Thiazolino 2-Pyridone Amide Inhibitors of Chlamydia trachomatis

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Thiazolino 2-pyridone amide inhibitors of Chlamydia trachomatis infectivity James A. D. Good, Jim Silver, Carlos Núñez-Otero , Wael Bahnan, K Syam Krishnan, Olli Salin, Patrik Engström, Richard Svensson, Per Artursson, Åsa Gylfe, Sven Bergström, and Fredrik Almqvist J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01759 • Publication Date (Web): 05 Feb 2016 Downloaded from http://pubs.acs.org on February 18, 2016

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Journal of Medicinal Chemistry

Thiazolino 2-pyridone amide inhibitors of Chlamydia trachomatis infectivity , Jim Silver

b,c,d

, Patrik Engström

b,c,d

James A. D. Good Olli Salin

b,d,e

a,b

, Carlos Núñez-Otero e, Wael Bahnan

, Richard Svensson

f,g

, Per Artursson

b,c,d

f,g

, K. Syam Krishnan a,b,

, Åsa Gylfe

b,d,e

, Sven

Bergström b,c,d,*, Fredrik Almqvist a,b,*.

a

Department of Chemistry, Umeå University, 901 87 Umeå, Sweden;

Research, Umeå University, 901 87 Umeå, Sweden; University, 901 87 Umeå, Sweden;

d

c

b

Umeå Centre for Microbial

Department of Molecular Biology, Umeå

Laboratory for Molecular Infection Medicine Sweden (MIMS),

Umeå University, 901 87 Umeå, Sweden; e Department of Clinical Microbiology, Umeå University, 901 85 Umeå, Sweden; f Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden; g The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Chemical Biology Consortium Sweden, Uppsala University, SE-751 23 Uppsala, Sweden. Abstract The bacterial pathogen Chlamydia trachomatis is a global health burden currently treated with broadspectrum antibiotics which disrupt commensal bacteria. We recently identified a compound through phenotypic screening that blocked infectivity of this intracellular pathogen without host cell toxicity (compound 1, KSK 120). Herein, we present the optimization of 1 to a class of thiazolino 2-pyridone amides that are highly efficacious (EC50 ≤100 nM) in attenuating infectivity across multiple serovars of C. trachomatis without host cell toxicity. The lead compound 21a exhibits reduced lipophilicity versus 1 and did not affect the growth or viability of representative commensal flora at 50 µM. In microscopy studies, a highly active fluorescent analogue 37 localized inside the parasitiphorous inclusion, indicative

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of a specific targeting of bacterial components. In summary, we present a class of small molecules to enable the development of specific treatments for C. trachomatis. Keywords: Chlamydia trachomatis, infectivity inhibition, peptidomimetic, 2-pyridones, KSK 120. Introduction Chlamydia trachomatis is the most common bacterial sexually transmitted infection (STI) and the leading infectious cause of blindness globally, and thus is a major burden to public health.1, 2

The

urogenital serovars D-K are associated with serious sequelae including pelvic inflammatory disease, ectopic pregnancy and infertility,3 whilst the less common invasive serovars LGV L1-L3 cause lymphogranuloma venereum, which results in serious disseminated infections of the lymphatic system.4 The ocular serovars A-C are responsible for trachoma, with conjunctival damage from repeated trachoma infections ultimately blinding those affected.2

More than 100 million new cases were

estimated of C. trachomatis STIs in 2008, while >1 million people are estimated to have lost their vision as a result of the damage accrued through repeated trachoma.1,

2

This Gram-negative obligate

intracellular pathogen is characterized by a biphasic developmental cycle that switches between an infectious elementary body (EB) and the replicative intracellular reticulate body (RB).5, 6 Infection is initiated by EBs which adhere to and invade the host cell, transition to the RB form, and replicate within a vacuole known as the inclusion. RBs subsequently differentiate back to infectious EBs, which after lysis of the inclusion and exiting the host cell, propagate the infection. This distinctive lifecycle offers multiple opportunities for therapeutic intervention, yet currently C. trachomatis infections are treated with broad-spectrum antibiotics that also disturb the commensal microbiota.7 This perturbation has been recognized to have long-lasting effects on the composition of commensal flora, affording opportunities for opportunistic pathogens to colonize and facilitating the transfer of resistance.8-10


Therapies

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specifically affecting C. trachomatis infections without affecting the commensal microbiota would therefore be of considerable clinical relevance.

Recently, we undertook a phenotypic screen for compounds that affect the infection phenotype of C. trachomatis by assessing bacterial distribution within the inclusion as a marker for attenuated infectivity.11 This identified a novel compound which inhibited C. trachomatis infectivity, without comprising host cell viability, by apparently disrupting the glucose metabolism of this energy parasite (compound 1, KSK 120 Figure 1).11, 12 The primary effect was not antibiotic, instead the key therapeutic effect of 1 was the production of progeny that were no longer able to initiate a new round of infection. In infectivity assays, where infected HeLa cells were treated with 10 µM of 1, and after 44 hours post infection (hpi), the bacteria collected and challenged to infect fresh HeLa cells, compound 1 attenuated the infection ≥10,000 fold against C. trachomatis serovars A, D and LGV-L2.11 Our preliminary investigations into the mode of action suggest 1 disrupts the glucose 6-phosphate (G6-P) metabolic pathway of C. trachomatis. Mutational analysis of C. trachomatis serially passaged with 1 identified several point mutations in genes involved in the G6-P pathway, and in particular in uhpC which encodes the inner-membrane hexose phosphate transporter UhpC that acquires G6-P from the host cell.13 As an obligate intracellular parasite, C. trachomatis relies extensively upon nutrients derived from the host cell, with G6-P a principal source of carbon. Pathways for the utilization of alternative carbons sources and sugar interconversion are present at the genomic level,14 however C. trachomatis has been found to be transcriptionally unresponsive to changes in nutrient availability in the surrounding milieu, thus rendering it susceptible to disruption to its energy supply.15, 16 As G6-P is only available intracellularly, and thus not to extracellular bacteria, these data indicated disrupting this pathway could be an effective strategy for targeting C. trachomatis without affecting commensal microbiota, with 1 a useful tool for studying the fundamental processes driving C. trachomatis infectivity.11,


12

In the present study, we


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investigated the therapeutic potential of 1 and developed a series of thiazolino 2-pyridone amides with improved efficacy and physicochemical properties, to obtain a new lead series of small molecule inhibitors against C. trachomatis infectivity.


