall, which provides a permeability barrier that limits the uptake of many antibiotics. As a result of these factors, effective therapy requires prolonged treatment with 3–4 antibiotics. A combination of poor patient compliance and the inevitable evolution of drug resistance has resulted in the development of multidrug resistant tuberculosis (MDR-TB) defined as resistance to both the first-line agents isoniazid and rifampin. Further, co-infection with HIV/AIDS is a deadly combination and over one-third of HIV-related deaths are due to TB or related mycobacterial infections. Despite the tremendous advances in TB chemotherapy made in the 20^th century, TB has now reemerged as one of the most significant threats to global public health, thus there is a great demand for new drugs to combat TB. The Global Alliance for TB Drug Development (http://www.tballiance.org) recommends that an ideal new TB drug should shorten the duration of effective treatment, improve the treatment of MDR-TB, and provide more effective treatment of latent TB infection. Additionally, drugs that are specific antitubercular agents as opposed to broad-spectrum antibiotics may be advantageous to prevent disturbance to beneficial commensal bacteria as well as minimize the potential development of cross-resistance.

Iron is an essential micronutrient for almost all known organisms including M. tuberculosis where it serves as an obligate cofactor for numerous metalloproteins.² A particular exception is Borrelia burgdorferi, the causative agent of Lyme disease, which apparently has evolved to utilize manganese instead of iron.³ Notwithstanding this anomaly, iron acquisition is critical for bacterial pathogenesis and bacteria have evolved a variety of mechanisms to obtain this vital nutrient. The most common mechanism involves the synthesis, secretion, and reuptake of small molecule iron-chelators termed siderophores.² In a mammalian host, siderophore production is crucial since the concentration of free iron is approximately 10⁻²⁴ M, which is far too low to support bacterial colonization and growth.⁴ Thus, inhibition of siderophore biosynthesis represents a logical strategy for the development of a new class of antibiotics.⁵ An alternate strategy pioneered by Miller and co-workers involves antagonism of siderophore function using synthetic siderophore analogues that likely cannot be taken up by bacterial siderophore transport system.³²

M. tuberculosis produces two series of structurally related siderophores, collectively known as the mycobactins, that are critical for virulence and growth (Figure 1).^{6, 7} Mycobactin biosynthesis is initiated by MbtA, an adenylate-forming enzyme that catalyzes a two-step reaction and is responsible for incorporating salicylic acid into the mycobactins (Figure 1).⁸ MbtA first binds its substrates salicylic acid and ATP then catalyzes their condensation to afford acyladenylate 2 and pyrophosphate. The acyladenylate remains tightly bound whereas pyrophosphate dissociates. Next, MbtA binds the N-terminal aryl carrier domain of MbtB and catalyzes the transfer of the salicyl moiety onto the nucleophilic sulfur atom of the phosphopantetheinyl cofactor of MbtB to afford thioester tethered-MbtB 3 that is elaborated to the mycobactins by a mixed nonribosomal peptide synthetase polyketide synthase (NRPS-PKS) assembly line.⁸

Figure 1.

Biosynthesis of the mycobactins and carboxymycobactins.⁶ The depicted lipid side chain is a representative as both 4 and 5 are isolated as a suite of compounds with various length lipid residues.

Acyladenylates have been shown to bind several orders of magnitude more tightly than the substrate acids since they simultaneously occupy both substrate binding pockets.^{9, 10} Thus acyladenylate analogues that incorporate a stabile linker as a bioisostere of the labile acylphosphate function provide potent adenylation enzyme inhibitors. The general inhibitor scaffold is comprised of four domains (aryl, linker, glycosyl, and base) as depicted in Figure 2. The most crucial portion of the inhibitor scaffold is the linker domain since this must be metabolically stable and appropriately position both the aryl and nucleoside moieties in their respective binding pockets. We have previously explored both the molecular geometry and polarity of the linker pharmacophore with the preparation of β-ketophosphonate, acylsulfamate, acylsulfamide, sulfamate, β-ketosulfonamide, α,α-difluoro-β-ketosulfonamide, acyltriazole, and vinylsulfonamide linkages as surrogates for the labile acylphosphate linkage.^11–13 Inhibitors incorporating the acylsulfamate and acylsulfamide linkages were found to be the most potent with low nanomolar apparent inhibition constants and possessed submicromolar antitubercular activity against whole-cell M. tuberculosis rivaling the first-line agent isoniazid.^{11, 14} Next, we systematically examined the glycosyl domain and found that both the 3′-hydroxy and 4′-ribofuranose ring oxygen were dispensable for bioactivity while modifications making the sugar either more or less flexible were detrimental.¹⁵ In this article, we explore the importance of the aryl ring of the bisubstrate inhibitor scaffold.

Figure 2.

Bisubstrate inhibitors of MbtA. The expanded portion of the figure shows the Ar modifications described herein.

Results and Discussion

Chemistry

Since NRPS adenylation domains exhibit a fairly strict substrate specificity, bisubstrate inhibitors containing a number of conservative aryl modifications were prepared to explore the importance of the ortho-hydroxy group, to define the steric requirements of the shallow aryl acid binding pocket of MbtA, and to assess the potential for interacting with specific residues within the active site such as C240. Additionally, a number of heteroaryl, cycloalkyl, alkyl, and aminoalkyl modifications were targeted. The syntheses of 6^{11, 14, 16}, 7^{11, 16}, 8¹¹, 9¹⁶, 21¹⁶, and 22¹⁷ were disclosed previously, but 8 and 21 were synthesized by alternate routes in improved overall yields. The other twenty-two inhibitors were synthesized through three related synthetic routes as described below.

Salicyl acid derivatives 12–20 and 23 were prepared according to the method described by Tan, Quadri and co-workers from the corresponding acids 34a–j by direct activation with CDI to afford intermediate cyclic anhydrides 35a–j, which were not isolated but subsequently reacted with either protected 5′-O-(sulfamoyl)adenosine derivatives 36 or 37 employing either DBU or Cs₂CO₃ as base to afford 38a–j (Scheme 1).¹⁴ Catalytic hydrogenation of 38h provided p-aminosalicyl derivative 39. Final deprotection of the isopropylidene acetal (80% aq TFA) or TBS ethers (TBAF) furnished bisubstrate inhibitors 12–20 and 23. Anthranoyl analogue 8 was prepared analogously from isatoic anhydride and 36 (not shown, see Experimental Section). The tert-butyldimethylsilyl (TBS) protected nucleosides were preferred for compounds, such as nicotinyl derivative 23 that decomposed under acidic conditions. Compounds 15–18 and 23, which were initially isolated as the tetrabutylammonium salts, were converted to the corresponding sodium salts by ion-exchange with a strong cation exchange resin in the sodium form.

Scheme 1^a.

^aReaction conditions: (a) CDI, MeCN, 60 °C; (b) 36 or 37, DBU or Cs₂CO₃, DMF; (c) 80% aq TFA, 0 °C; or TBAF, THF; (d) H₂, Pd/C.

Direct CDI activation of aryl acids lacking a 2-hydroxy group was much less efficient. In these cases the acyl imidazolidate that forms in situ is substantially less reactive than the cyclic anhydride species (see compound 35, Scheme 1) formed with salicylic acid derivatives. For most other aryl-, heteoaryl-, and alkyl carboxylic acids (40a–k) the corresponding preformed N-hydroxysuccinimide (NHS) esters provided superior coupling yields. Thus, coupling of NHS esters 41a–k to 36 or 37 mediated by either DBU or Cs₂CO₃ afforded acylsulfamate derivatives 42a–k (Scheme 2).¹⁸ Catalytic hydrogenation of 42c provided 2,3-dihydroxybenzoyl derivative 43. In general Cs₂CO₃ as base is favored since in a few instances, such as 42a, DBU salts of the coupled products were obtained – the DBU could not removed by an acid wash, ion-exchange, or by chromatography (co-eluting with 1% Et₃N). Deprotection of the isopropylidene acetal (80% aq TFA) or the TBS ethers (TBAF) of 42a–b, 42d–k, and 43 yielded the final bisubstrate inhibitors 10, 11, 21, 24, 25, 27–29, and 31–33. Compounds 27–29, and 31, which were initially isolated as tetrabutylammonium salts, were converted to the corresponding sodium salts by ion-exchange chromatography. The acetonide protected nucleosides were preferred for compounds, such as 4-azido-2,3,5,6-tetrafluorphenyl derivative 32 that decomposed under the fluoride mediated conditions (TBAF or HF·pyr) required for TBS deprotection.

Scheme 2^a.

^aReaction conditions: (a) DCC, NHS, THF; (a) 36 or 37, DBU or Cs₂CO₃, DMF; (c) 80% aq TFA or TBAF, THF; (d) H₂, Pd/C.

Attempts to prepare a morpholine derivative from the corresponding NHS ester 44 and 5′-O-sulfamoyladenosine derivative 37 did not afford the anticipated product 45, but rather 46 that was deprotected with TBAF to provide 47 in 56% overall yield from 44 (Scheme 3). The β-alanine fragment in 47 was confirmed by HRMS and NMR.¹⁹ The β-alanine moiety of compound 46 is hypothesized to arise from a sequential series of reactions as depicted in Scheme 3 below. Initial nucleophilic attack of sulfamate 37 onto the succinimidyl carbonyl of 44 followed by opening of the imide affords acylhydroxamate intermediate 48, which in turn undergoes Lössen rearrangement to provide isocyanate 49 and expulsion of the carbamic acid of morpholine. Decarboxylation of the carbamic acid affords morpholine that condenses with isocyanate 49 to provide the observed β-alanine urea derivative 46. Precedent exists for opening of the succinimide by a nucleophile.²⁰ Failure to obtain the desired product arises from the attenuated electrophilicity of the N-(morpholinyl)carbonyl group, which favors addition at the more reactive imide carbonyls.

Scheme 3^a.

Based on the above synthetic considerations, morpholinyl analogue 26 was successfully prepared by simply interchanging the electrophile. Thus, treatment of chlorosulfonylisocyanate 51 with morpholine 50 provided chlorosulfonylurea 52 (Scheme 4). This intermediate was not isolated, but condensed with 2′,3′-O-isopropylideneadenosine 53 to afford 54, subsequent acetonide deprotection (80% aq TFA) and ion-exchange to the sodium salt afforded 26.

Scheme 4^a.

^aReaction conditions: (a) CH₂Cl₂, pyr; (b) 53, MeCN; (c) 80% aq TFA; (d) ion-exchange, 4% overall.

Treatment of 5′-O-(sulfamoyl)adenosine derivative 36 with carbonyl diimidazole afforded [(imidazolyl)carbonyl]sulfamate 55 (Scheme 5). This intermediate was not isolated but treated with methanol and triethylamine, subsequent acetonide deprotection (80% aq TFA) and ion-exchange to the sodium salt afforded 30.

Scheme 5^a.

^aReaction conditions: (a) MeCN, CDI; (b) MeOH, Et₃N; (c) 80% aq TFA; (d) ion-exchange, 64% overall.

Bicyclic chromone 58 was prepared as both structural and modeling studies suggested that the ortho-hydroxy group of 6 internally hydrogen bonds with the sulfamate nitrogen (Figure 4).^{12, 21} This analogue simultaneously explores modifications to both the aryl and linker domains of the inhibitor scaffold and was synthesized from 2′,3′-O-isopropylideneadenosine 53 by interconversion of the 5′-alcohol to an azide, Boc protection of the N⁶-amine of adenine, and hydrogenation to afford 56 (Scheme 6).²² CDI mediated coupling of chromone-3-carboxylic acid provided 57 that was deprotected with aqueous TFA to furnish 58.

Scheme 6^a.

^aReaction conditions: (a) ref. 21, 92%; (b) Boc₂O, NaH, DMF/THF (4:1), 16 h, 76%; (c) H₂ (1 atm), Pd/C, MeOH, 95%; (d) i) CDI, DBU, DMF ii) chromone-3-carboxylic acid; (e) 80% aq TFA, 20% (2 steps).

Finally, acylurea analogue 63 was prepared in order to examine the importance of the sulfonyl group of the linker domain of 6 (Figure 2). Acylurea 63 was prepared from 2-benzyloxybenzamide 59 by treatment with oxalyl chloride to provide intermediate acylisocyanate 60 (Scheme 7). After complete removal of the solvent and excess oxalyl chloride, a solution of 5-aminoadenosine derivative 56 in acetonitrile was added to provide 61, which was sequentially deprotected with aqueous TFA to 62 and hydrogenated to afford acylurea 63.

Scheme 7^a.

^aReaction conditions: (a) (COCl)₂, CH₂Cl₂, 50 °C, 3 h; (b) 52, MeCN, 16 h, 88%; (c) H₂, Pd/C, 51%; (d) 80% aq TFA, 70%.

Enzyme Assay

Enzyme assays were performed at 37 °C with recombinant MbtA expressed in E. coli in a buffer of 75 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 2 mM DTT, 250 μM salicylic acid, 10 mM ATP, and 1 mM PPi.¹⁵ The initial rates of pyrophosphate exchange (≤ 10% reaction) were monitored using an enzyme concentration (typically 5–10 nM) by measuring the amount of [³²P]ATP formed after addition of [³²P]PP_i. The enzyme concentration was determined by active-site titration with inhibitor 6. The apparent inhibition constants (K_i^app) were determined by fitting the concentration-response plots either to the Hill equation (eq 1, see Experimental Section) or to the Morrison equation (eq 2, see Experimental Section). The Morrison equation was employed in cases where the inhibitors exhibited tight-binding behavior (K_i^app ≤ 100·[E]). This class of inhibitors has been shown to exhibit reversible and competitive inhibition toward MbtA with respect to both salicylic acid and ATP.¹⁵ Attempts to determine the true inhibition constants (K_i^app) have been hampered by the tight-binding nature of the inhibitors, which has precluded assessment of the K_i by traditional steady-state kinetic methods.²³ The K_i of the parent inhibitor 6 is 6.6 nM (measured at 250 μM salicylic acid, 10 mM ATP). If one accounts for the competitive substrate salicylic acid, which is held at 250 μM or 120·K_M, then the K_i is 55 pM. However, this analysis still neglects the effect of the nonvaried substrate ATP, which is held at 10 mM, or 55·K_M, thus the true K_i for 6 is estimated at approximately 1 pM, a value that has been independently confirmed by isothermal calorimetry (Daniel Wilson, Thomas Anderson, unpublished results). All of the K_i^app values reported are uncorrected for substrate concentrations and represent an upper limit of the true dissociation constant. Although the K_i^app reported herein are not a measure of the true inhibitor potency, the differences are reflective of free energy differences associated with inhibitor binding to MbtA presuming equivalent modalities of inhibition.