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Results & Discussion Synthesis The thiazolino 2-pyridone scaffold was constructed by the cyclocondensation of ∆2-thiazoline (R)-2 and acyl Meldrum’s acid 3 by our previously developed method (Scheme 1).17, 18 Methyl ester hydrolysis of (R)-4 with lithium hydroxide gave carboxylic acid 5, and subsequent amide coupling using propylphosphonic anhydride afforded amide derivatives 6, 8-9 and 11-19.19,

20

These coupling

conditions did not give satisfactory conversion for the 2-pyridyl and 4-pyrimidyl analogues 7 and 10, which were instead prepared using TBTU or HATU with microwave irradiation (MWI) respectively.21 The C-6 carboxylic acid derivative 20 was prepared by formylation and oxidation under PinnickLindgren conditions with sodium chlorite,22,

23

with oxidation of the thioether attenuated through the

presence of DMSO as a scavenger (Scheme 2).24

Introduction of the C-6 amino substituent into the 2-

pyridone ring was achieved by nitration followed by reduction with zinc and acetic acid to afford derivatives 21a-c.25

This approach afforded moderate yields which varied considerably between

substrates (21a-c, 37-69%), so we sought an alternative route. Nitration of methyl ester 4 followed by reduction reliably introduced the C-6 amine, and subsequent hydrolysis and amide coupling under forcing conditions with HATU and MWI afforded derivatives 21d and 25 (Scheme 3). Attempts to perform the same amide coupling for 21d under conventional conditions (HATU, DIPEA, CH2Cl2, rt)26 gave only limited conversion. We envisaged that introducing a bromomethyl substituent into the C-7 position of the 2-pyridone would enable efficient late stage variation of the 1-naphthyl group by transition metal cross-coupling reactions. However, as the requisite ketene precursors required for this brominated intermediate were previously found to be difficult to reliably prepare and handle due to their high reactivity,27 we prepared the



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corresponding chloromethyl analogue 28 instead (Scheme 4).28

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The propylphosphonic anhydride

mediated condensation of chloroacetic acid 26 with Meldrum’s acid provided the chloromethyl ketene source 27 in excellent yields, and using 27 in the subsequent cyclocondensation afforded the key intermediate 28 in near quantitative yields. The Suzuki-Miyaura cross-coupling of 28 with various aromatic and heteroaromatic substrates proceeded efficiently, including with sterically hindered 2,3disubstituted phenylboronic acids. Subsequent hydrolysis and amide coupling furnished amides 31a-h and 32, and amination in the C-6 position gave 33a and 33b. Upon reflection, switching from a bromomethyl scaffold to the chloromethyl intermediate 28 resulted in a more tractable and efficient synthesis,27 and preparing acyl Meldrum’s acid 27 with propylphosphonic anhydride provided additional practical advantages. DCC, the previously employed coupling agent, is toxic and sensitizing, with the cyclohexyl urea byproduct obtained requiring extensive washing and filtration to remove.28 For the preparation of 27, propylphosphonic anhydride was more efficient without such toxicity, and the byproducts were readily removed by aqueous work-up. The majority of final compounds were prepared as racemates, as the LiOH hydrolysis conditions employed [LiOH (1 M aqueous solution, 2 eq) in THF (~0.025 M)] caused substantial epimerization of the C-3 stereocenters of 5 and 30a-g (Scheme 1 and Scheme 4). The key carboxylic acid intermediate 5 was ascertained to be essentially racemic (e.r. 47:53) by derivatizing to the corresponding methyl ester 4 with TMS-diazomethane.29 Similarly, the carboxylic acids 30c and 30e from the Suzuki-Miyaura coupling route (Scheme 4) had e.r. of 48:52 and 43:57 respectively. We undertook a revised synthesis to prepare enantioenriched samples of the thiazolino 2-pyridone amide 6a (Scheme 5). The methyl esters (R)-4 and (S)-4 were first prepared with e.r >9:1 by using 0.2 equivalents of TFA in the cyclocondensation.18 Reducing the stoichiometry of TFA from 1 equivalents enhanced the enantioselectivity of the transformation ((R)-4: 1 eq TFA: e.r.11:89; 0.2 eq TFA: e.r. 8:92),


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but with the penalty of abrogating the yield ((R)-4: 1 eq TFA: 84%; 0.2 eq TFA: 49%). For the hydrolysis, using fewer equivalents of lithium hydroxide [LiOH (0.1 M aq. soln., 1 eq) in THF/MeOH (3:7; 0.025 M)] had previously proved effective in preparing enantioenriched 5.30 With the lower stoichiometry we found reproducibility an issue, with reactions often proceeding sluggishly and opted instead for the mild LiBr/NEt3 hydrolysis procedure developed by Matsson et al.31 which has been reported to proceed without epimerization on a process scale.32 This afforded acids (R)-5 and (S)-5 with e.r. conservatively estimated >8:2, as determined by derivatization to the corresponding esters (R)4 and (S)-4. Next applying the optimized enantioselective propylphosphonic anhydride amide coupling procedure developed by Dunetz et al. afforded the amides (R)-6a and (S)-6a with e.r. of 85:15 and 90:10 respectively.20 HATU also proved equally efficient and stereoselective for the preparation of (R)-6a from (R)-5 (e.r. 85:15).26 We prepared a fluorescent analogue 37, containing a BODIPY fluorophore using our recently developed route to fluorescent 2-pyridone peptidomimetics.33 Compound 37 was prepared from the BODIPY containing methyl ester 36 by NEt3/LiBr hydrolysis at 50 °C followed by amide coupling (Scheme 6).



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Biological Evaluation

The anti-infective activity of new compounds was determined by a reinfection assay that quantified the number of infectious bacteria remaining following treatment. In summary, HeLa cells infected with serovar LGV-L2 were treated with compound and incubated for 48 h, lysed, and the collected bacteria used to reinfect fresh HeLa cells. The level of reinfection relative to untreated DMSO controls was subsequently quantified by counting the number of new inclusion forming units (IFUs), corresponding to the number of viable infectious bacteria. We determined the minimum concentration required to reduce the relative level of infection by 95% for each compound, a metric we termed Reinfect95, and evaluated at fixed concentrations of 10, 5, 2.5, 1, 0.5, 0.25 and 0.1 µM. This approach enabled the assessment of compound activity with relative throughput. Cytotoxicity was recorded against HeLa cells at 10 µM, the highest tested concentration, in a resazurin assay to ensure that any observed effects were not mediated through non-specific toxicity.

The starting point for lead optimization, 1, abrogated C. trachomatis infectivity with an estimated EC50 ≈ 1.25 µM against the LGV-L2 serovar.11 However, the high lipophilicity of 1 (calc. Log P = 6.00) from the C-2/C-3 double bond, attendant planarity, and 1-naphthyl moiety in the C-7 position was likely detrimental to drug-like properties, and we therefore focused our efforts on these features (schematic, Figure 1).34 Compound 1 also exhibited slight host cell toxicity in the resazurin assay at 10 µM in uninfected cells, in contrast with previous results obtained at the same concentration in an XTT assay,11 suggesting the therapeutic index could be increased (Table 1). To address the planarity we switched to a dihydrothiazolino 2-pyridone core, and the racemic analogue 6a proved equipotent with 1 in the reinfection assay without host cell toxicity (Table 1). No difference in activity was distinguishable between the corresponding enantiomers (R)-6a and (S)-6a, and the majority of the remaining


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compounds were prepared racemically. Next, we proceeded to examine the importance of the amide moiety and scope of permissible amide substituents. Heterocyclic amides 7-11 were designed to increase the polarity of the amide moiety. Pyridyl regioisomers 7-9 were only weakly active and affected host cell viability, whilst switching to the thiazolo amide 9 also attenuated activity compared to the phenyl amide 6a. Interestingly, the pyrimidine analogue 10 displayed comparable anti-infective activity to 6a, albeit with slight host cell toxicity. The aliphatic amides 12-14 were all weakly active compared to the phenyl substituted 6a, implying an aromatic substituent was preferable for efficacy (e.g. N-cyclohexyl 14 versus N-phenyl 6a).

The benzylic amide 15 was comparably active to 6a, indicating some steric

freedom for the amide functionality, but the tertiary N-methyl, N-phenyl derivative 16 proved less effective. Various tertiary carbocyclic amides were also screened (17-19), however none were effective at 10 µM.

The thiazolino secondary N-phenyl amide 6a displayed efficacy comparable to 1 without affecting host cell viability, so was selected as the new lead candidate. Introducing a meta-methyl substituent imparted a small improvement in efficacy (6e versus 6a, Table 2), and other small lipophilic modifications were tolerated, indicating the availability of the phenyl ring for substitution. Introducing more polar substituents to reduce the lipophilicity of 6a gave varying results. The m-CONHMe and p-sulfonamide substituted amides 6i and 6o proved ineffective, but introducing a p-CONH2 substituent in 6n gave comparable biological activity to 6a and reduced lipophilicity (6a: comp. Log P = 5.44; 6n: comp. Log P = 4.78).

One strategy to reduce the lipophilicity of the 6a was to replace the C-7 CH2-naphthyl, and we next explored the importance of this moiety for anti-infective efficacy. Removing the aromatic moiety abrogated anti-infective activity in the methyl substituted analogue 35, whereas truncating to a benzyl



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maintained efficacy (31a versus 6a, Table 3). Various benzylic substituents displayed equivalent efficacy in the reinfection assay (31b-d), with disubstituted benzyl surrogates for the naphthyl group also effective, however at 10 µM several analogues displayed slight toxicity in the resazurin assay (e.g. 31b and 31f, HeLa cell viability ≈ 85%). As an alternative to truncating the naphthyl, changing to a heterocyclic quinoline was investigated, however this was not well tolerated (31h). Combining the p-Cl benzyl or a 2,3-dimethylbenzyl with a p-tolyl amide gave analogues 32a and 32b which were both more effective in reducing C. trachomatis infectivity than the parent benzylic or p-tolyl analogues 31b, 31f and 6l. Compounds 32a and 32b abrogated the infection at 1 µM, thereby demonstrating that a reduction in lipophilicity could be achieved through truncating the naphthyl without compromising antiinfective efficacy.

We next examined introducing substituents into the C-6 position, and adding a carboxylate abolished activity (20, Table 4).

Reversing the electrostatics and introducing an amino substituent into the

pyridone improved the anti-infective efficacy by an order of magnitude in the Reinfect95 assay, whilst simultaneously increasing the hydrophilicity of the central scaffold (21a versus 6a). Extending further to a CH2-morpholino analogue 34 diminished efficacy in comparison to the unsubstituted derivative 6a. As compound 21a possessed a promising combination of increased efficacy and hydrophilicity over 6a, we focused on combining this modification with the most advantageous C-3 amides and C-7 substituents. The m-tolyl amide 6e had improved efficacy over the phenyl analogue 6a, but appending the C-6 amine had no further effect on activity (21b, Table 4). When the C-6 amine was introduced into the p-tolyl amide 6l, this afforded a substantial improvement in activity, with 21c displaying comparable activity to the most active compound 21a. Adding a C-6 amine substituent to the p-benzamide analogue (21d) abolished anti-infective activity compared to the parent compound 6i rather than improving efficacy, but the amino substituent was tolerated for the pyrimidine analogue 25 versus the unsubstituted


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10. For the p-chlorobenzyl, p-tolyl amido analogue 33a, appending the C-6 amine decreased activity compared with 32a, and for the related 2,3-dimethylbenzyl derivative 33b this amine had no effect on efficacy versus 32b. However, we noted that one improvement afforded by this modification was to obviate the slight toxicity evident for the corresponding non-aminated analogues 32a and 32b (Table 3). In accordance with this observation, while the C-6 amine substituent did not uniformly improve efficacy, a key benefit was to reduce the lipophilicity of the lead series. We reason that for those analogues where this modification imparted a substantial increase in efficacy (e.g. 21a versus 6a), this change in activity may relate to an improvement in physicochemical properties rather than intrinsic activity.

We proceeded to assess the most promising analogues more rigorously by evaluating their effect on host cell viability at 25 µM. In agreement with the earlier results at 10 µM, compound 1 clearly reduced host cell viability at this higher concentration of 25 µM (HeLa cell viability ≈ 73%, Table 5). The pyrimidine analogue 25 also slightly affected HeLa cell viability at this concentration, and was not pursued further. All other new compounds were non-toxic to the host cell at 25 µM. To more accurately determine the efficacy of the most promising lead analogues, we evaluated the EC50 values of 6e, 21a-c and 33a-b against serovar LGV-L2. Compound 21a was the most effective, with an EC50 ≈ 59 nM, representing a ≥4-fold increase in efficacy over the starting point for optimization 1 (EC50 ≈ 254 nM). Given 21a was non-toxic to the host cell at 25µM, this preliminary toxicity assessment suggests an excellent therapeutic index. The m-tolyl amide 6e was also extremely potent with EC50 ≈ 102 nM, but with higher lipophilicity represented a less attractive compound for further development.

The related m-tolyl

analogue 21b, incorporating the C-6 amine substituent was less active (EC50 ≈ 147 nM), and was equipotent with the corresponding p-tolyl analogue 21c (EC50 ≈ 140 nM). Analogues 33a and 33b combining a p-tolyl amide with a substituted benzyl in the C-7 position had comparable activity to 1.



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Assessing the efficacy of compounds 21a-c and 33a-b in the sexually transmitted serovar D confirmed that their activity was not serovar-specific, with infectivity ablated correspondingly across both serotypes, indicating the compounds can be generally applicable for treating C. trachomatis STIs.