Aryl Ring SAR Study

To explore the significance of the ortho-hydroxy group, a systematic series of analogues was prepared bearing substitution at the 2-position. Several important trends emerged from this series. Deletion of the hydroxy afforded benzoyl analogue 7 and resulted in a concomitant 14-fold loss of binding affinity (Table 1). Substitution of the ortho-hydroxy group reduced potency in all cases but by widely varying amounts: by 6-fold for 2-fluoro 9, 117-fold for 2-amino 8, 1100 fold for 2-nitro 11, and 2700-fold for 2-chloro 10. Docking studies of the parent ligand 6 into a homology model of MbtA as well as quantum mechanics calculations free from the constraints of the active site show that 6 adopts a coplanar conformation that is stabilized by an internal hydrogen bond where the phenol acts as a hydrogen bond donor to the sulfamate nitrogen (pK_a ~ 1), which is deprotonated (Figure 3).¹¹ The X-ray cocrystal structure of DhbE with adenylated 2,3-dihydroxybenzoic acid also shows a similar coplanar arrangement.²¹ Molecular mechanics simulations of 10 and 11 show that the bulky nitro- and chloro-substituents disfavor the required coplanar conformation and additionally are unable to form an internal hydrogen bond. The 117-fold loss of activity when the aryl hydroxy is replaced by an amino group suggests that the carboxamide side chain of N235 in MbtA must present the amino group to the inhibitor (for ease of interpretation using the DhbE X-ray structure, residues are numbered as in DhbE). The 2-fluoro analog, whch notably lacks the metabolically labile phenol function, displayed the smallest destabilization of binding, consistent with its ability to adopt a coplanar arrangement in quantum calculations. Unfortunately, the bicyclic chromone analogue 58 that mimics the postulated planar conformation of 6 was inactive. The related acyclic acylurea derivative 63 exhibited a profound 329-fold loss in potency relative to acylsulfamide 64 (Table 2). Modeling suggests that twisting of the urea functionality of 63 is required for binding, partially explaining its decreased activity relative to 6. Cyclization as in 58 imposes even greater energy penalties for this twisting, which likely explains the further loss of potency of this compound.

Table 1.

Compound, R =	KIapp (μM)a	MIC99 (μM)b (iron-deficient)	MIC99 (μM)c (iron-rich)	Compound, R =	KIapp (μM)a	MIC99 (μM)b (iron-deficient)	MIC99 (μM)c (iron-rich)
	0.0066 ± 0.00015d	0.39	1.56
	0.092 ± 0.007	12.5	50		2.9 ± 0.4	> 200	> 200
	0.77 ± 0.12	>200	> 200		0.175 ± 0.019	12.5	50
	0.038 ± 0.008	12.5	50	26
	18.1 ± 1.7	>200	> 200
	7.32 ± 0.9	>200	> 200		> 100	> 200	> 200
	0.061 ± 0.003	50	> 200
	0.012 ± 0.0006	12.5	50		49.8 ± 2.0	> 200	> 200
	0.020 ± 0.0014	12.5	50		17.3 ± 2.8	> 200	> 200
	0.012 ± 0.0016	0.098	0.39	29
	0.021 ± 0.001	6.25–12.5	50		> 100	> 200	> 200
	0.014 ± 0.0007	1.56	25
	4.38 ± 0.44	>200	> 200	30
	0.040 ± 0.004	1.56	25
	0.0073 ± 0.0006	0.78	3.13		2.5 ± 0.4	> 200	> 200
21	0.137 ± 0.012	>200	> 200	31	> 100	> 200	> 200
22	> 100	>200	> 200	32	> 100	> 200	> 200
23	3.7 ± 0.5	>200	> 200	33	36.5 ± 2.2	> 200	> 200

^aValue ± std. error;

^bgrown in GAST media without added Fe³⁺;

^cgrown in GAST media supplemented with Fe³⁺;

^dsee ref. 15.

Figure 3.

Schematic diagram of key nucleoside/protein interactions for analog 7 based on docking.

Table 2.

Compound, R =	KIapp (μM)a	MIC99 (μM)b (iron-deficient)	MIC99 (μM)c (iron-rich)
1	0.0066 ± 0.0015d	0.39e	1.56
64	0.0038 ± 0.0006d	0.19e	0.39
63	1.25 ± 0.05	200	> 200
58	> 100	12.5±25	50

^aValue ± std. error;

^bgrown in GAST media without added Fe³⁺;

^cgrown in GAST media supplemented with Fe³⁺;

^dsee ref. 15;

^esee ref. 11.

Analogues 12–14 were prepared to define the steric requirements of the shallow binding pocket and to identify potential sites for further modification. A homology model of MbtA reveals a shallow hydrophobic binding pocket for salicylic acid consisting of F236, C240, G306, V329, G331, and L337. The chlorine atom is isosteric with a methyl group and importantly can be modified by standard palladium-mediated coupling reactions. The SAR from this chloro-scan showed that substitution at the 4-position of the aryl ring was most tolerated resulting in a modest 2-fold decrease in potency relative to 6 whereas substitution at the 3- and 5-positions reduced inhibitor potency 3- and 9-fold respectively (Table 1). Since the optimal position was found to be the 4-position, several additional substituents (F, Br, CH₃, CF₃, and NH₂) were examined at this position. None of the analogues in this series (15–19) exceeded the potency of 4-Cl 13 and strongly electron-withdrawing substituents were strongly disfavored. Thus, 4-CF₃ 18 exhibited a remarkable 313-fold loss of potency relative to the 4-CH₃ 17. Additionally, modeling suggested that a 6-substituent might be able to accept a poor-geometry hydrogen bond from the backbone amide of G307. The only 6-substituted compound in the series, 6-fluorosalicyl 20, showed activity nearly identical to the parent compound.

Since many siderophores contain an aryl-capped residue derived from 2,3-dihydroxybenzoic acid, inhibitor 21 was evaluated against MbtA and found to possess a 20-fold loss in potency relative to the parent compound 6. Bacillus subtilis uses the adenylating enzyme DhbE in the synthesis of the 2,3-dihydroxybenzoic acid-capped siderophore bacillibactin and compounds 6 and 21 were shown to possess K_I^app values toward DhbE of 106 and 85 nM respectively; however, this analysis neglected the tight-binding nature of the inhibitors and thus underestimated both the inhibitor potency as well as selectivity.^{16, 24} MbtA shares 42% overall sequence identity to DhbE, but significantly higher homology in the ligand binding site. Of the 21 residues contacting the adenylate ligand in the X-ray cocrystal structure of DhbE (within 4 Å), 16 are identical in MbtA and the remaining 5 residues represent conservative changes (Y236F, S240C, A308S, V337L, T411S).²¹ In DhbE, the 3-hydroxy hydrogen bonds to S240, which is a cysteine in MbtA and therefore a weaker hydrogen-bonding partner. In addition, the nearby V337L substitution reduces the space available for the meta-hydroxyl. Therefore residues S240 and V337 probably contribute to the specificity differences between DhbE and MbtA. Bacillus anthracis incorporates native substrate 3,4-dihydroxybenzoic acid to make the siderophore petrobactin.²⁵ Accordingly, compound 22 was prepared to probe the MbtA active site compatibility for 3,4-dihydroxybenzoic acid. 3,4-Dihydroxybenzoyl analogue 22 was a modest nanomolar inhibitor of AsbC (IC₅₀ = 250 nM¹⁷), but displayed no activity toward MbtA indicating important active site difference between the two adenylation enzymes.

Incorporation of a nitrogen at the 3-position in pyridyl analogue 23–25 was explored because of the S240C and V337L substitutions. A nitrogen internal to the aryl ring would avoid any steric issues present with a 3-hydroxyl group, and molecular modeling of 24 and 25 showed that C240 can potentially donate a hydrogen bond to the 3-nitrogen. The carbon to nitrogen substitutions in 2-Cl pyridyl 25 provided an 103-fold increase in potency relative to 2-Cl phenyl 10, while 2-F pyridyl 24 decreased activity 76-fold relative to 2-F phenyl 9. Additionally, the 2-OH pyridyl 23 suffered a 560-fold loss in potency relative to 6, a result that can be partially reconciled since 23 exists exclusively as the keto tautomer. The lack of any apparent trend with 24 and 25 suggests that multiple effects are responsible for the observed activity.

In order to explore the tolerance for nonaromatic groups, a small set of alkyl derivatives 27–29 were prepared. The trend from this small series shows that larger alkyl substituents led to an increase activity. This series was not further explored systematically since the best compound 28 exhibited a 2600-fold loss in potency relative to 6. In docking studies, all low energy conformations of 28 showed unfavorable contacts between the cyclohexyl hydrogens and residues Phe236 and Gly331, which are on opposite faces of the aryl binding pocket, suggesting the pocket is not tall enough to accommodate saturated ring systems. However, methoxy analogue 30 was found to exhibit a greater than 40-fold increase in activity relative to methyl analogue 29 and this unexpected increase in activity warrants further investigation.

Incorporation of a methylene spacer between the linkage carbonyl and the phenyl group in benzyl analogue 31 led to complete loss of activity (>1000-fold decrease in potency relative to 7). 4-Azido-2,3,5,6-tetrafluorophenyl derivative 32 was prepared as a potential photoaffinity probe, but found to be completely inactive.²⁶ Finally, the phenylalanine derived inhibitor 33 was evaluated to probe the selectivity of MbtA toward aminoacyl adenylate inhibitors. Surprisingly, compound 33 exhibited moderate micromolar activity suggesting that the amino group of 33 is able to favorably interact with MbtA.

Inhibition of M. tuberculosis H37Rv

Compounds 6–33, 58, and 63–64 were evaluated for whole-cell activity against M. tuberculosis H37Rv under iron-limiting and iron-rich conditions. The minimum inhibitory concentrations (MIC₉₉) that inhibited >99% of cell growth are shown in tables 1–2. The MIC₉₉ for 6–8 and 64 have been previously reported under iron-deficient conditions, but the MIC₉₉ under iron-rich conditions have not yet been disclosed.¹¹ In general, the MIC₉₉ values were approximately 100-fold greater than the corresponding K_I^app values. The strong correlation between in vitro enzyme inhibition and whole-cell biological activity provides support for the designed mechanism of action. 4-Fluorosalicyl derivative 15 displayed the most potent activity with an impressive MIC₉₉ of 0.098 μM under iron-deficient conditions, which is 4-fold more potent that the parent compound 6. Although the 2-hydroxy group is required for optimal activity, the finding that this can be replaced with a fluoro in analogue 9 and still maintain respectable biological activity may be useful to increase the metabolic stability due to potential glucuronidation of the phenol. Interestingly, chromone derivative 58 displayed modest activity with an MIC₉₉ of 12.5–25 μM under iron deficient conditions despite no apparent enzyme inhibition. However, it should be recognized that the inhibition studies were conducted under supersaturating concentrations of substrates, which underestimate compound potency by more than 1000-fold..

Another consideration is inhibitor selectivity since siderophore production is not essential under iron-rich conditions, suggesting that activity under iron-rich conditions is due to off-target binding and subsequent inhibition of alternate biochemical pathways.¹⁴ The parent inhibitor 6 has an MIC₉₉ of 0.39 μM under iron deficient conditions and 1.56 μM under iron-rich conditions. The ratio of MIC₉₉s of iron-rich/iron-deficient (MIC₉₉^Fe+/MIC₉₉^Fe−) is a measure of the inhibitor selectivity and the selectivity ratio for 6 is only 4. By contrast, the first-line antitubercular agent isoniazid, which disrupts cell-well biosynthesis, was found to be equipotent under iron-rich and iron-deficient conditions (MIC₉₉^Fe+ = MIC₉₉^Fe− = 0.19 μM). p-Aminosalicyl derivative 19 displayed improved selectivity showing a 16-fold increase in potency under iron-deficient conditions, suggesting that this modification reduced off-target binding. Compound 6 was previously shown to exhibit moderate activity against Yerinia pseudotuberculosis with an MIC₉₉ of 20 μM and > 400 μM under iron–deficient and iron–rich conditions respectively.¹⁵ Bioinformatic analysis of M. tuberculosis and Y. pseudotuberculosis suggests that the observed phenotypic difference between these organisms elicited by 6 may be due to the presence of over thirty functionally related fatty acid adenylating (FAAD) enzymes found in M. tuberculosis, but not Y. pseudotuberculosis.²⁷ Many of these FAAD enzymes catalyze essential reactions in the biosynthesis M. tuberculosis lipids and may represent potential off-target proteins inhibited by this class of adenylation inhibitors.²⁸

Conclusion

A series of 5′-O-[(N-acyl)sulfamoyl]adenosines was prepared and evaluated for inhibition of MbtA and activity against whole-cell M. tuberculosis under iron-deficient and iron-rich conditions. A comprehensive and systematic evaluation of the acyl group revealed that a benzoyl substituent is required for antitubercular activity and potent enzyme inhibition. Modifications of the benzoyl group indicated that a hydroxy or fluoro group at C-2 is necessary, and that small substituents at C-4 are tolerated. 4-Fluorosalicyl 15 was found to exhibit the most antitubercular activity while p-aminosalicyl 19 was the first analogue that exhibited improved selectivity against M. tuberculosis under iron-rich conditions. Overall, the collective SAR demonstrated that the aryl domain is poorly tolerant to modification in accord with the fairly strict specificity of NRPS adenylation enzymes; however minor modifications were found to provide incremental enhancement in both potency and selectivity of this new class of adenylation inhibitors.