An important attribute for potential small molecule therapeutic targeting C. trachomatis infections is their lack of impact on commensal flora. We assessed the tolerance of the bacterial species Lactobacillus crispatus and Escherichia coli and the fungi Candida albicans as representatives from the commensal flora. All species grew normally when treated with 21a at 50 µM. We also assessed the lipophilicity of 21a in comparison to 1, due to its defining relationship on multiple drug-like properties.34 We found a clear improvement in 21a versus 1, with the C-6 amine non-basic (1: Log P = 5.0; 21a: Log D7.4 = 4.3, pKa = 2.4). However, given the optimal range for small molecule drugs has been proposed to lie between Log P/D = 1-3, this indicates further improvements can be achieved to enhance the druggability of the lead series.34

We previously used a fluorescent analogue to gain insight into the distribution of 1, which suggested localization to the bacterial inclusion.11 However, the previously employed fluorescent analogue incorporated a carboxylic acid in the C-3 position instead of an amide and was only weakly effective, with the imaging performed at high concentration (100 µM). While an interesting preliminary observation, to address these limitations in the method we prepared the fluorescent analogue 37 designed to mimic the potent analogue m-tolyl amide 6e but containing a BODIPY fluorophore (Scheme 6). Introducing the BODIPY fluorophore in place of the C-7 naphthyl was well tolerated, and 37 attenuated C. trachomatis infectivity >95% at 2.5 µM. In subsequent microscopy studies, compound 37 localized to the intracellular bacterial inclusion (Figure 2). In contrast, an inactive BODIPY control compound 38 did not localize inside the inclusions or HeLa cell, indicating the observed accumulation


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of 37 was not an artefact mediated by the BODIPY fluorophore. Bypassing bacterial membranes is a challenging aspect of bacterial drug discovery,35 this demonstrates 37 effectively penetrates and localizes to the intracellular bacterial inclusion, and implicit within this observation is this occurs due to a specific association with components hosted inside the chlamydial inclusion.

We further interrogated the relevance of this localization to the lead series by performing coadministration experiments with 37, 6e and 21a. In these experiments, infected HeLa cells were treated with 37, or 37 with either 6e or 21a at the same time (Figure 3). The intensity of the fluorescence within the inclusion was significantly reduced upon co-administration of 37 with either 6e or 21a. We interpret this data as indicating that 6e and 21a outcompete 37, which is no longer able to bind to targets inside the inclusion, thereby reducing the observed fluorescence localized to the inclusion. In total, the data indicate the lead series specifically targets a component hosted within the bacterial inclusion, rather than the host cell. Taken together with host cell tolerance for high concentrations of the lead series and lack of effect on representative commensal flora, this demonstrates the potential of this class of compounds to be developed further.

Conclusions

As clinicians transition to a post-antibiotic era with challenging multi-drug resistance occurring with increasing frequency in clinically relevant pathogens,36,

37

conserving our antibiotic arsenal by

developing new and complementary therapeutics is becoming increasingly important. The challenge of identifying novel antimicrobials with whole cell activity has been widely recognized in recent years, with industrial HTS approaches with both target-based and phenotypic screening generating few viable lead series, and novel broad spectrum antibiotics scarce.35,

38

In this study, we developed non-toxic



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thiazolino 2-pyridone amide inhibitors affecting C. trachomatis infectivity with potent efficacy in whole-cell bacterial assays, without disturbing the growth of commensal bacteria. We are not aware of similar inhibitors of this pathogen that can effectively block the infection cycle of C. trachomatis which do not act as conventional antibiotics. The present study provides proof-of-concept for the chemical tractability of this series, with improvements realized in efficacy and hydrophilicity, the latter a key attribute in the successful development of small molecule therapeutics.34 Our preliminary investigations into the mode of action of screening hit 1 suggested disruption to the G6P metabolic pathway reduced infectivity, and further studies are ongoing into the mode and mechanism of action of 21a. The localization of the highly active fluorescent analogue 37 suggests specific association with an inclusion component necessary for the production of infectious progeny. In summary, we present a class of small molecules to be developed for the treatment of C. trachomatis infections.


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Table 1 – Exploring the amido NR1R2 substituents.

Cmpd

R1

R2

Reinfect95 (µM)

HeLa Viability 10 µM (%)

1a

Ph

H

2.5

87 ± 3

6a

Ph

H

2.5

100 ± 4

(R)-6a

Ph

H

2.5

100 ± 4 b

(S)-6a

Ph

H

2.5

100 ± 4 b

7

2-Pyridine

H

10

90 ± 1

8

3-Pyridine

H

10

77 ± 6

9

4-Pyridine

H

5

69 ± 8

10

2,4-Pyrimidine

H

2.5

92 ± 2

11

2-(1,3-Thiazole)

H

5

96 ± 3

12

Me

H

10

93 ± 4

13

Me

OMe

>10

88 ± 4

14

Cyclohexyl

H

10

88 ± 3

15

Benzyl

H

2.5

93 ± 6

16

Ph

Me

5

96 ± 4

17

>10

86 ± 5

18

>10

96 ± 4

19

>10

90 ± 3

Notes: a Unsaturated across C-2/C-3 bond; b determined for 6a.



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Table 2 – Optimization of the N-phenyl substituent.

Cmpd

R

Reinfect95 (µM)


6a

H

2.5

100 ± 4

6b

2-F

2.5

96 ± 3

6c

3-F

2.5

96 ± 3

6d

3-Cl

2.5

100 ± 2

6e

3-Me

1

99 ± 4

6f

3-Et

2.5

100 ± 3

6g

3-CF3

2.5

95 ± 3

6h

3-OMe

2.5

98 ± 3

6i

3-CONHMe

10

93 ± 3

6j

4-F

2.5

98 ± 2

6k

4-Cl

2.5

94 ± 3

6l

4-Me

2.5

97 ± 3

6m

4-OMe

2.5

100 ± 3

6n

4-CONH2

2.5

98 ± 5

6o

4-SO2NH2

10

95 ± 3


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Table 3 – Variation of the 1-naphthyl moiety in the C-7 position.

Cmpd

R1

R2

Reinfect95 (µM)


35

H

H

10

99 ± 2

31a

Ph

H

2.5

96 ± 4

31b

H

2.5

87 ± 1

31c

H

2.5

94 ± 4

32a

4-Me

1

89 ± 3

31d

H

2.5

103 ± 8

31e

H

2.5

91 ± 4

32b

4-Me

1

95 ± 3

31f

H

2.5

85 ± 3

31g

H

2.5

91 ± 4

6a

H

2.5

100 ± 4



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31h

H

10


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91 ± 3

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Table 4 – Functionalization of the C-6 position.

Cmpd

R1

R2

R3

Reinfect95 (µM)


20

1-Naphthyl

CO2H

H

>10

103 ± 6

21a

1-Naphthyl

NH2

H

0.25

101 ± 3

(R)-34

1-Naphthyl

H

5

99 ± 3

21b

1-Naphthyl

NH2

1

98 ± 3

21c

1-Naphthyl

NH2

0.25

98 ± 2

21d

1-Naphthyl

NH2

>10

103 ± 1

25

1-Naphthyl

NH2

2.5

99 ± 9

33a

NH2

2.5

101 ± 2

33b

NH2

1

101 ± 2



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Table 5 – Determination of EC50 values.

Cmpd

Serovar LGV-L2 EC50 (nM)a

Serovar D EC50 (nM)a


1

254 (185-347)

n.d.

73 ± 4

6e

102 (79-132)

n.d.

100 ± 2

21a

59 (38-93)

58 (45-76)

98 ± 2

21b

147 (80-270)

124 (71-220)

97 ± 2

21c

140 (116-170)

99 (54-184)

98 ± 1

25

n.d.

n.d.

88 ± 1

33a

266 (187-377)

270 (201-362)

102 ± 2

33b

281 (192-410)

274 (181-414)

102 ± 2

Notes: a 95% confidence intervals in parentheses. n.d. = not determined.


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Experimental Biology

Cell culture and C. trachomatis propagation The HeLa229 cell line (CCL-2.1; ATCC) was propagated at 37 °C (5% CO2) in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 2 mM L-glutamine. C. trachomatis serovar L2 454/Bu (VR902B; ATCC) and C. trachomatis serovar D strain UW-3 (VR-885; ATCC), were grown in HeLa cells. EBs were purified as described by Caldwell et al. and stored in SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid).39

Determination of EC50 values evaluation HeLa cells were infected with C. trachomatis serovar LGV-L2 at an MOI of 0.3. At 1 hpi, RPMI media containing the different dilutions of the tested compounds in DMSO were added. The different compounds were added in a halving serial dilution starting at a concentration of 2 µM in an 8 step decrement reaching 0.0156 µM. After 44-48 h incubation, the cells were osmotically lysed by the addition of cold sterile distilled water to release infectious EB progeny. 5×SPG was added to equalize the osmotic pressure to a 1×SPG isotonic condition. An equal amount of HBSS (Hanks balanced salt solution) (Gibco/Invitrogen) was added to the lysate (yielding a 1:1 dilution) and 10-fold serial dilutions of the resulting mixture was used to infect fresh HeLa cells. At 1 hpi, the inoculum was replaced with RPMI media and the infection allowed to progress for 44-48 hours before fixation and staining. Fixation was performed by adding methanol for 5 minutes and the cells were subsequently washed with phosphate buffered saline (PBS). The chlamydial inclusions were stained by a primary rabbit antiChlamydia antibody (generated in-house)40 and a secondary donkey anti-rabbit FITC-labelled antibody



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(Jackson ImmunoResearch). The DNA of the cells and Chlamydia was stained by the addition of DAPI. The stained cells were analyzed by an ArrayScan automated scanner (ArrayScan VTI HCS, Thermo Scientific). Data were presented as the relative numbers of IFUs in treated infections compared to the numbers of IFUs in DMSO-treated control infections. EC50 values were calculated from at least three independent experiments. The data analysis and statistical testing were performed using nonlinear regression (curve fit) in GraphPad Prism v.5.

Reinfect95 assay The Reinfect95 value is defined as the concentration at which a compound consistently reduced the number of IFUs produced by 95% relative to DMSO-treated controls. This value was determined from at least two independent experiments, each performed in triplicate. The infection assays were performed in a similar manner as described for the EC50 evaluation 5. In brief, HeLa cells were infected with C. trachomatis serovar LGV-L2 at an MOI of 0.1-0.5 as described above. We noted the effect of the compounds was consistent across the MOI range utilized. Compounds were added to the cells under the conditions stated previously. All compounds were evaluated at 2.5 µM, and dependent on the observed activity, either evaluated at 5 µM or 10 µM, or conversely at 1, 0.5, 0.25 or 0.1 µM. For the reinfections, the infected cells were osmotically lysed, and 5× SPG was added to the lysates, as described above, to create isotonic conditions. The lysates were serially diluted (10-fold steps) and used to infect fresh HeLa cells. The number of IFUs generated under treatment by each compound was analyzed by ArrayScan and compared to the DMSO-treated control infections.

Resazurin cell toxicity assay


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HeLa cells were seeded in RPMI medium at a density of 7000 cells per well in 96 well plates. The outer wells were left without cells (only media, to serve as controls). The next day, the RPMI medium was removed and was replaced with colorless DMEM media (Gibco) supplemented with 10% FBS and 2 mM L-glutamine. The tested compounds were added in triplicates at a final concentration of 10 µM to the cells. As a control, 25 µM of compound was added to the cell-free outer wells to monitor for compound mediated reduction of resazurin. After 48 h of incubation at 37 °C, 10 µl of 400 µM of resazurin reagent was added to the wells, which were further incubated at 37 °C in the dark for 3 h. Fluorescence was read using a Tecan Safire plate reader (Tecan) with excitation at 535 nm and emission at 595 nm. The values reported are the mean from three individual experiments, each performed in triplicate. The standard deviation includes all nine replicates from the three individual experiments. Statistical analysis of mean and standard deviation was performed in Microsoft Excel.

Bacterial growth experiments E. coli (strain MW100) was grown in Luria-Bertani broth overnight in a shaking incubator at 37 °C. The overnight cultures were diluted to an OD600 = 0.05 in LB supplemented with either DMSO, or 50 µM of 1, 6e or 21a. The cultures were allowed to grow for 8 hours while the OD600 was monitored every hour. Experiments were performed in triplicate for each compounds, and growth assessed visually and by OD measurements. Candida albicans (SC314) was grown in medium overnight in a shaking incubator at 30 °C. The cultures were then diluted to OD600 0.05 and allowed to grow until early log phase (OD600 0.2). Compounds 21a and 6e were added to the growing cultures at 10 and 50 µM and the cultures were allowed to grow until saturation. Samples were taken from the growing cultures every 2 h and assessed microscopically for fungal morphology (ratio of yeast:hyphal cells). Lactobacillus crispatus (CCUG



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27076A) was cultured in deMan Rogosa Sharpe broth at 37 °C in 5% CO2. Overnight cultures were diluted 1:20 and allowed to grow for 48 h with 50 µM of 21a or 1% DMSO.