Experimental Section

General Procedures

All commercial reagents (Sigma-Aldrich, Acros), 4-bromosalicylic acid (ABCR), 4-trifluormethylsalicylic acid (TCI America) were used as provided unless otherwise indicated. An anhydrous solvent dispensing system (J. C. Meyer) using two packed columns of neutral alumina was used for drying THF, Et₂O, and CH₂Cl₂ while two packed columns of molecular sieves were used to dry DMF and the solvents were dispensed under argon. Anhydrous grade DME, MeOH, and MeCN were purchased from Aldrich. Pyridine was freshly distilled from KOH, Et₃N was distilled from CaH₂. Flash chromatography was performed with Silia P grade silica gel 60 (Silicycle) with the indicated solvent system. All reactions were performed under an inert atmosphere of dry Ar or N₂ in oven-dried (150 °C) glassware. ¹H and ¹³C NMR spectra were recorded on a Varian 600 MHz spectrometer. Proton chemical shifts are reported in ppm from an internal standard of residual chloroform (7.26 ppm) or methanol (3.31 ppm), and carbon chemical shifts are reported using an internal standard of residual chloroform (77.3 ppm) or methanol (49.1 ppm). Proton chemical data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, br = broad), coupling constant, integration. High resolution mass spectra were obtained on Agilent TOF II TOF/MS instrument equipped with either an ESI or APCI interface. High resolution mass spectra were obtained on Agilent TOF II TOF/MS instrument equipped with either an ESI or APCI interface. Analytical HPLC were obtained on a Agilent 1100 Series HPLC system with a PDA detector. Optical rotations were measured on Rudolph Autopol III polarimeter. Melting points were measured on electrothermal Mel-Temp manual melting point apparatus and are uncorrected.

General Procedure for CDI Mediated Acylation.^{14, 18}

A solution of 34a–j (3.0 mmol, 3.0 equiv) and CDI (3.6 mmol, 3.6 equiv) in DMF (10 mL) was stirred at 60 °C for 2 h. The solution was cooled to rt and a mixture of either 36¹¹ or 37¹⁴ (1.0 mmol, 1.0 equiv) and either DBU or Cs₂CO₃ (1.5 mmol, 1.5 equiv) was then added and the reaction stirred a further 3 h at rt. The reaction mixture was concentrated in vacuo and purified by flash chromatography to afford the title compound.

General Procedure for Synthesis of NHS Esters

Method A: To a solution of carboxylic acid 40a–e and 40j-k, (1.0 mmol, 1.0 equiv) in THF (10 mL) at 0 °C was added N-hydroxysuccinimide (1.0 mmol, 1.0 equiv) and DCC (1.0 mmol, 1.0 equiv). The resulting mixture was stirred for 30 min at 0 °C then 2 h at rt. The reaction mixture was filtered to remove the DCU precipitate and the filtrate was concentrated under reduced pressure. Purification by flash chromatography afforded the desired N-hydroxysuccinimdyl aroyl ester. Method B: To a solution of acid chloride/anhydride 40f–i (1.0 mmol, 1.0 equiv) in THF (10 mL) at 0 °C was added N-hydroxysuccinimide (1.0 mmol, 1.0 equiv) and pyridine (1.0 mmol, 1.0 equiv). The resulting mixture was stirred for 30 min at 0 °C then 2 h at rt. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The product was used without further purification.

General Procedure for NHS-Ester Mediated Acylation.^{11, 18}

To a solution of N-hydroxysuccinimdyl ester 41a–k (1.0 mmol) in DMF (10 mL) at 0 °C was added 36 or 37 (1.5 mmol, 1.5 equiv) and DBU or Cs₂CO₃ (2.0 mmol, 2.0 equiv). The reaction mixture was warmed to rt and stirred 16 h. The reaction was concentrated under reduced pressure and the residue taken up in EtOAc and filtered. The solids were washed with additional EtOAc (100 mL) and the combined filtrate was concentrated. Purification by flash chromatography (EtOAc/MeOH/Et₃N) afforded the title compound.

General Procedure for TFA deprotection

To a solution of 5′-O-[N-acyl(sulfamoyl)]-2′,3′-O-isopropylideneadenosine triethylammonium salt (0.2 mmol) was added 80% aq TFA (2.5 mL). The resulting solution was stirred for 30 min at 0 °C then concentrated under reduced pressure. Purification by flash chromatography (EtOAc/MeOH/Et₃N) afforded the title compound.

General Procedure for TBS deprotection

To a solution of 5′-O-[N-acyl(sulfamoyl)]-2′,3′-O-di-(tert-butyldimethylsilyl)adenosine triethylammonium salt (0.1 mmol, 1 equiv) in THF (5 mL) at rt was added TBAF (1.0 M in THF, 0.25 mmol, 2.5 equiv). The reaction mixture was stirred 30 min then concentrated in vacuo. Purification by flash chromatography (EtOAc/MeOH/Et₃N) afforded the title compound.

General Procedure for Ion-Exchange

A solution of a nucleoside tetrabutylammonium or triethylammonium salt (0.25 mmol, 1 equiv) in H₂O (0.5 mL) was added to a short column (10 × 50 mm, Dowex 50WX8-100-Na+) and incubated for 10 min before eluting with H₂O (20 mL). The fractions containing the product were lyophilized to afford the sodium salt as a flocculant white solid. The Dowex cation exchange resin was converted to the sodium form by sequentially washing the column with MeOH (50 mL), H₂O (50 mL), 1 N aq NaOH (25 mL), and H₂O (100 mL).

5′-O-[N-(2-Aminobenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine triethylammonium salt

To a solution of isatoic anhydride (47 mg, 0.284 mmol, 1.1 equiv) in DMF (1.0 mL) at rt was added 36 (100 mg, 0.26 mmol, 1.0 equiv) and Cs₂CO₃ (169 mg, 0.52 mmol, 2.0 equiv). The reaction was stirred 12 h at rt then concentrated in vacuo. Purification by flash chromatography (100:25:1 EtOAc/MeOH/TEA) afforded the title compound (135 mg, 86%). ¹H NMR, ¹³C NMR, and HRMS agreed with literature values.¹¹ This compound was deprotected as described.¹¹

N-Hydroxysuccinimdyl 2-chlorobenzoate (41a)

This was prepared from 2-chlorobenzoic acid (780 mg, 5.0 mmol) using the general procedure for NHS ester synthesis. Purification by flash chromatography (1:1 Hexanes/EtOAc) afforded the title compound (880 mg, 70%): R_f 0.5 (1:1 Hexanes/EtOAc); ¹H NMR (600 MHz, CDCl₃) δ 2.91 (s, 4H), 7.39 (ddd, J = 7.8, 6.0, 1.8 Hz, 1H), 7.53–7.57 (m, 2H), 8.10 (dd, J = 7.8, 1.8 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 25.9, 124.8, 127.1, 131.8, 132.6, 134.8, 135.8, 160.4, 169.2; MS (ESI+) calcd for C₁₁H₉ClNO₄ [M+H]⁺ 254.0, found 254.0.

5′-O-[N-(2-Chlorobenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine (42a)

This was prepared from 41a (126 mg, 0.50 mmol) using the general procedure for NHS-ester mediated acylation. Purification by flash chromatography (100:10:1 EtOAc/MeOH/TEA) afforded the title compound (58 mg, 22%): R_f 0.2 (100:10:1 EtOAc/MeOH/TEA); [α]_D²¹ − 102 (c 2.79, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.36 (s, 3H), 1.59 (s, 3H), 4.35 (d, J = 1.8 Hz, 2H), 4.58 (d, J = 1.8 Hz, 1H), 5.20 (d, J = 6.0 Hz, 1H), 5.37 (dd, J = 6.0, 3.0 Hz, 1H), 6.24 (d, J = 3.0 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 8.18 (s, 1H), 8.46 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 24.4, 26.3, 68.9, 82.1, 84.5, 84.6, 90.7, 114.1, 118.9, 126.4, 128.9, 129.6, 129.8, 130.7, 139.5, 140.3, 149.3, 152.8, 156.1, 174.7; HRMS (APCI+) calcd for C₂₀H₂₂ClN₆O₇S [M+H]⁺ 525.0959, found 525.0953 (error 1.1 ppm).

5′-O-[N-(2-Chlorobenzoyl)sulfamoyl]adenosine (10)

This was prepared from 42a (29 mg, 0.050 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography afforded the title compound (22 mg, 93%): R_f 0.18 (100:15:2 MeOH/EtOAc/TEA); [α]_D²¹ −18.7 (c 0.83, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 4.37 (br s, 1H), 4.49 (t, J = 4.8 Hz, 1H), 4.62–4.70 (m, 3H), 6.11 (d, J = 4.8 Hz, 1H), 7.34–7.37 (m, 1H), 7.44–7.47 (m, 3H), 8.36 (s, 1H), 8.51 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 70.3, 71.5, 74.5, 82.4, 89.3, 119.3, 127.1, 128.9, 130.2, 130.9, 132.4, 133.5, 142.7, 144.3, 148.9, 150.8, 165.7; HRMS (ESI+) calcd for C₁₇H₁₈ClN₆O₇S [M+H]⁺ 485.0641, found 485.0649 (error 0.2 ppm).

N-Hydroxysuccinimdyl 2-nitrobenzoate (41b)

This was prepared from 2-nitrobenzoic acid (835 mg, 5.0 mmol) using the general procedure for NHS ester synthesis. Purification by flash chromatography (6:4 Hexanes/EtOAc) afforded the title compound (1.10 g, 85%): R_f 0.2 (6:4 Hexanes/EtOAc); ¹H NMR (600 MHz, CDCl₃) δ 2.89 (s, 4H), 7.76–7.79 (m, 2H), 7.92–7.95 (m, 1H), 8.06–8.08 (m, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 25.9, 123.2, 124.8, 130.9, 133.6, 133.7, 147.9, 161.2, 168.7; MS (ESI+) calcd for C₁₁H₉N₂O₆ [M+H]⁺ 265.0, found 264.9.

2′,3′-O-Isopropylidene-5′-O-[N-(2-nitrobenzoyl)sulfamoyl]adenosine triethylammonium salt (42b)

This was prepared from 41b (133 mg, 0.50 mmol) using the general procedure for NHS-ester mediated acylation. Purification by flash chromatography (100:10:1 EtOAc/MeOH/TEA) afforded the title compound (75 mg, 28%): R_f 0.17 (100:10:1 EtOAc/MeOH/TEA); [α]_D²¹ −142 (c 2.38, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.23 (t, J = 7.2 Hz, 9H), 1.36 (s, 3H), 1.59 (s, 3H), 3.13 (q, J = 7.2 Hz, 6H), 4.32–4.35 (m, 2H), 4.58 (br s, 1H), 5.18 (d, J = 6.0, 1H), 5.37–5.39 (m, 1H), 6.23 (d, J = 2.4, 1H), 7.52 (t, J = 7.2 Hz, 1H), 7.62 (t, J = 8.4 Hz, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 8.45 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.0, 24.4, 26.3, 46.7, 68.8, 82.2, 84.5, 84.6, 90.7, 114.1, 118.9, 123.4, 129.3, 129.7, 132.6, 135.5, 140.2, 148.1, 149.3, 152.8, 156.1, 172.6; HRMS (ESI+) calcd for C₂₀H₂₂N₇O₉S [M+H]⁺ 536.1199, found 536.1201 (error 0.4 ppm).

5′-O-[N-(2-Nitrobenzoyl)sulfamoyl]adenosine triethylammonium salt (11)

This was prepared from 42b (55 mg, 1.0 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (100:25:2.5 EtOAc/MeOH/TEA) afforded the title compound (11 mg, 22%): R_f 0.15 (100:25:2.5 EtOAc/MeOH/TEA); [α]_D²¹ −99.7 (c 0.290, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.24 (t, J = 7.8 Hz, 9H), 3.14 (q, J = 7.8 Hz, 6H), 4.31–4.34 (m, 1H), 4.40 (t, J = 3.6 Hz, 2H), 4.45 (dd, J = 3.6 Hz, 1H), 4.72 (t, J = 6.0 Hz, 1H), 6.09 (d, J = 6.0 Hz, 1H), 7.52 (t, J = 8.4, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.68 (d, J = 7.2 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 8.49 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.0, 46.7, 68.5, 71.3, 74.9, 83.5, 87.9, 118.9, 123.4, 129.3, 129.5, 132.7, 135.7, 139.8, 147.9, 149.7, 152.7, 156.1, 172.7; HRMS (APCI+) calcd for C₁₇H₁₈N₇O₉S [M+H]⁺ 496.0876, found 496.0904 (error 5.6 ppm).

5′-O-[N-(3-Chloro-2-hydroxybenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine (38a)

This was prepared from 3-chlorosalicylic acid (170 mg, 1.0 mmol) using the general procedure for CDI mediated acylation. Purification by flash chromatography (100:5:1 EtOAc/MeOH/TEA) afforded the title compound (83 mg, 15%): R_f 0.15 (100:5:1 EtOAc/MeOH/TEA); [α]_D²¹ −32.3 (c 2.00, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.31 (s, 3H), 1.55 (s, 3H), 4.32–4.34 (m, 2H), 4.54 (br s, 1H), 5.09–5.11 (m, 1H), 5.34 (dd, J = 6.0, 3.0 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H), 6.71 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 8.14 (s, 1H), 8.39 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 24.3, 26.2, 68.9, 82.1, 84.5, 84.6, 90.8, 114.1, 118.0, 119.0, 120.8, 121.3, 128.7, 133.3, 140.2, 149.2, 152.7, 156.1, 156.8, 172.9; HRMS (ESI+) calcd for C₂₀H₂₂ClN₆O₈S [M+H]⁺ 541.0903, found 541.0903 (error 0 ppm).

5′-O-[N-(3-Chloro-2-hydroxybenzoyl)sulfamoyl]adenosine triethylammonium salt (12)

This was prepared form 38a (30 mg, 0.060 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (100:15:1 EtOAc/MeOH/TEA) afforded the title compound (20 mg, 65%): R_f 0.17 (100:15:1 EtOAc/MeOH/TEA); [α]_D²¹ −32.8 (c 0.570, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.28 (t, J = 7.2 Hz, 9H), 3.18 (q, J = 7.2 Hz, 6H), 4.34–4.35 (m, 1H), 4.39–4.46 (m, 3H), 4.74 (t, J = 5.4 Hz, 1H), 6.11 (d, J = 6.0 Hz, 1H), 6.76 (t, J = 7.8 Hz, 1H), 7.40 (dd, J = 7.8, 1.2 Hz, 1H), 7.91 (dd, J = 7.8, 1.2 Hz, 1H), 8.20 (s, 1H), 8.53 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.1, 46.6, 68.5, 71.1, 74.8, 83.3, 87.9, 117.9, 118.9, 120.9, 121.3, 128.7, 133.1, 139.8, 149.6, 152.6, 156.0, 156.7, 172.6; HRMS (ESI+) calcd for C₁₇H₁₈ClN₆O₈S [M+H]⁺ 501.0595, found 501.0585 (error 2.0 ppm).