Labelling, immunofluorescence, and fluorescent imaging HeLa cells were seeded at a density of 150,000 cells per well onto 10 mm glass coverslips in a 24 well plate. The following day, the media was removed from the cells and a suspension of C. trachomatis serovar LGV-L2 in HBSS was added to the cells to achieve an infection ratio of 1 bacterium per 2 cells (MOI 0.5). Sixty minutes after infection, the HBSS was removed and fresh media was added to the cells, supplemented with the appropriate compound at the designated concentration. For the competition experiments, both 37 as well as the competing compounds were added together directly after infection at the stated concentrations and left on the cells for the duration of the experiment. When the experiments were completed, the cells were washed with PBS, fixed with 4% paraformaldehyde (for 15 minutes) and permeabilized with 0.1% Triton X-100. The cells were then blocked with 1% BSA for 1 h at room temperature and stained with an anti-MOMP to visualize the bacteria. A secondary antibody conjugated to a Rhodamine fluorophore (Jackson Immunoresearch Laboratories 711-295-152) was used stain the cells and the slides were then mounted onto pre-cleaned slides using DAKO Fluorescence mounting medium. Imaging was performed in a Nikon Eclipse C1 Plus scanning laser confocal microscope using a 60× oil immersion objective, using the automated EZ-C1 software. The images were acquired using a Nikon Digital sight DS-U2 Camera controller and a Nikon C1-SHV camera. Projection images were generated from the acquired Z-planes (typically 9-10 planes with a step size = 0.3 µm), and data analysis was performed using Image J image analysis software.41 All color adjustments required to generate the images presented in this study were performed in an identical manner on all the images. For the quantification of fluorescence intensity, the fluorescence of 37 (BODIPY fluorophore, green channel)


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was measured in arbitrary units, and was divided by the area of the respective inclusion to normalize for inclusion size. Adobe Photoshop and Illustrator (Adobe Systems) were used to compile the images and present the data.

comp. Log P Computed Log P values were calculated in MOE.42 Log P/D7.4 and pKa determination The logP/D7.4 measurements were performed utilizing a miniaturized shake-flask method in 2 mL HPLC glass vials. In brief, 1-octanol (pre-saturated with 0.1 M potassium phosphate pH 7.4) and potassium phosphate (pre-saturated with 1-octanol) was mixed 1:3 and 1:6 to a final volume of 1.2 mL. 2 µL from a 10 mM DMSO stock of compound and Ketoconazole (control, logD7.4 = 3.90) were added in separate vials. The vials were rotated (Kisker orbital shaker, 600 rpm) overnight at rt. After the incubation the vials were centrifuged at 3500 rpm for 30 min to separate the phases. The octanol phase was carefully transferred to a separate vial with a pipette. For UPLC-MS/MS analysis, an aliquot of the sample in buffer or organic phase was diluted, depending on the predicted partitioning (2-10 000× dilution), in 60% acetonitrile containing Warfarin as a general internal standard. Quantification was performed using UHPLC-MS/MS and an external standard curve between 1-1000 nM. UHPLC–MS/MS measurements used a Waters XEVO TQ triple-quadrupole mass spectrometer (electrospray ionization, ESI) coupled to a Waters Acquity UPLC (Waters Corp.). For chromatographic separation a general gradient was used (1% mobile phase B to 90% over 2 min total run) on a BEH C18 1.7 µm column, 2 mm × 50 mm (Waters Corp.). Mobile phase A consisted of 5% acetonitrile and 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The flow rate was 0.5 mL/min. The pKa measurements were performed on a Sirius T3 automated instrument from Sirius Analytical Ltd. (East Sussex, UK) equipped with a D-PAS (dip probe absorption spectroscopy) lamp for



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spectrophotometric titrations and pH-electrode for potentiometric titrations. The spectrophotometric titrations were performed using 2–5 µL 10 mM DMSO compound stock of which was added to 25 µL of phosphate buffer. The phosphate buffer was added because the amount of sample used is not enough to act as its own buffering system while performing the titration. Due to apparent limited solubility these titrations also required methanol as cosolvent (varied between 20-50 % methanol (wt/wt)). The instrument then added a predetermined volume of ionic-strength-adjusted (ISA) water (1.5 mL), or a combination of ISA and ISA containing 80% methanol in the potentiometric titrations. A pH-metric titration from high to low pH was performed (pH 2-12). During the titration, the instrument collected a UV–vis spectrum by using the D-PAS technique to establish a titration curve. The electrode was calibrated using a blank titration from pH 1.8 to pH 12.0 before every individual determination. The measurements were performed under argon to minimize the effect of dissolved CO2. Precipitation was continuously monitored at 500 nm. The temperature was controlled throughout the experiment at 25 ± 1 °C.

Chemistry General Reagents and solvents were used as received from commercial suppliers unless otherwise noted. Triethylamine, N,N-diisopropylethylamine and pyridine were passed through activated alumina oxide and dried over activated 3 Å molecular sieves prior to use. CH2Cl2, THF and DMF were dried in a solvent drying system (CH2Cl2 and THF drying agent: neutral alumina; DMF drying agent: activated molecular sieves equipped with an isocyanate scrubber) and collected fresh prior to every reaction. Zinc dust was activated prior to each reaction by stirring in aqueous HCl (1 M) for 5 min, filtering and


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washing successively with H2O, EtOH, acetone, Et2O and drying thoroughly under reduced pressure at 80 °C. Anhydrous reactions were carried out in oven dried glassware under a nitrogen atmosphere. Microwave reactions were performed using a microwave synthesizer in sealed vessels with temperature monitoring by an internal IR probe. Reaction progress was monitored by TLC and LC-MS. TLC was performed on aluminum backed silica gel plates (median pore size 60 Å) and detected with UV light at 254 nm. Flash column chromatography was performed using an automated chromatography system on silica gel with average particle diameter 50 µM (range 40-65 µM, pore diameter 53 Å) and detection at 254 nm; eluents are given in brackets. Preparative HPLC was performed with a Nucleodur® C18 HTec column (25 cm × 21.5 mm; particle size 5 µM) using a flow rate of 18 mL/min. Optical rotations were measured with a polarimeter at 25 °C at 589 nm. 1H and 13C NMR spectra were recorded on a 400 or 600 MHz spectrometer at 298 K and calibrated by using the residual peak of the solvent as the internal standard (CDCl3: δH = 7.26 ppm; δC = 77.16 ppm; DMSO-d6: δH = 2.50 ppm; δC = 39.52 ppm).

19

F

NMR spectra were recorded on a 400 MHz spectrometer at 298 K with CF3CO2H as an external standard (δF = −76.55 ppm). HRMS was performed on a mass spectrometer with ESI-TOF (ESI+); sodium formate was used as the calibration chemical. Compound purity was assessed by LC-MS using an Agilent 1290 Infinity binary LC System and an Agilent 6150 quadrapole LC-MS with UV detection at 210 nM and 254 nM. All compounds evaluated in biological assays were ≥95% pure.

BODIPY

control 38 was purchased from a commercial supplier. Compound 1 was prepared as previously described.11 ∆2-Thiazolines (R)-2 and (S)-2, naphthyl Meldrum’s acid 3 and compounds 22 and 23 were prepared according to published procedures.17, 25, 43 Data in agreement with the literature.17, 25, 29

Chiral HPLC



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The e.r. of (R)-2 and (S)-2 were verified using a Chiralcel OD-H column (25 cm × 4.6 mm; 5 µM) with a gradient of hexane/2-propanol (85:15) and flow rate of 1 ml min-1.18 All other compounds were analyzed with a (S,S)-Whelk-O 1 column (25 cm × 4.6 mm; 10 µM) with hexane/CH2Cl2/2-propanol (58/40/2) and flow rate of 1.5 mL min-1. UV detection was at 254 nM. See Supporting Material for chromatographs.

Procedures General procedure for the alkylation of carboxylic acids for chiral HPLC Carboxylic acid analogues were derivatized to their corresponding methyl carboxylates with TMSdiazomethane for chiral HPLC analysis using the following representative procedure.44 The acid 5 (5 mg, 0.013 mmol) was dissolved in anhydrous methanol/benzene (1:4, 0.5 mL) under nitrogen, and TMS-diazomethane (2 M in hexanes, 13 µL, 0.026 mmol) added and stirred at rt for 30 min. The reaction was quenched with AcOH (30 uL), concentrated, and repeatedly concentrated (3×) from CHCl3 to remove residual benzene and acetic acid. The crude residue was dissolved in the HPLC mobile phase, syringe filtered and analyzed by chiral HPLC to ascertain the e.r. Methyl

(3R)-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-

a]pyridine-3-carboxylate ((R)-4) With 1 eq. TFA: The following serves as a general procedure for the cyclocondensation reaction between ∆2-thiazolines and acyl Meldrum’s acid derivatives. The title compound was prepared by adaptation of the procedure reported by Chorell et al.18 TFA (385 µL, 5 mmol) was added to a solution of thiazoline (R)-2 (997 mg, 5.00 mmol) and naphthyl meldrum's acid 3 (3.903 g, 12.50 mmol) in 1,2-dichloroethane (15 mL) and heated by MWI at 120 °C for 3 min. After cooling to rt, the reaction mixture was quenched with saturated aqueous NaHCO3 solution (20 mL) and extracted with CH2Cl2 (3×20 mL). The combined organic extracts were washed successively with H2O


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and brine (100 mL each), dried (Na2SO4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO2, 15-100% EtOAc in heptane) subsequent freeze drying (H2O:MeCN; ~3:1) afforded an off-white solid as the product (1.648 g, 84%). e.r. = 11:89. Data in agreement with the literature.29 With 0.2 eq. TFA: The title compound was prepared by adaptation of the procedure reported by Chorell et al.18 TFA (77 µL, 1 mmol) was added to a solution of thiazoline (R)-2 (997 mg, 5.00 mmol) and naphthyl Meldrum’s acid 3 (3.903 g, 12.50 mmol) in anhydrous 1,2 dichloroethane (15 mL) and heated by MWI at 120 °C for 2 min 20 sec. After cooling to rt, the reaction mixture was diluted with CH2Cl2 (~5 mL), washed successively with saturated aqueous NaHCO3 solution (2×10 mL) and brine (10 mL), dried (Na2SO4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO2, 10-100% EtOAc in heptane) subsequent freeze drying (H2O:MeCN; ~3:1) afforded the product as an off-white solid (964 mg, 49%). [α]D20 = +154 (c = 0.5, CHCl3). e.r. = 7:93. Data in agreement with the literature.29 Methyl

(3S)-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-

a]pyridine-3-carboxylate ((S)-4)

The title compound was prepared as described for (R)-4 with

thiazoline (S)-2 (598 mg, 3.00 mmol), naphthyl Meldrum’s acid 3 (2.342 g, 7.50 mmol) and TFA (46 µL, 0.60 mmol) in anhydrous 1,2 dichloroethane (9 mL). Purification by flash chromatography (SiO2, 10-100% EtOAc in heptane) subsequent freeze drying (H2O:MeCN; ~3:1) afforded the product as an off-white solid (588 mg, 50%). [α]D20 = +178 (c = 0.5, CHCl3). e.r. = 96:4. Data in agreement with the literature.29 8-Cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-3carboxylic acid (5) The following serves as a representative procedure for the LiOH mediated



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hydrolysis of methyl carboxylates. The title compound was prepared by adaption of the procedure reported by Chorell et al.45 LiOH (1 M aqueous, 8 mL, 8.00 mmol) was added dropwise to a stirred solution of the (R)-4 (1.565 g, 4.00 mmol) in THF (150 mL) at 0 °C. The solution was allowed to attain rt and stirred for 48 h. The reaction mixture was acidified with aqueous HCl (1 M) to circa pH 1, and extracted with EtOAc (3×75 mL). The organic extracts were washed successively with ice-cold water and brine (50 mL each), dried (Na2SO4), and concentrated under reduced pressure. The residue was triturated with ice-cold Et2O and heptane (2:1; 2×) and freeze-dried (H2O:MeCN; ~3:1) to afford the crude product as a white solid which was used without further purification (1.301 g, 86%). e.r. = 47:53 (as determined by derivatization to 4).

1

H NMR (100 MHz, DMSO-d6) δ = 0.59-0.67 (m, 1H), 0.72-

0.80 (m, 1H), 0.85-0.97 (m, 2H), 1.68-1.76 (m, 1H), 3.50 (dd, J = 1.8, 11.9 Hz, 1H), 3.80 (dd, J = 9.1, 11.9 Hz, 1H), 4.40 (d, J = 17.4 Hz, 1H), 4.50 (d, J = 17.4 Hz, 1H), 5.25 (s, 1H), 5.34 (dd, J = 1.7, 9.1 Hz, 1H), 7.38 (d, J = 6.8 Hz, 1H), 7.48-7.56 (m, 3H), 7.85-7.91 (m, 2H), 7.94-7.99 (m, 1H), 13.33 (br s, 1H).