5′-O-[N-(4-Chloro-2-hydroxybenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine triethylammonium salt (38b)

This was prepared from 4-chlorosalicylic acid (43 mg, 0.25 mmol) using the general procedure for CDI mediated acylation. Purification by flash chromatography (100:10:1 EtOAc/MeOH/TEA) afforded the title compound (94 mg, 70%): R_f 0.15 (100:10:1 EtOAc/MeOH/TEA); [α]_D²¹ −88.4 (c 1.86, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.24 (t, J = 7.8 Hz, 9H), 1.34 (s, 3H), 1.58 (s, 3H), 3.09 (q, J = 7.8 Hz, 6H), 4.29 (dd, J = 10.8, 4.2 Hz, 1H), 4.32 (dd, J = 10.8, 3.6 Hz, 1H), 4.53–4.54 (m, 1H), 5.11 (dd, J = 6.0, 1.8 Hz, 1H), 5.37 (dd, J = 6.0, 3.0 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 6.72–6.80 (m, 2H), 7.83 (d, J = 9.0 Hz, 1H), 8.14 (s, 1H), 8.41 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.3, 24.2, 26.2, 46.6, 68.8, 82.2, 84.51, 84.53, 90.8, 114.1, 115.9, 116.6, 118.3, 118.4, 131.6, 138.3, 140.2, 149.2, 152.8, 156.1, 161.7, 172.6; MS (ESI+) calcd for C₂₀H₂₂ClN₆O₈S [M+H]⁺ 541.09, found 541.08.

5′-O-[N-(4-Chloro-2-hydroxybenzoyl)sulfamoyl]adenosine triethylammonium salt (13)

This was prepared from 38b (35 mg, 0.060 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (100:15:1 EtOAc/MeOH/TEA) afforded the title compound (19 mg, 60%): R_f 0.14 (100:15:1 EtOAc/MeOH/TEA); [α]_D²¹ −23.3 (c 1.81, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.24 (t, J = 7.8 Hz, 9H), 3.13 (q, J = 7.8 Hz, 6H), 4.30 (dd, J = 6.6, 3.0 Hz, 1H), 4.34–4.42 (m, 3H), 4.70 (t, J = 5.4 Hz, 1H), 6.07 (d, J = 5.4 Hz, 1H), 6.74 (dd, J = 8.4, 1.2 Hz, 1H), 6.79 (d, J = 1.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 8.16 (s, 1H), 8.47 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.1, 46.7, 60.3 68.5, 71.2, 74.8, 83.3, 88.1, 116.6, 118.3, 119.0, 131.5, 138.3, 139.9, 149.7, 152.7, 156.1, 161.7, 172.6; HRMS (ESI+) calcd for C₁₇H₁₈ClN₆O₈S [M+H]⁺ 501.0595, found 501.0592 (error 0.6 ppm).

5′-O-[N-(5-Chloro-2-hydroxybenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine (38c)

This was prepared from 5-chlorosalicylic acid (86 mg, 0.50 mmol) using the general procedure for CDI mediated acylation. Purification by flash chromatography (100:5:1.5 EtOAc/MeOH/TEA) afforded the title compound (33 mg, 12%): R_f 0.18 (100:5:1.5 EtOAc/MeOH/TEA); [α]_D²¹ −96.0 (c 1.26, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.33 (s, 3H), 1.56 (s, 3H), 4.30–4.33 (m, 2H), 4.53 (br s, 1H), 5.10 (d, J = 6.0 Hz, 1H), 5.35 (dd, J = 6.0, 2.4 Hz, 1H), 6.19 (d, J = 2.4 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 7.22 (dd, J = 8.4, 2.4 Hz, 1H), 7.84 (d, J = 2.4 Hz, 1H), 8.13 (s, 1H), 8.39 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 24.2, 26.2, 60.3, 68.9, 82.1, 84.5, 84.6, 90.8, 104.7, 114.1, 118.5, 120.5, 122.7, 129.3, 132.8, 140.2, 152.7, 156.0, 159.5, 172.4; HRMS (ESI+) calcd for C₂₀H₂₂ClN₆O₈S [M+H]⁺ 541.0903, found 541.0914 (error 2.0 ppm).

5′-O-[N-(5-Chloro-2-hydroxybenzoyl)sulfamoyl]adenosine triethylammonium salt (14)

This was prepared from 38c (28 mg, 0.050 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (100:20:1.5 EtOAc/MeOH/TEA) afforded the title compound (15 mg, 59%): R_f 0.15 (100:20:1.5 EtOAc/MeOH/TEA); [α]_D²¹ −23.3 (c 1.72, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.17 (t, J = 7.2 Hz, 9H), 2.90 (q, J = 7.2 Hz, 6H), 4.31–4.42 (m, 4H), 4.68 (t, J = 5.4 Hz, 1H), 6.06 (d, J = 5.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.87 (s, 1H), 8.15 (s, 1H), 8.44 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.8, 46.3, 60.4, 68.5, 71.1, 74.8, 83.2, 88.3, 116.8, 118.0, 118.4, 120.6, 129.4, 132.8, 139.9, 152.7, 156.1, 159.5, 172.4; HRMS (ESI+) calcd for C₁₇H₁₈ClN₆O₈S [M+H]⁺ 501.0595, found 501.0594 (error 0.2 ppm).

5′-O-[N-(4-Fluoro-2-hydroxybenzoyl)sulfamoyl]adenosine sodium salt (15)

This was prepared from 4-fluorosalicylic acid (16 mg, 0.10 mmol) using the general procedure for CDI mediated acylation to afford 38d. The crude product was used directly for the next step.

Crude 38d prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (25 mg, 48% overall yield): R_f 0.21 (8:2 EtOAc/MeOH); [α]_D²¹ −0.05 (c 0.33, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 4.29–4.31 (m, 1H), 4.35 (dd, J = 10.8, 3.0 Hz, 1H), 4.38–4.40 (m, 2H), 4.69 (t, J = 5.4 Hz, 1H), 6.07 (d, J = 6.0 Hz, 1H), 6.47–6.50 (m, 2H), 7.94 (t, J = 7.8 Hz, 1H), 8.15 (s, 1H), 8.49 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 69.5, 72.4, 76.0, 84.6, 89.2, 104.1 (d, ²J_C-F = 23.4), 106.5 (d, ²J_C-F = 21.8), 120.1, 133.44, 133.51, 141.1, 150.9, 153.8, 157.2, 164.1, 167.2 (d, J_C-F = 247.8), 174.0; HRMS (ESI−) calcd for C₁₇H₁₆FN₆O₈S [M−H]⁻ 483.0739, found 483.0758 (error 3.9 ppm).

5′-O-[N-(4-Bromo-2-hydroxybenzoyl)sulfamoyl]adenosine sodium salt (16)

This was prepared from 4-bromosalicylic acid (60 mg, 0.28 mmol) using the general procedure for CDI mediated acylation to afford 38e. The crude product was used directly for the next step.

Crude 38e prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (116 mg, 74% overall yield): R_f 0.23 (8:2 EtOAc/MeOH); [α]_D²¹ −0.13 (c 0.53, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 4.31–4.32 (m, 1H), 4.35–4.37 (m, 1H), 4.40–4.42 (m, 2H), 4.70 (t, J = 5.4 Hz, 1H), 6.08 (d, J = 6.0 Hz, 1H), 6.91 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 6.97 (d, J = 1.8, 1H), 7.81 (d, J = 7.8 Hz, 1H), 8.17 (s, 1H), 8.50 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 69.7, 72.4, 76.1, 84.6, 89.3, 119.9, 120.2, 120.9, 122.5, 127.8, 132.8, 141.1, 150.9, 153.94, 157.3, 162.9, 174.1; HRMS (ESI−) calcd for C₁₇H₁₆BrN₆O₈S [M−H]⁻ 542.9939, found 542.9945 (error 1.1 ppm).

5′-O-[N-(2-Hydroxy-4-methylbenzoyl)sulfamoyl]adenosine sodium salt (17)

This was prepared from 4-methylsalicylic acid (76 mg, 0.50 mmol) using the general procedure for CDI mediated acylation to afford 38f. The crude product was used directly for the next step.

Crude 38f prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (113 mg, 45% overall yield): R_f 0.20 (8:2 EtOAc/MeOH); [α]_D²¹ −0.15 (c 0.56, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 2.27 (s, 3H), 4.31–4.32 (m, 1H), 4.34–4.37 (m, 1H), 4.39–4.42 (m, 2H), 4.71 (t, J = 5.4 Hz, 1H), 6.08 (d, J = 6.0 Hz, 1H), 6.59–6.61 (m, 2H), 7.81 (d, J = 7.8 Hz, 1H), 8.17 (s, 1H), 8.51 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 20.7, 68.6, 71.5, 75.2, 83.7, 88.3, 117.1, 119.3, 119.5, 130.4, 140.2, 144.4, 150.0, 152.9, 156.4, 161.1, 174.4 (missing 1 C); HRMS (ESI−) calcd for C₁₈H₁₉N₆O₈S [M−H]⁻ 479.0990, found 479.1009 (error 4.0 ppm).

5′-O–[N-(4-Trifluoromethyl-2-hydroxybenzoyl)sulfamoyl]adenosine sodium salt (18)

This was prepared from 4-trifluoromethylsalicylic acid (20.6 mg, 0.10 mmol) using the general procedure for CDI mediated acylation to afford 38g. The crude product was used directly for the next step.

Crude 38g prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (27 mg, 48% overall yield): R_f 0.25 (8:2 EtOAc/MeOH); [α]_D²¹ −0.04 (c 0.25, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 4.30–4.33 (m, 1H), 4.37–4.45 (m, 3H), 4.70 (t, J = 5.4 Hz, 1H), 6.09 (d, J = 6.0 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 7.04 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 8.17 (s, 1H), 8.51 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 68.8, 71.5, 75.2, 83.7, 88.3, 114.0, 114.3, 119.3, 123.0, 131.4, 136.3, 140.2, 150.0, 153.0, 156.4, 161.3, 172.5 (missing 1 aryl C and CF₃); HRMS (ESI−) calcd for C₁₈H₁₆F₃N₆O₈S [M−H]⁻ 533.0707, found 533.0714 (error 1.1 ppm).

4-Benzyloxycarbonylamino-2-hydroxybenzoic acid (34h)

To a solution of 4-aminosalicylic acid (2.0 g, 13.1 mmol) in THF (40 mL) was added saturated aq NaHCO₃ (40 mL) followed by benzyl chloroformate (2.04 mL, 14.4 mmol, 1.1 equiv). The reaction was stirred for 16 h at rt then concentrated to remove THF. The remaining aqueous layer was acidified with 6 N HCl to pH 3–4 then extracted with EtOAc (3× 50 mL). The combined organic layers were dried (Na₂SO₄) and concentrated to afford the title compound (2.30 g, 61%) as a light brown solid: ¹H NMR (600 MHz, CD₃OD) δ 5.17 (s, 2H), 6.91 (dd, J = 9.0, 1.8 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H), 7.29–7.39 (m, 5H), 7.72 (d, J = 9.0 Hz, 1H), 9.57 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 66.6, 105.2, 107.2, 109.3, 127.9, 128.0, 128.3, 131.1, 136.6, 145.9, 154.0, 163.1, 172.0; HRMS (ESI−) calcd for C₁₅H₁₂NO₅ [M−H]⁻ 286.0721, found 286.0727 (error 2.1 ppm)

5′-O-[N-(4-Benzyloxycarbonylamino-2-hydroxybenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine (38h)

This was prepared from 34h (71 mg, 0.25 mmol) using the general procedure for CDI mediated acylation. Purification by flash chromatography (100:7 MeOH/EtOAc) afforded the title compound (75 mg, 38%) as a white solid: R_f 0.20 (100:7 MeOH/EtOAc); [α]_D²¹ 86.1 (c 1.29, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 1.32 (s, 3H), 1.56 (s, 3H), 4.30 (t, J = 4.2 Hz, 2H), 4.54 (br s, 1H), 5.10 (d, J = 5.4 Hz, 1H), 5.15 (s, 2H), 5.35 (br s, 1H), 6.20 (d, J = 3.0 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.34 (t, J = 7.2 Hz, 2H), 7.38 (d, J = 7.2 Hz, 2H), 7.66 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 8.13 (s, 1H), 8.40 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 24.2, 26.2, 66.4, 68.7, 82.2, 84.5, 90.8, 105.6, 108.8, 114.1, 119.0, 121.4, 127.8, 127.9, 128.3, 130.9, 135.1, 136.8, 140.2, 143.9, 149.2, 152.7, 154.2, 156.1, 161.8, 173.9; HRMS (ESI+) calcd for C₂₈H₃₀N₇O₁₀S [M+H]⁺ 656.1769, found 656.1707 (error 9.4 ppm).

5′-O-[N-(4-Amino-2-hydroxybenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine triethylammonium salt (39)

To a solution of compound 38h (120 mg, 0.18 mmol) in MeOH (5 mL) was added 10% Pd/C (12 mg, 10% by wt) and the reaction stirred under H₂ (1 atm) for 12 h. The reaction mixture was filtered through a plug of Celite. Purification by flash chromatography (100:10:1 MeOH/EtOAc/TEA) afforded the title compound (84 mg, 89%) as a white solid: R_f 0.10 (100:10:1 MeOH/EtOAc/TEA); [α]_D²¹ −153 (c 1.88, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.18 (t, J = 7.2 Hz, 9H), 1.33 (s, 3H), 1.58 (s, 3H), 2.97 (q, J = 7.2 Hz, 6H), 4.27 (d, J = 3.6 Hz, 2H), 4.55 (br s, 1H), 5.11 (d, J = 6.0 Hz, 1H), 5.37 (dd, J = 6.0, 2.4 Hz, 1H), 6.02 (d, J = 2.4 Hz, 1H), 6.11 (dd, J = 8.4, 1.8 Hz, 1H), 6.20 (d, J = 1.8 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 8.15 (s, 1H), 8.48 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.6, 24.2, 26.2, 46.4, 68.6, 82.3, 84.4, 84.5, 90.8, 100.3, 106.2, 108.9, 114.1, 118.9, 131.5, 131.7, 140.2, 149.3, 152.8, 153.8, 156.1, 162.7; HRMS (ESI+) calcd for C₂₀H₂₄N₇O₈S [M+H]⁺ 522.1402, found 522.1385 (error 3.3 ppm).

5′-O-[N-(4-Amino-2-hydroxybenzoyl)sulfamoyl]adenosine (19)

This was prepared from 39 (50 mg, 0.090 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (100:30:3 MeOH/EtOAc/TEA) afforded the title compound (15 mg, 35%) as a white solid: R_f 0.10 (100:30:3 MeOH/EtOAc/TEA); [α]_D²¹ −55.1 (c 3.75, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.18 (t, J = 7.2 Hz, 9H), 3.17 (q, J = 7.2 Hz, 6H), 4.29–4.40 (m, 4H), 4.72 (t, J = 6.0 Hz, 1H), 6.02 (br s, 1H), 6.08 (d, J = 6.0 Hz, 1H), 6.10 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 8.16 (s, 1H), 8.50 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.0, 46.4, 68.2, 71.3, 74.9, 83.5, 88.0, 100.3, 106.2, 109.0, 118.9, 131.7, 139.9, 149.7, 152.7, 153.8, 156.1, 162.7, 177.8; HRMS (ESI−) calcd for C₁₇H₁₈N₇O₈S [M−H]⁻ 480.0943, found 480.0941 (error 0.4 ppm).

5′-O-[N-(6-Fluoro-2-hydroxybenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine triethylammonium salt (38i)

This was prepared from 6-fluorosalicylic acid (70 mg, 0.50 mmol) using the general procedure for CDI mediated acylation. Purification by flash chromatography (100:7.5:1 EtOAc/MeOH/TEA) afforded the title compound (157 mg, 60%): R_f 0.15 (100:7.5:1 EtOAc/MeOH/TEA); [α]_D²¹ −52.8 (c 0.420, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.23 (t, J = 7.2 Hz, 9H), 1.35 (s, 3H), 1.58 (s, 3H), 3.08 (q, J = 7.2 Hz, 6H), 4.31 (dd, J = 10.8, 4.2 Hz, 1H), 4.35 (dd, J = 10.8, 4.2 Hz, 1H), 4.54 (dd, J = 4.2, 1.8 Hz, 1H), 5.14 (dd, J = 3.0, 1.8 Hz, 1H), 5.37 (dd, J = 6.0, 3.0 Hz, 1H), 6.22 (d, J = 3.0 Hz, 1H), 6.44–6.50 (m, 1H), 6.58–6.62 (m, 1H), 7.17–7.22 (m, 1H), 8.15 (s, 1H), 8.42 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ8.2, 24.3, 26.3, 46.6, 68.9, 82.2, 84.5, 90.8, 106.1 (d, J_C-F = 24 Hz), 111.9, 112.6, 114.1, 119.0, 131.8 (d, J_C-F = 11.7 Hz), 132.3 (d, J_C-F = 12.2 Hz), 140.2, 149.3, 152.8, 156.1, 162.0 (d, J = 131 Hz), 164.2, 170.7 (d, J_C-F = 2.7 Hz); HRMS (ESI+) calcd for C₂₀H₂₂FN₆O₈S [M+H]⁺ 525.1198, found 525.1215 (error 3.2 ppm).

5′-O-[N-(6-Fluoro-2-hydroxybenzoyl)sulfamoyl]adenosine triethylammonium salt (20)

This was prepared from 38i (50 mg, 0.10 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (100:16:3 EtOAc/MeOH/TEA) afforded the title compound (36 mg, 76%): R_f 0.09 (100:16:3 EtOAc/MeOH/TEA); [α]_D²¹ −47.0 (c 0.340, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.14 (t, J = 7.2 Hz, 9H), 2.85 (q, J = 7.2 Hz, 6H), 4.33 (dd, J = 6.6, 3.0 Hz, 1H), 4.38–4.45 (m, 3H), 4.69 (t, J = 5.4 Hz, 1H), 6.08 (d, J = 5.4 Hz, 1H), 6.47 (dd, J = 11.4, 8.4 Hz, 1H), 6.60 (d, J = 8.4 Hz, 1H), 7.16–7.21 (m, 1H), 8.15 (s, 1H), 8.41 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.9, 46.2, 68.6, 71.1, 74.8, 83.2, 88.3, 106.1 (d, J_C-F = 24 Hz), 109.8 (d, J_C-F = 9.0 Hz), 112.7 (d, J_C-F = 3.3 Hz), 119.1, 132.4 (d, J_C-F = 12.3 Hz), 139.9, 149.5, 152.7, 156.1, 162.0 (d, J_C-F = 125 Hz), 164.1, 171.1 (d, J = 3.3 Hz); HRMS (ESI+) calcd for C₁₇H₁₈FN₆O₈S [M+H]⁺ 485.0891, found 485.0906 (error 3.1 ppm).

N-Hydroxysuccinimdyl-2,3-dibenzyloxybenzoate (41c)

This was prepared from 2,3-dibenzyloxybenzoic acid²⁹ (1.00 g, 2.99 mmol, 1.0 equiv) using the general procedure for NHS ester synthesis. Purification by flash chromatography (4:1 EtOAc/hexanes) afforded the title compound (0.80 g, 62%): R_f 0.85 (EtOAc); ¹H NMR (600 MHz, CDCl₃) δ 4.49 (m, 4H), 5.13 (s, 4H), 7.12 (t, J = 8.4 Hz, 1H), 7.20–7.30 (m, 4H), 7.32–7.39 (m, 4H), 7.39–7.44 (m, 3H), 7.85 (dd, J = 8.4, 1.2 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 25.9, 71.6, 76.1, 120.4, 121.3, 123.9, 124.4, 127.8, 128.2, 128.4, 128.5, 128.8, 128.9, 136.5, 137.2, 150.1, 153.2, 160.9, 169.4.

5′-O-[N-(2,3-Dibenzyloxybenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine triethylammonium salt (42c)

This was prepared from 41c (837 mg, 1.88 mmol, 2.50 equiv) using the general procedure for NHS mediated acylation. Purification by flash chromatography (95:5:1 MeOH/EtOAc/Et₃N) afforded the title compound (460 mg, 77%): R_f 0.30 (1:5 MeOH/EtOAc); [α]²⁰_D −43.5 (c 0.540, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.08 (t, J = 7.2 Hz, 9H), 1.32 (s, 3H), 1.57 (s, 3H), 2.77 (q, J = 7.2 Hz, 6H), 4.24 (d, J = 3.6 Hz, 2H), 4.40–4.48 (m, 1H), 5.04–5.12 (m, 5H), 5.29 (dd, J = 5.4, 3.0 Hz, 1H), 6.19 (d, J = 3.0 Hz, 1H), 7.00–7.06 (m, 2H), 7.06–7.10 (m, 1H), 7.14–7.22 (m, 3H), 7.26–7.36 (m, 3H), 7.38–7.46 (m, 4H), 8.17 (s, 1H), 8.44 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 10.2, 25.6, 27.5, 47.3, 69.8, 72.0, 76.6, 83.2, 85.6, 85.7, 91.8, 115.2, 116.1, 120.2, 121.8, 125.1, 128.6, 128.9, 129.0, 129.1, 129.5, 129.6, 137.7, 138.5, 139.4, 141.4, 146.8, 150.5, 153.5, 153.9, 157.3, 176.4; HRMS (ESI−) calcd for C₃₄H₃₃N₆O₉S [M−H]⁻ 701.2035, found 701.2082 (error 6.7 ppm).

5′-O-[N-(2,3-Dihydroxybenzoyl)sulfamoyl]adenosine triethylammonium salt (21)

Compound 42c (0.45 g, 0.56 mmol) was treated with 10% Pd/C (90 mg, 20% by wt) at rt under H₂ (1 atm) in MeOH (20 mL) for 12 h. The reaction mixture was filtered through Celite and concentrated to afford 43. The crude product was used directly for the next step.

Compound 43 prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash chromatography (25:75:1 MeOH/EtOAc/Et₃N) afforded the title compound (287 mg, 88%) as a white solid: mp 138–140 °C; R_f 0.2 (1:1 MeOH/EtOAc); [α]²⁰_D −23.5 (c 0.480, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.24 (t, J = 7.8 Hz, 9H), 3.12 (q, J = 7.8 Hz, 6H), 4.26–4.50 (m, 4H), 4.71 (t, J = 4.8 Hz, 1H), 6.08 (d, J = 5.4 Hz, 1H), 6.61 (t, J = 7.8 Hz, 1H), 6.84 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 8.16 (s, 1H), 8.50 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.3, 47.8, 69.6, 72.4, 76.0, 84.6, 89.2, 118.6, 119.3, 120.2, 120.9, 121.9, 141.1, 146.1, 146.8, 150.9, 153.9, 157.3, 175.0; HRMS (ESI+) calcd for C₁₇H₁₉N₆O₉S [M+H]⁺ 483.0929, found 483.0917 (error 2.5 ppm).

2′,3′-OO, -bis(t-Butyldimethylsilyl)-5′-O-[N-(2-pyridon-1-yl)sulfamoyl]adenosine triethylammonium salt (38j)

This was prepared from 2-hydroxynicotinic acid (83 mg, 0.60 mmol) using the general procedure for CDI mediated acylation. Purification by flash chromatography (100:10:2 MeOH/EtOAc/TEA) afforded the title compound (62 mg, 15%): R_f 0.27 (100:10:2 MeOH/EtOAc/TEA); [α]_D²¹ −148 (c 4.20, MeOH); ¹H NMR (600 MHz, CD₃OD) δ −0.39 (s, 3H), −0.07 (s, 3H), 0.13 (s, 3H), 0.15 (s, 3H), 0.69 (s, 9H), 0.95 (s, 9H), 1.27 (t, J = 7.2 Hz, 9H), 3.18 (q, J = 7.2 Hz, 6H), 4.31 (br s, 1H), 4.44–4.47 (m, 3H), 4.87 (dd, J = 7.2, 4.8 Hz, 2H), 6.10 (d, J = 7.2 Hz, 1H), 6.76 (br s, 1H), 7.98 (br s, 1H), 8.18 (s, 1H), 8.34 (d, J = 6.0 Hz, 1H), 8.57 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ −5.1, −4.2, −4.17, −4.11, 9.4, 18.7, 19.0, 26.3, 26.5, 47.8, 69.7, 74.8, 77.5, 86.1, 88.6, 105.9, 114.1 (br), 120.3, 141.6 (br), 142.9, 148.0 (br), 151.1, 154.0, 157.5, 166.0 (br), 172.6; HRMS (ESI−) calcd for C₂₈H₄₄N₇O₈SSi₂ [M−H]⁻ 694.2516, found 694.2548 (error 4.6 ppm).

5′-O-[N-(2-Pyridon-1-yl)sulfamoyl]adenosine triethylammonium salt (23)

This was prepared from 38j (35 mg, 0.050 mmol) using the general procedure for TBAF deprotection. Purification by flash chromatography (90:5:7 MeOH/EtOAc/TEA) afforded the title compound (6.1 mg, 87%): R_f 0.03 (90:5:7 MeOH/EtOAc/TEA); [α]_D²¹ −105 (c 0.760, MeOH); ¹H NMR (600 MHz, DMSO-d₆) δ 1.15 (t, J = 6.6 Hz, 9H), 2.95 (q, J = 6.6 Hz, 6H), 4.11–4.17 (m, 2H), 4.19 (dd, J = 7.8, 4.2 Hz, 1H), 4.24 (dd, J = 10.8, 4.2 Hz, 1H), 4.60 (q, J = 6.0 Hz, 1H), 5.38 (d, J = 4.2 Hz, 1H), 5.53 (d, J = 6.0 Hz, 1H), 5.92 (d, J = 6.0 Hz, 1H), 6.82 (br s, 1H), 7.31 (br s, 2H), 8.11 (s, 1H), 8.12–8.18 (br m, 1H), 8.39 (s, 1H); ¹³C NMR (150 MHz, DMSO-d₆) δ 9.3, 45.7, 68.3, 70.7, 73.5, 82.4, 86.9, 115.3, 118.9, 139.1 (br), 139.3, 149.6, 151.4 (br), 152.7, 156.1, 165.4, 169.6 (missing one aryl carbon); HRMS (ESI−) calcd for C₁₆H₁₆N₇O₈S [M−H]⁻ 466.0787, found 466.0767 (error 4.3 ppm).

N-Hydroxysuccinimdyl 2-fluoropyridine-3-carboxylate (41d)

This was prepared from 2-fluoronicotinic acid (300 mg, 2.1 mmol) using the general procedure for NHS ester synthesis. Purification by flash chromatograpy (1:1 EtOAc/Hexane) afforded the title compound (250 mg, 50%): R_f 0.18 (1:1 EtOAc/Hexane); ¹H NMR (600 MHz, CDCl₃) δ 2.91 (s, 4H), 7.38 (t, J = 6.0 Hz, 1H), 8.49–8.52 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 23.3, 107.0 (d, ²J_C-F = 25.1 Hz), 119.4 (d, ⁴J_C-F = 5.0 Hz), 141.4, 151.4 (d, ³J_C-F = 15.6 Hz), 155.9 (d, ³J_C-F = 15.6 Hz), 159.0 (d, ¹J_C-F = 251 Hz), 166.4; HRMS (ESI+) calcd for C₁₀H₈FN₂O₄ [M+H]⁺ 239.0463, found 239.0469 (error 2.5 ppm).

5′-O-{N-[(2-Fluoropyridin-3-yl)carbonyl]sulfamoyl}-2′,3′-O-isopropylideneadenosine (42d)

This was prepared from 41d (100 mg, 0.40 mmol) using the general procedure for NHS ester mediated acylation. Purification by flash chromatography (1:7 MeOH/EtOAc) afforded the title compound (58 mg, 27%): R_f 0.17 (1:7 MeOH/EtOAc); [α]_D²¹ −63.0 (c 2.50, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.36 (s, 3H), 1.59 (s, 3H), 4.32 (d, J = 3.6 Hz, 2H), 4.56–4.58 (m, 1H), 5.15 (dd, J = 6.0, 2.4 Hz, 1H), 5.37 (dd, J = 6.0, 3.0 Hz, 1H), 6.22 (d, J = 3.0 Hz, 1H), 7.26–7.30 (m, 1H), 8.16 (s, 1H), 8.17–8.23 (m, 2H), 8.45 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 24.3, 26.3, 68.7, 82.1, 84.5, 84.6, 90.7, 114.1, 118.9, 121.4, 140.2, 142.2, 148.2, 148.3, 149.3, 152.7, 156.1, 170.1 (d, ¹J_C-F = 252 Hz), 173.7; HRMS (ESI−) calcd for C₁₉H₁₉FN₇O₇S [M−H]⁻ 508.1056, found 508.1059 (error 0.6 ppm).

5′-O-{N-[(2-Fluoropyridin-3-yl)carbonyl]sulfamoyl}adenosine triethylammonium salt (24)

This was prepared from 42d (30 mg, 0.060 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (100:20:1.5 MeOH/EtOAc/TEA) afforded the title compound (14 mg, 50%): R_f 0.17 (100:20:1.5 MeOH/EtOAc/TEA); [α]_D²¹ −69.6 (c 2.55, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.24 (t, J = 7.2 Hz, 9H), 3.13 (q, J = 7.2 Hz, 6H), 4.31–4.32 (m, 1H), 4.32–4.43 (m, 3H), 4.70 (t, J = 4.8 Hz, 1H), 6.08 (d, J = 5.4 Hz, 1H), 7.27–7.29 (m, 1H), 8.16 (s, 1H), 8.18 (d, J = 4.2 Hz, 1H), 8.24–8.27 (m, 1H), 8.50 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.1, 46.5, 68.4, 71.2, 74.8, 83.4, 87.9, 118.9, 121.5 (d, J_C-F = 4.5 Hz), 122.1 (d, J_C-F = 25.7 Hz), 139.9, 142.2 (d, J_C-F = 2.7 Hz), 148.3 (d, J_C-F =14.0 Hz), 149.6, 152.7, 156.1, 160.6 (d, J_C-F = 243 Hz), 170.1 (d, J_C-F = 6.2 Hz); HRMS (ESI−) calcd for C₁₆H₁₅FN₇O₇S [M−H]⁻ 468.0743, found 468.0740 (error 0.6 ppm);.

N-Hydroxysuccinimdyl 2-chloropyridine-3-carboxylate (41e)

This was prepared from 2-chloronicotinic acid (780 mg, 5.00 mmol) using the general procedure for NHS ester synthesis. Purification by flash chromatography (1:1 EtOAc/Hexane) afforded the title compound (660 mg, 52%): R_f = 0.21 (1:1 EtOAc/Hexane); ¹H NMR (600 MHz, CDCl₃) δ 2.92 (s, 4H), 7.41 (t, J = 7.8 Hz, 1H), 8.42 (dd, J = 7.8, 1.8 Hz, 1H), 8.63 (dd, J = 7.8, 1.8 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 25.9, 122.1, 122.4, 141.4, 151.7, 153.9, 159.6, 168.9; HRMS (ESI+) calcd for C₁₀H₈ClN₂O₄ [M+H]⁺ 255.0167, found 255.0163 (error 1.6 ppm).

5′-O-{N-[(2-Chloropyridin-3-yl)carbonyl]sulfamoyl}-2′,3′-O-isopropylideneadenosine triethylammonium salt (42e)

This was prepared from 41e (50 mg, 0.20 mmol) using the general procedure for NHS mediated acylation. Purification by flash chromatography (100:10:1 EtOAc/MeOH/TEA) afforded the title compound (86 mg, 82%): R_f 0.15 (100:10:1 EtOAc/MeOH/TEA); [α]_D²¹ = −94.3 (c 0.340, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.27 (t, J = 7.8 Hz, 9H), 1.36 (s, 3H), 1.59 (s, 3H), 3.16 (q, J = 7.8 Hz, 6H), 4.33–4.35 (m, 2H), 4.57 (dd, J = 5.4, 2.4 Hz, 1H), 5.20 (dd, J = 6.0, 2.4 Hz, 1H), 5.39 (dd, J = 6.0, 3.0 Hz, 1H), 6.24 (d, J = 3.0 Hz, 1H), 7.35 (dd, J = 7.8, 4.8 Hz, 1H), 7.90 (dd, J = 7.8, 1.8 Hz, 1H), 8.19 (s, 1H), 8.32 (dd, J = 4.8, 1.8 Hz, 1H), 8.45 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.0, 24.4, 26.3, 46.7, 68.9, 82.1, 84.5, 84.6, 90.7, 114.2, 119.0, 122.7, 136.1, 138.3, 140.2, 147.3, 149.0, 149.3, 152.8, 156.1, 172.3; HRMS (ESI+) calcd for C₁₉H₂₁Cl N₇O₇S [M+H]⁺ 526.0906, found 526.0909 (error 0.6 ppm).

5′-O-{N-[(2-Chloropyridin-3-yl)carbonyl]sulfamoyl}adenosine triethylammonium salt (25)

This was prepared from 42e (40 mg, 0.080 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (100:20:1 EtOAc/MeOH/TEA) afforded the title compound (21 mg, 53%): R_f 0.10 (100:20:1 EtOAc/MeOH/TEA); [α]_D²¹ −64.7 (c 0.340, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.26 (t, J = 7.8 Hz, 9H), 3.15 (q, J = 7.8 Hz, 6H), 4.33 (dd, J = 6.6, 3.0 Hz, 1H), 4.39–4.46 (m, 3H), 4.73 (t, J = 6.0 Hz, 1H), 6.09 (d, J = 4.8 Hz, 1H), 7.35 (dd, J = 7.2, 4.2 Hz, 1H), 7.94 (dd, J = 7.2, 1.8 Hz, 1H), 8.19 (s, 1H), 8.31 (dd, J = 4.2, 1.8 Hz, 1H), 8.49 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 8.1, 46.7, 68.5, 71.2, 74.8, 83.4, 88.1, 119.0, 122.6, 136.2, 138.3, 139.9, 147.3, 148.9, 149.7, 152.7, 156.1, 172.4; HRMS (ESI+) calcd for C₁₆H₁₇ClN₇O₇S [M+H]⁺ 486.0593, found 486.0598 (error 1.0 ppm).

5′-O-{N-[(Morpholino)carbonyl]sulfamoyl}adenosine sodium salt (26)

Morpholine (54 μL, 3.0 mmol, 1.0 equiv) was added dropwise to a solution of chlorosulfonyl isocyanate (54 μL, 3.0 mmol, 1.0 equiv) in CH₂Cl₂ (15 mL) at −20 °C. The reaction mixture was stirred at −15 °C to −20 °C for 4 h, then the solvent was evaporated in vacuo and the crude product 52 was used for the subsequent step without any further workup.

To the above intermediate 52 was added a solution of 2′,3′-O-isopropylideneadenosine (155 mg, 2 mmol, 0.66 equiv) and pyridine (48 μL, 3 mmol, 1 equiv) in MeCN (38 mL). The resulting solution was stirred at rt for 20 h. The solvent was evaporated in vacuo and purification by flash column chromatography (70:30:1 EtOAc/MeOH/TEA) afforded 2′,3′-O-isopropylidene-5′-O{N-[(morpholino)carbonyl]sulfamoyl}adenosine triethylammonium salt 54 that was approximately 50–75% pure based on ¹H NMR.

Crude 54 prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash column chromatography (70:30:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (12 mg, 4% overall yield): R_f 0.29 (6:4 EtOAc/MeOH); [α]_D²¹ −0.06 (c 0.22, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 3.40–3.60 (m, 4H), 3.58–3.60 (m, 4H), 4.25–4.30 (m, 3H), 4.41–4.43 (m, 1H), 4.70 (t, J = 5.4 Hz, 1H), 6.09 (d, J = 6.0 Hz, 1H), 8.19 (s, 1H), 8.54 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 49.7, 68.1, 68.8, 72.5, 76.3, 85.0, 89.1, 120.2, 141.4, 151.0, 154.0, 157.4, 162.4; HRMS (ESI−) calcd for C₁₅H₂₀N₇O₈S [M−H]⁻ 458.1099, found 458.1106 (error 1.4 ppm).

2′,3′-OO, -bis(t-Butyldimethylsilyl)-5′-O-{N-[(cyclopentyl)carbonyl]sulfamoyl}adenosine triethylammonium salt (42f)

Cyclopentanecarbonyl chloride (920 μL, 7.54 mmol) was converted to the NHS ester 41f using the general procedure B for NHS ester synthesis. The crude NHS ester was used directly for the next step.

The title compound was prepared from 41f (126 mg, 0.60 mmol) using the general procedure for NHS ester mediated acylation. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) afforded the title compound (270 mg, 67% overall yield): R_f 0.23 (6:4 EtOAc/MeOH); [α]_D²¹ −64.7 (c 0.34, MeOH); ¹H NMR (600 MHz, CD₃OD) δ −0.37 (s, 3H), −0.01 (s, 3H), 0.16 (s, 3H); 0.17 (s, 3H), 0.71 (s, 9H), 0.98 (s, 9H), 1.23 (t, J = 7.2 Hz, 9H), 1.54–1.57 (m, 2H), 1.70–1.72 (m, 2H), 1.80–1.84 (m, 4H), 2.65 (p, J = 8.4 Hz, 1H), 3.03 (q, J = 7.2 Hz, 6H), 4.29–4.34 (m, 3H), 4.45 (d, J = 4.2 Hz, 1H), 4.84–4.86 (m, 1H, obscured by CD₃OD: assigned by gCOSY as H-2′), 6.12 (d, J = 7.2 Hz, 1H), 8.18 (s, 1H), 8.57 (s, 1H); ¹³C NMR (600 MHz, CD₃OD) δ −5.2, −4.2, −4.14 (2C), 9.8, 18.8, 19.0, 26.3, 26.5, 27.1, 31.9, 32.0 47.8, 69.1, 75.4, 77.7, 86.6, 88.3, 120.2, 141.7, 151.2, 154.0, 157.5, 186.9; MS (ESI−) calcd for C₂₈H₄₉N₆O₇SSi₂ [M−H]⁻ 669.3, found 669.3.

5′-O-{N-[(Cyclopentyl)carbonyl]sulfamoyl}adenosine sodium salt (27)

This was prepared from 42f (270 mg, 0.40 mmol) using the general procedure for TBS protection. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (96 mg, 55%): R_f 0.25 (8:2 EtOAc/MeOH); [α]_D²¹ −0.23 (c 0.49, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 1.52–1.54 (m, 2H), 1.66–1.68 (m, 2H), 1.72–1.77 (m, 2H), 1.80–1.83 (m, 2H), 2.63 (p, J = 8.4 Hz, 1H), 4.24–4.31 (m, 3H), 4.38–4.39 (m, 1H), 4.69 (t, J = 6.0 Hz, 1H), 6.08 (d, J = 6.0 Hz, 1H), 8.19 (s, 1H), 8.48 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 27.1, 27.1, 31.3, 31.9, 69.2, 72.5, 76.2, 84.8, 89.3, 120.3, 141.3, 151.0, 154.0, 157.4, 186.3; HRMS (ESI−) calcd for C₁₆H₂₁N₆O₇S [M−H]⁻ 441.1197, found 441.1183 (error 3.2 ppm).

2′,3′-OO, -bis(t-Butyldimethylsilyl)-5′-O-{N-[(cyclohexyl)carbonyl]sulfamoyl}adenosine triethylammonium salt (42g)

Cyclohexanecarbonyl chloride (930 μL, 6.82 mmol) was converted to the NHS ester 41g using the general procedure B for NHS ester synthesis. The crude NHS ester was used directly for the next step.

The title compound was prepared from 41g (88 mg, 0.39 mmol) using the general procedure for NHS ester mediated acylation. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) afforded the title compound (230 mg, 87%); R_f 0.22 (6:4 EtOAc/MeOH); ¹H NMR (600 MHz, CD₃OD) δ −0.37 (s, 3H), −0.01 (s, 3H), 0.16 (s, 3H), 0.17 (s, 3H), 0.71 (s, 9H), 0.98 (s, 9H), 1.23 (t, J = 7.2 Hz, 9H), 1.28–1.33 (m, 3H), 1.43–1.46 (m, 2H), 1.65–1.66 (m, 1H), 1.75–1.76 (m, 2H), 1.86–1.88 (m, 2H), 2.16 (tt, J = 11.4, 3.0 Hz, 1H), 3.06 (q, J = 7.2 Hz, 6H), 4.30–4.33 (m, 3H), 4.45 (d, J = 4.2 Hz, 1H), 4.84–4.86 (m, 1H, obscured by CD₃OD: assigned by gCOSY as H-2′), 6.11 (d, J = 7.8 Hz, 1H), 8.18 (s, 1H), 8.56 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ −5.3, −4.33, −4.28 (2C), 9.7, 18.6, 18.9, 26.2, 26.4, 27.0, 27.4, 31.25, 31.29, 47.6, 69.0, 74.9, 77.5, 86.4, 88.2, 120.1, 141.5, 151.1, 153.9, 157.3, 185.8; MS (ESI−) calcd for C₂₉H₅₁N₆O₇SSi₂ [M−H]⁻ 683.3, found 683.3.

5′-O-{N-[(Cyclohexyl)carbonyl]sulfamoyl}adenosine sodium salt (28)

This was prepared from 42g (230 mg, 0.33 mmol) using the general procedure for TBS protection. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (82 mg, 51%): R_f 0.22 (8:2 EtOAc/MeOH); [α]_D²¹ −0.26 (c 0.50, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 1.17–1.30 (m, 3H), 1.37–1.42 (m, 2H), 1.63–1.65 (m, 1H) 1.73–1.74 (m, 2H), 1.82–1.84 (m, 2H), 2.14 (tt, J= 11.4, 3.0 Hz, 1H), 4.23–4.30 (m, 3H), 4.37–4.39 (m, 1H), 4.69 (t, J = 6.0 Hz, 1H), 6.08 (d, J = 6.0 Hz, 1H), 8.19 (s, 1H), 8.48 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 27.2, 27.3, 31.27, 31.28, 69.2, 72.6, 76.2, 84.8, 89.3, 120.3, 141.3, 151.0, 153.9, 157.4, 186.4; HRMS (ESI−) calcd for C₁₇H₂₃N₆O₇S [M−H]⁻ 455.1354, found 455.1348 (error 1.3 ppm).

2′,3′-OO, -bis(t-Butyldimethylsilyl)-5′-O-[(N-(acetyl)sulfamoyl]adenosine triethylammonium salt (42h)

Acetic anhydride (920 μL, 9.8 mmol) was converted to the NHS ester 41h using the general procedure B for NHS ester synthesis. The crude NHS ester was used directly for the next step.

The title compound was prepared from 41h (96 mg, 0.61 mmol) using the general procedure for NHS ester mediated acylation. Purification by flash chromatography (70:30:1 EtOAc/MeOH/TEA) afforded the title compound (350 mg, 93%): R_f 0.20 (85:15 EtOAc/MeOH); ¹H NMR (600 MHz, CD₃OD) δ −0.35 (s, 3H), −0.01 (s, 3H), 0.16 (s, 3H), 0.17 (s, 3H), 0.71 (s, 9H), 0.98 (s, 9H), 1.29 (t, J = 7.2 Hz, 9H), 1.96 (s, 3H), 3.17 (q, J = 7.2 Hz, 6H), 4.46 (d, J = 4.2 Hz, 1H), 4.30–4.35 (m, 3H), 4.82–4.84 (1H, m, obscured by CD₃OD: assigned by gCOSY as H-2′), 6.12 (d, J = 7.2 Hz, 1H), 8.19 (s, 1H), 8.56 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ −5.2, −4.24, −4.20, −4.17, 9.5, 18.8, 19.0, 26.2, 26.3, 26.5, 47.9, 69.2, 74.9, 77.7, 86.4, 88.3, 120.2, 141.6, 151.2, 154.0, 157.5, 180.4; MS (ESI−) calcd for C₂₄H₄₃N₆O₇SSi₂ [M−H]⁻ 615.2, found 615.2.

5′-O-[N-(Acetyl)sulfamoyl]adenosine sodium salt (29)

This was prepared from 42h (350 mg, 0.57 mmol) using the general procedure for TBS protection. Purification by flash chromatography (80:20:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (148 mg, 74%): R_f 0.26 (7:3 EtOAc/MeOH); [α]_D²¹ −0.25 (c 0.56, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 1.95 (s, 3H), 4.26–4.33 (m, 3H), 4.38–4.39 (m, 1H), 4.66 (t, J = 6.0 Hz, 1H), 6.08 (d, J = 6.0 Hz, 1H), 8.19 (s, 1H), 8.49 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 26.2, 69.2, 72.4, 76.2, 84.7, 89.3, 120.3, 141.2, 151.0, 154.0, 157.4, 180.8, HRMS (ESI−) calcd for C₁₆H₂₁N₆O₇S [M−H]⁻ 387.0728, found 387.0722 (error 1.5 ppm).

5′-O-{N-[(Methoxy)carbonyl]sulfamoyl}adenosine sodium salt (30)

A solution of 36 (80 mg, 0.2 mmol, 1.0 equiv) and CDI (65 mg, 0.4 mmol, 2 equiv) in MeCN (10 mL) was stirred at rt for 2 h. Next, MeOH (3 mL) and Et₃N (55 μL, 0.4 mmol, 2.0 equiv) were added and the reaction was stirred for an additional 2 h, then the reaction mixture was concentrated in vacuo. Purification by flash column chromatography (80:20:1 EtOAc/MeOH/Et₃N) afforded 2′,3′-O-isopropylidene-5′-O-{N-[(methoxy)carbonyl]sulfamoyl}adenosine triethylammonium salt.

2′,3′-O-isopropylidene-5′-O-{N-[(methoxy)carbonyl]sulfamoyl}adenosine triethylammonium salt prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash chromatography (70:30:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (84 mg, 64% overall yield): R_f 0.20 (7:3 EtOAc/MeOH); [α]_D²¹ −0.16 (c 0.58, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 3.56 (s, 3H), 4.23–4.31 (m, 3H), 4.36–4.38 (m, 1H), 4.65 (t, J = 5.4 Hz, 1H), 6.08 (d, J = 6.0 Hz, 1H), 8.18 (s, 1H), 8.48 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 51.8, 68.2, 71.4, 75.3, 83.8, 88.3, 119.3, 140.2, 145.0, 153.0, 156.4, 161.2; HRMS (ESI−) calcd for C₁₇H₂₃N₆O₇S [M−H]⁻ 403.0677, found 403.0690 (error 3.1 ppm).

5′-O-{N-[(Benzyl)carbonyl]sulfamoyl}adenosine sodium salt (31)

Phenylacetic acid (1.00 g, 7.3 mmol) was converted to the NHS ester 41i using the general procedure A for NHS ester synthesis. The crude NHS ester was used directly for the next step.

Compound 41i (85 mg, 0.37 mmol) prepared above was treated with 36 using the general procedure for NHS ester mediated acylation to afford crude 2′,3′-O,O -bis(t-butyldimethylsilyl)-5′-O-{N-[(benzyl)carbonyl]sulfamoyl}triethylammonium salt that was used directly for the next step.

2′,3′-OO, -bis(t-Butyldimethylsilyl)-5′-O-{N-[(benzyl)carbonyl]sulfamoyl}triethylammonium salt prepared above was deprotected using the general procedure for TBS deprotection. Purification by flash chromatography (75:25:1EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (110 mg, 65% overall yield: R_f 0.29 (7:3 EtOAc/MeOH); [α]_D²¹ −0.14 (c 0.43, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 3.48 (s, 2H), 4.18–4.20 (m, 1H), 4.22–4.26 (m, 2H), 4.29–4.30 (m, 1H), 4.62 (t, J = 5.4 Hz, 1H), 6.06 (d, J = 6.0 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.8 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 8.19 (s, 1H), 8.45 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 47.2, 69.3, 72.4, 76.2, 84.6, 89.3, 120.3, 127.3, 129.3, 130.4, 138.5, 141.2, 150.9, 154.0, 157.4, 181.1; HRMS (ESI−) calcd for C₁₈H₁₉N₆O₇S [M−H]⁻ 463.1041, found 463.1046 (error 1.1 ppm).

5′-O–[N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)sulfamoyl]adenosine triethylammonium salt (32)

N-hydroxysuccinimidyl-4-azidotetrafluorobenzoate²⁶ (235 mg, 1.0 mmol, 1.0 equiv) was treated with 36 using the general procedure for NHS ester mediated acylation. Purification by flash column chromatography (80:20:1 EtOAc/MeOH/Et₃N) afforded 5′-O–[N-(4-azido-2,3,5,6-tetrafluorobenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine triethylammonium salt (141 mg, 39%).

5′-O–[N-(4-azido-2,3,5,6-tetrafluorobenzoyl)sulfamoyl]-2′,3′-O-isopropylideneadenosine triethylammonium salt (105 mg, 0.2 mmol) prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash column chromatography (80:20:1 EtOAc/MeOH/Et₃N) afforded the title compound (62 mg, 47%): R_f 0.29 (8:2 EtOAc/MeOH); [α]_D²¹ −0.11 (c 0.37, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 1.29 (t, J = 7.8 Hz, 9H), 3.21 (q, J = 7.8 Hz, 6H), 4.32–4.42 (m, 4H), 4.70 (t, J = 5.4 Hz, 1H), 6.09 (d, J = 5.4 Hz, 1H), 8.19 (s, 1H), 8.48 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.3, 48.1, 69.9, 72.5, 76.1, 84.7, 89.3, 120.3, 141.2, 151.0, 154.0, 157.4, 166.1 (missing 3 aryl carbons and C=O); HRMS (ESI−) calcd for C₁₇H₁₂F₄N₉O₇S [M−H]⁻ 562.0522, found 562.0548 (error 4.6 ppm).

N-Hydroxysuccinimdyl N-(t-butyloxycarbonyl)-L-phenylalanine (41k)

This was prepared from N-(t-butyloxycarbonyl)-L-phenylalanine (795 mg, 3.00 mmol) using the general procedure for NHS ester synthesis. Purification by flash chromatography (2:5 EtOAc/Hexane) afforded the title compound (800 mg, 74%): R_f 0.33 (3:1 Hexane/EtOAc); [α]_D²¹ −37.1 (c 0.730, MeOH); ¹H NMR (600 MHz, CDCl₃) δ 1.39 (s, 9H), 2.84 (s, 4H), 3.18 (dd, J = 13.2, 5.4 Hz, 1H), 3.30 (dd, J = 13.2, 5.4 Hz, 1H), 4.90 (t, J = 5.4 Hz, 1H), 7.25–7.33 (m, 5H); ¹³C NMR (150 MHz, CDCl₃) δ 25.8, 28.2, 38.4, 52.8, 80.8, 127.6, 128.9, 129.9, 134.9, 154.8, 167.9, 168.7; MS (ESI+) C₁₃H₁₅N₂O₄ [M+H] calcd 263.1, found 263.1.

5′-O-{N-[N-(t-Butyloxycarbonyl)-L-phenylalanyl]sulfamoyl}-2′,3′-O-isopropylideneadenosine (42k)

This was prepared from 41k (363 mg, 1.00 mmol) using the general procedure for NHS mediated acylation. Purification by flash chromatography (100:10:1 EtOAc/MeOH/TEA) afforded the title compound (600 mg, 95%): R_f 0.16 (10:1 EtOAc/MeOH); [α]_D²¹ − 19.1 (c 1.00, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.33 (s, 9H), 1.36 (s, 3H), 1.58 (s, 3H), 2.84 (dd, J = 13.2, 9.0 Hz, 1H), 3.10 (dd, J = 13.2, 4.8 Hz, 1H), 4.16–4.23 (m, 3H), 4.47–4.51 (m, 1H), 5.06 (d, J = 6.0 Hz, 1H), 5.32 (br s, 1H), 6.21 (br s, 1H), 7.11–7.22 (m, 5H), 8.19 (s, 1H), 8.41 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 25.1, 26.3, 27.5, 57.9, 80.5, 82.0, 84.4, 84.6, 85.3, 85.9, 90.7, 92.3, 114.1, 118.9, 126.2, 128.1, 129.4, 139.4, 140.3, 149.1, 149.3, 152.7, 156.0; HRMS (ESI+) calcd for C₂₇H₃₆N₇O₉S 634.2295, found 634.2309 (error 3.0 ppm).

5′-O-[N-(L-Phenylalanyl)sulfamoyl]adenosine sodium salt (33)

This was prepared from 42k (200 mg, 0.32 mmol) using the general procedure for TFA deprotection. Purification by HPLC (Microsorb^™ C18 Dynamax^™ preparative HPLC column, 41.4 × 250 mm, pore size 100 Å, 20–80% A in 45 min, A = 40 mM TEAB buffer, B = 70% CH₃CN in H₂O, flow rate = 40 mL/min, monitored at 254 nm) and lypholization of the pooled fractions followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (90 mg, 58%): t_R = 12.7 min; [α]_D²¹ −7.5 (c 0.74, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 2.87 (dd, J = 13.2, 5.4 Hz, 1H), 3.17 (dd, J = 13.2, 5.4 Hz 1H), 3.64 (t, J = 6.6 Hz, 1H), 4.22–4.29 (m, 3H), 4.36 (t, J = 4. 2 Hz, 1H), 4.62 (t, J = 4.8 Hz, 1H), 6.07 (d, J = 5.4 Hz, 1H), 7.13–7.15 (m, 1H), 7.22–7.25 (m, 4H), 8.18 (s, 1H), 8.50 (s, 1H); ¹³C NMR (150 MHz, CD3OD) δ 41.6, 58.5, 67.9, 70.9, 75.1, 83.2, 88.3, 119.0, 126.2, 128.2, 129.3, 138.4, 140.1, 149.6, 152.7, 156.1, 181.6 (missing 2C’s); HRMS (ESI+) calcd for C₁₉H₂₄N₇O₇S 494.1458 found 494.1489 (error 0.6 ppm).

5′-O-[N-(3-{[(Morpholino)carbonyl]amino}propanoyl)sulfamoyl]adenosine sodium salt (47)

Morpholinocarbonyl chloride (0.77 mg, 6.7 mmol) was converted to the NHS ester 44 using the general procedure B for NHS ester synthesis. The crude NHS ester was used directly for the next step.

Compound 44 (82 mg, 0.36 mmol) prepared above was treated with 37 using the general procedure for NHS mediated acylation. The crude product 46 was used directly for the next step.

Compound 46 prepared above was deprotected using the general procedure for TBS deprotection. Purification by flash chromatography (70:30:1 EtOAc/MeOH/TEA) followed by conversion to the sodium salt using the general procedure for ion-exchange afforded the title compound (107 mg, 56% overall yield): R_f 0.21 (6:4 EtOAc/MeOH); [α]_D²¹ −0.08 (c 0.42, CH₃OH); ¹H NMR (600 MHz, CD₃OD) δ 2.40 (t, J = 6.6 Hz, 2H), 3.29 (t, J = 4.8 Hz, 4H), 3.42 (t, J = 6.6 Hz, 2H), 3.58 (t, J = 4.8 Hz, 4H), 4.27–4.33 (m, 3H), 4.39 (t, J = 4.2 Hz, 1H), 4.66 (t, J = 5.4 Hz, 1H), 6.08 (d, J = 5.4 Hz, 1H), 8.20 (s, 1H), 8.51 (s, 1H), ¹³C NMR (150 MHz, CD₃OD) δ 38.8, 40.3, 45.2, 67.7, 69.2, 72.4, 76.3, 84.6, 89.4, 120.3, 141.3, 150.9, 154.0, 157.4, 160.2, 181.4; HRMS (ESI−) calcd for C₁₈H₂₅N₈O₉S [M−H]⁻ 529.1470, found 529.1489 (error 3.5 ppm).

5′-Azido-N⁶-butyloxylcarbonyl-5′-deoxy-2′,3′-O-isopropylideneadenosine

To a solution of 5′-azido-5′-deoxy-2′,3′-O-isopropylideneadenosine²² (146 mg, 0.44 mmol, 1.0 equiv) in THF/DMF (4:1, 10 mL) was added sodium hydride (60% dispension in mineral oil, 20 mg, 0.5 mmol, 1.15 equiv) at rt. The resulting solution was stirred 30 min then solid di-tert-butyl dicarbonate (106 mg, 0.48 mmol, 1.1 equiv) was added in one portion. The reaction was monitored by TLC until complete consumption of the amine was observed (3 h). The solution was diluted with CH₂Cl₂ (20 mL) and washed with saturated aq NaHCO₃ (20 mL). The aqueous phase was back extracted with CH₂Cl₂ (2× 10 mL) and the combined organic phase was washed with H₂O (20 mL), saturated aq NaCl (30 mL), dried (Na₂SO₄) and concentrated. Purification by flash chromatography (4:6 Hexane/EtOAc) afforded the title compound (144 mg, 60%): R_f 0.55 (1:2 Hexane/EtOAc); [α]_D²¹ −33.1 (c 0.972, CH₃OH); ¹H NMR (600 MHz, CDCl₃) δ 1.35 (s, 3H), 1.53 (s, 9H), 1.58 (s, 3H), 3.54 (d, J = 5.4 Hz, 2H), 4.36 (dd, J = 3.6, 5.4 Hz, 1H), 5.01 (dd, J = 6.6, 3.6 Hz, 1H), 5.42 (dd, J = 6.6, 2.4 Hz, 1H), 6.13 (d, J = 2.4 Hz, 1H), 8.09 (s, 1H), 8.74 (s, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 25.5, 27.3, 28.3, 52.4, 82.0, 82.6, 84.2, 85.8, 90.9, 115.1, 122.3, 141.8, 149.8, 150.3, 150.4, 153.2; MS (APCI+) calcd for C₁₈H₂₅O₅N₈ [M+H]⁺ 433.2, found 433.2.

5′-Amino-N⁶-t-butoxycarbonyl-5′-deoxy-2′,3′-O-isopropylideneadenosine (56)

To a solution of 5′-azido-N⁶-tert-butoxycarbonyl-5′-deoxy-2′,3′-O-isopropylideneadenosine (150 mg, 0.35 mmol, 1.0 equiv) in MeOH (5.0 mL) was added 10% Pd/C (10 mg, 7% by wt) in MeOH (0.5 mL) and the reaction stirred under H₂ (2.5 atm) for 12 h. The reaction mixture was filtered through a plug of Celite. Purification by flash chromatography (100:17:1 EtOAc/MeOH/TEA) afforded the title compound 132 mg, 94%). ¹H NMR, ¹³C NMR, and HRMS agreed with literature values.¹¹

5′-Amino-5′-N-[(chromon-3-yl)carbonyl]adenosine (58)

To a solution of chromone 3-carboxylic acid (74 mg, 0.39 mmol) and CDI (63.3 mg, 0.39 mmol, 1.0 equiv) in DMF (6 mL) was stirred at 60 °C for 2 h. The solution was cooled to rt and a mixture of 56 (100 mg, 0.32 mmol, 0.8 equiv) and DBU (92 μL, 0.62 mmol, 1.5 equiv) were then added and the reaction stirred overnight at rt. The reaction mixture was concentrated in vacuo and purified by flash chromatography to afford 57. The crude product was used directly for the next step.

Crude 57 prepared above was deprotected using the general procedure for TFA deprotection. Purification by flash chromatography (60:40 EtOAc/MeOH) afforded the title compound (29 mg, 20% overall yield): R_f 0.22 (7:3 EtOAc/MeOH); [α]_D²¹ −31.7 (c 0.11, MeOH); ¹H NMR (600 MHz, DMSO) δ 3.97 (br s, 1H), 4.08 (dd, J = 5.4, 4.8 Hz, 1H), 4.16 (dd, J = 6.6, 3.6 Hz, 1H), 4.22 (br s, 1H), 4.67–4.71 (m, 1H), 5.43 (br s, 1H), 5.57 (br s, 1H), 5.92 (d, J = 6.0 Hz, 1H), 7.26–7.30 (m, 4H), 7.63 (t, J = 7.8 Hz, 1H), 7.88 (t, J = 7.8 Hz, 1H), 8.10 (br s, 1H), 8.33 (s, 1H), 8.43–8.45 (m, 1H), 10.39 (br s, 1H); ¹³C NMR (150 MHz, DMSO) δ 51.7, 70.8, 72.7, 82.1, 87.7, 96.0, 116.9, 119.3, 120.2, 123.9, 125.3, 134.3, 139.9, 149.3, 152.6, 154.2, 156.1, 162.6, 163.2, 179.4; MS (APCI−) calcd for C₂₀H₁₈N₆O₆ [M−H]⁻ 437.1215, found 437.1378.

2-Benzyloxybenzamide (59)

To a solution of 2-hydroxybenzamide (2.74 g, 20 mmol, 1.0 equiv) and K₂CO₃ (3.30 g, 24 mmol, 1.2 equiv) in acetone (100 mL) was added benzyl bromide (2.80 mL, 24 mmol, 1.2 equiv) and the reaction heated at reflux for 16 h. After cooling to rt, the solution was filtered and the filtrate was concentrated under reduced pressure. The crude solid was recrystallized from acetone to afford the title compound (2.89 g, 62%) as a white solid: ¹H NMR (CDCl₃, 600 MHz) δ 5.19 (s, 2H), 5.92 (br s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 7.09 (t, J = 8.4 Hz, 1H), 7.36–7.48 (m, 6H), 7.73 (br s, 1H), 8.23 (d, J = 7.8 Hz, 1H); ¹³C NMR (CDCl₃, 150 MHz) δ 71.5, 112.9, 121.3, 121.8, 128.1, 129.0, 129.2, 132.9, 133.6, 135.8, 157.4, 167.2; HRMS (APCI+) calcd for C₁₄H₁₄NO₂ [M+H]⁺ 228.1019, found 228.1042 (error 10.0 ppm).

5′-Amino-5′-N-{[(2-benzyloxybenzoyl)amino]carbonyl}-N⁶-t-butoxycarbonyl-5′-deoxy-2′,3′-O-isopropylideneadenosine (61)

To a stirred solution of 59 (114 mg, 0.50 mmol, 1.0 equiv) in CH₂Cl₂ (20 mL) oxalyl chloride (129 μL, 1.5 mmol, 3 equiv) was added dropwise at rt. The solution was then heated at 50 °C for 3 h. After cooling to rt, the reaction was concentrated in vacuo to afford a light yellow solid. Next, a solution of 56 (244 mg, 0.6 mmol, 1.2 equiv) in CH₃CN (10 mL) was added and the resulting mixture was stirred 16 h at rt. The crude reaction was concentrated in vacuo and the residue was partitioned between CH₂Cl₂ (25 mL) and H₂O (25 mL). The aqueous layer was extracted with CH₂Cl₂ (2×25 mL). The combined organic extracts were dried (Na₂SO₄) and concentrated under reduced pressure. Purification by flash chromatography (7:3 EtOAc/Hexane) afforded the title compound (290 mg, 88%) as a white solid: R_f 0.10 (6:4 EtOAc/Hexane); ¹H NMR (CDCl₃, 600 MHz) δ 1.35 (s, 3H), 1.54 (s, 9H), 1.59 (s, 3H), 3.64–3.71 (m, 2H), 4.41–4.44 (m, 1H), 4.99 (dd, J = 6.0, 3.6 Hz, 1H), 5.28 (s, 2H), 5.37 (dd, J = 6.0, 3.0 Hz, 1H), 6.12 (d, J = 3.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 7.35–7.37 (m, 1H), 7.40–7.43 (m, 4H), 7.48 (dt, J = 8.4, 1.2 Hz, 1H), 8.05 (br s, 1H, NH), 8.12 (dd, J = 7.8, 1.2 Hz, 1H), 8.18 (s, 1H), 8.74 (s, 1H), 8.85 (t, J = 6.0 Hz, 1H, NH), 9.97 (s, 1H, NH); ¹³C NMR (CDCl₃, 150 MHz) δ 25.7, 27.5, 28.3, 41.5, 71.8, 81.9, 82.5, 84.2, 85.3, 90.4, 113.5, 115.2, 119.9, 122.1, 122.3, 127.7, 129.1, 129.2, 132.9, 134.9, 135.0, 141.7, 149.7, 150.2, 150.7, 153.4, 153.9, 157.2, 165.9; HRMS (ESI+) calcd for C₃₃H₃₈N₇O₈ [M+H]⁺ 660.2776, found 660.2667 (error 16.5 ppm).

5′-Amino-5′-N-{[(2-benzyloxybenzoyl)amino]carbonyl}-5′-deoxyadenosine (62)

This was prepared from 61 (100 mg, 0.15 mmol) using the general procedure for TFA deprotection. Purification by flash chromatography (500:3 EtOAc/MeOH) afforded the title compound (55 mg, 70%): R_f 0.3 (500:3 EtOAc/MeOH); ¹H NMR (CD₃OD, 600 MHz) δ 3.64 (dt, J = 14.4, 4.8 Hz, 1H), 3.76 (dt, J = 14.4, 6.0 Hz, 1H), 4.18 (q, J = 4.8 Hz, 1H), 4.40 (t, J = 5.4 Hz, 1H), 4.72 (t, J = 5.4 Hz, 1H), 5.31 (s, 2H), 5.99 (d, J = 5.4 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.36 (t, J = 7.8 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H), 7.54 (t, J = 7.2 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 8.15 (s, 1H), 8.29 (s, 1H), 8.96 (br t, J = 6.0 Hz, 1H, 5′-NH); ¹³C NMR (CD₃OD, 150 MHz) δ 40.7, 71.2, 71.4, 74.0, 83.2, 89.2, 113.8, 119.4, 120.5, 121.5, 127.6, 128.3, 128.7, 131.6, 134.6, 135.8, 140.7, 149.4, 151.8, 154.7, 155.5, 157.2, 166.6; HRMS (ESI−) calcd for C₂₅H₂₄N₇O₆ [M−H]⁻ 518.1794, found 518.1795 (error 0.1 ppm).

5′-Amino-5′-deoxy-5′-N-{[(2-hydroxybenzoyl)amino]carbonyl}adenosine (63)

To a solution of 62 (50 mg, 0.096 mmol) in MeOH (10 mL) was added 10% Pd/C (10 mg, 20% by wt) and the reaction stirred at rt under H₂ (1 atm). After 2 h, the reaction was filtered through Celite and the filtrate concentrated under reduced pressure. Purification by flash chromatography (10:2 EtOAc/MeOH) afforded the title compound (21 mg, 51%): R_f 0.12 (100:20 EtOAc/MeOH); ¹H NMR (CD₃OD, 600 MHz) δ 3.66 (dd, J = 14.4, 4.2 Hz, 1H), 3.80 (dd, J = 14.4 Hz, 4.8 Hz, 1H), 4.20 (q, J = 4.8 Hz, 1H), 4.42 (t, J = 4.8 Hz, 1H), 4.74 (t, J = 4.8 Hz, 1H), 6.00 (d, J = 4.8 Hz, 1H), 6.90 (ovlp t, J = 7.8 Hz, 1H), 6.91 (ovlp d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 8.16 (s, 1H), 8.32 (s, 1H); ¹³C NMR (CD₃OD, 150 MHz) δ 40.7, 71.2, 73.9, 83.3, 89.0, 116.7, 117.4, 119.3, 119.4, 130.9, 134.8, 140.4, 149.5, 152.7, 155.1, 156.1, 158.9, 167.5; HRMS (ESI−) calcd for C₁₈H₁₈N₇O₆ [M−H]⁻ 428.1324, found 428.1327 (error 0.7 ppm).

Enzyme kinetic studies. ATP/PPi Exchange Assay.³⁰

MbtA was expressed in E. coli and purified as described.¹⁵ The inhibition assays were performed as described in duplicate.¹⁵ In brief the reaction was initiated by adding 10 μL [³²P]PP_i with 7 nM MbtA in 90 μL reaction buffer (250 μM salicylic acid, 10 mM ATP, 1 mM PPi, 75 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 2 mM DTT) at 37°C in the presence of five different concentrations of the inhibitor. The reaction was terminated by the addition of 200 μL of quenching buffer (350 mM HClO₄, 100 mM PPi, 1.8 % w/v activated charcoal). The charcoal was pelleted by centrifugation and washed once with 500 μLH₂O and analyzed by liquid scintillation counting as described.³⁰ The K_I^app values were calculated using either the Hill Morrison (eq 1) or Morrison (eq 2) equations as described.^{15, 31}

$$v_{\text{i}}/v_0=\frac{1}{1+{([I]/{K_{\text{I}}}^{\text{app}})}^{\text{h}}}$$ (1)

$$v_{\text{i}}/v_0=\frac{([E]-[I]-{K_{\text{I}}}^{\text{app}})+\sqrt{{([E]-[I]-{K_{\text{I}}}^{\text{app}})}^2+4·[E][K_{\text{I}}^{\text{app}}]}}{2·[E]}$$ (2)

M. tuberculosis H37Rv MIC Assay

All compounds MICs were experimentally determined as previously described.¹¹ Minimum inhibitory concentrations (MICs) were determined in quadruplicate in iron-deficient GAST and GAST supplemented with 200 μM FeCl₃ according to the broth microdilution method using compounds from DMSO stock solutions or with control wells treated with an equivalent amount of DMSO. Isoniazid was used as positive controls while DMSO was employed as a negative control. All measurements reported herein used an initial cell density of 10⁴–10⁵ cells/assay and growth monitored at 10–14 days, with the untreated and DMSO-treated control cultures reaching an OD₆₂₀ 0.2~0.3. Plates were incubated at 37 °C (100 μL/well) and growth was recorded by measurement of optical density at 620 nm.

Docking Studies

The homology model and docking runs were configured as described¹², except that the side chains of residues 235, 240, 329, and 337 were allowed to move along with the ligand.

Supplementary Material

1File001. Supporting Information Available.

Table of HPLC purities of the inhibitors disclosed herein. This material is available free of charge via the Internet at http://pubs.acs.org.

ABBREVIATIONS

CDI

carbonyl diimidazole

DBU

1,8-diazabicyclo[5.4.0]undec-7-ene

MDR-TB

multidrug resistant tuberculosis MIC, minimum inhibitory concentration

NHS

N-hydroxysuccinimide

NRPS

nonribosomal peptide synthetase

PAS

para-aminosalicylic acid

PDB

protein data bank

PKS

polyketide synthase

SAR

structure activity relationships

TB

tuberculosis

TBAF

tetrabutylammonium fluoride

TBS

tert-butyldimethylsilyl

TFA

trifluoroacetic acid