13

C NMR (100 MHz, DMSO-d6) δ = 7.2, 7.4, 10.8, 31.3, 35.3, 62.5, 111.9, 113.4, 124.1, 125.7,

125.8, 126.4, 127.3, 127.6, 128.6, 131.6, 133.5, 134.6, 148.0, 156.3, 159.9, 169.6. HRMS (ESI+) (m/z): [M+H]+ calcd. for C22H20NO3S, 378.1158; found, 378.1156. (3R)-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine3-carboxylic acid ((R)-5) The title compound was prepared by adaptation of the method developed by Mattsson et al.31 The methyl ester (R)-4 (100 mg, 0.255 mmol) was dissolved in MeCN (2% water v/v, 1 mL). Triethylamine (107 µL, 0.766 mmol) and LiBr (222 mg, 2.553 mmol) were added and the mixture stirred at rt for 3 h. Upon completion, the reaction was cooled to 0 °C, and quenched with aqueous HCl (0.5 M, ~4 mL) and the aqueous layer extracted with EtOAc (2×10 mL). The combined organic extracts were washed with brine (2×5 mL) dried (Na2SO4), the solvent removed under reduced pressure, and the residue freeze dried (H2O:MeCN; ~3:1) to afford the product as an off-white solid,


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which was used without further purification (67 mg, 70%). e.r. = 10:90 (as determined by derivatization to (R)-4). Data in agreement with racemic 5. (3S)-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine3-carboxylic acid ((S)-5) The title compound was prepared as described for (R)-5 by adaptation of the method developed by Mattsson et al with ester (S)-5 (100 mg, 0.255 mmol).31 This afforded the crude product as an off-white solid, which was used without further purification (61 mg, 63%). e.r. = 85:15 (as determined by derivatization to 4). Data in agreement with the racemate 5. 8-Cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-N-phenyl-2,3-dihydro-5H-[1,3]thiazolo[3,2a]pyridine-3-carboxamide (6a) The title compound was prepared by adaptation of the procedure reported by Dunetz et al.20, 46 The carboxylic acid 5 (386 mg, 1.02 mmol) was dissolved in anhydrous MeCN/EtOAc (1:1, 11 mL) under an inert atmosphere, and after cooling the suspension to ~ -10 °C (NaCl/ice), pyridine (247 µL, 3.06 mmol) was added. The reaction mixture was stirred for 5 min, and aniline (140 µL, 1.53 mmol) added, followed by dropwise addition of T3P® (50% in EtOAc; 1.202 mL, 2.04 mmol). The reaction was stirred for 1 h, then allowed to warm to rt and stirred overnight. The reaction was cooled to 0 °C, quenched with aqueous HCl (1 M) and extracted with EtOAc (3×). The organic extracts were washed successively with H2O (3×) and brine, dried (Na2SO4) and the solvent removed under reduced pressure.

Purification by flash chromatography (SiO2, 0-30% EtOAc in

heptane) afforded the racemic amide as a white solid (450 mg, 97%). e.r. = 44:56. 1H NMR (400 MHz, CDCl3) δ = 0.69-0.76 (m, 1H), 0.88-0.99 (m, 2H), 1.03-1.11 (m, 1H), 1.69-1.77 (m, 1H), 3.54 (dd, J = 8.0, 11.1 Hz, 1H), 4.12-4.18 (m, 1H), 4.34 (d, J = 17.6 Hz, 1H), 4.45 (d, J = 17.6 Hz, 1H), 5.76-5.81 (m, 2H), 7.09-7.14 (m, 1H), 7.28-7.36 (m, 3H), 7.40-7.55 (m, 5H), 7.68-7.71 (m, 1H), 7.89-7.99 (m, 2H), 9.96 (s, 1H).

13

C NMR (100 MHz, CDCl3) δ = 7.1, 8.3, 11.5, 21.5, 29.7, 36.4, 64.7, 114.9, 115.7,

117.1, 120.6, 123.9, 125.2, 125.7, 125.9, 126.5, 127.9, 128.0, 128.9, 129.0, 132.1, 133.7, 134.1, 137.9,



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139.0, 148.6, 157.8, 162.7, 164.7. HRMS (ESI+) (m/z): [M+Na]+ calcd. for C28H24N2NaO2S, 475.1451; found, 475.1458.

(3R)-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-N-phenyl-2,3-dihydro-5H-[1,3]thiazolo[3,2a]pyridine-3-carboxamide ((R)-6a)

Via propylphosphonic anhydride: The title compound was

prepared by adaptation of the procedure reported by Dunetz et al.20 The carboxylic acid (R)-4 (25 mg, 0.066 mmol), aniline (69 µL, 0.076 mmol) and pyridine (150 µL, 0.186 mmol) were dissolved in anhydrous EtOAc (0.66 mL) under an inert atmosphere, and cooled to -15 °C. T3P® (50% in EtOAc; 79 µL, 1.32 mmol) was added dropwise, and the reaction mixture allowed to warm to 0 °C, and stirred for 16 h at 0 °C. The reaction was cooled to -15 °C and quenched slowly with aqueous HCl (0.5 M, 2 mL), allowed to warm to rt, and diluted with EtOAc (20 mL). The organic extract was washed successively with aqueous HCl (0.5 M, 3×), saturated aqueous NaHCO3 solution (2×), water (1×), and brine (1×, 5 mL each), dried (Na2SO4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO2, 0-60% EtOAc in heptane) and subsequent freeze drying (H2O:MeCN; ~3:1) afforded the amide as a white solid (21 mg, 71%). [α]D20 = -191 (c = 0.35, CHCl3). e.r. = 15:85. Data in agreement with the racemate 6a. Via HATU: The carboxylic acid (R)-4 (25 mg, 0.066 mmol) was dissolved in CH2Cl2 (0.66 mL) under an inert atmosphere and cooled to 0 °C. DIPEA (26 µL, 0.149 mmol), aniline (69 µL, 0.076 mmol) and HATU (29 mg, 0.076 mmol) were added, and the mixture allowed to warm to rt, and stirred for 6 h. The reaction mixture was cooled to 0 °C and diluted with CH2Cl2 (10 mL), quenched with aqueous HCl (0.5 M, ~5 mL), and washed successively with aqueous HCl (0.5 M, 1×), water (2×), and brine (1×, 5 mL each), dried (Na2SO4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO2, 0-60% EtOAc in heptane) and subsequent freeze drying (H2O:MeCN; ~3:1)


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afforded the product as a white solid (21 mg, 71%). e.r. = 15:85. Data in agreement with the racemate 6a. (3S)-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-N-phenyl-2,3-dihydro-5H-[1,3]thiazolo[3,2a]pyridine-3-carboxamide ((S)-6a) The title compound was prepared as described for (R)-6a from the carboxylic acid (S)-5 (25 mg, 0.066 mmol).20 Purification by flash chromatography (SiO2, 0-60% EtOAc in heptane) and subsequent freeze drying (H2O:MeCN; ~3:1) afforded the amide as a white solid (23 mg, 77%). [α]D20 = 223 (c = 0.35, CHCl3). e.r. = 15:85. Data in agreement with the racemate 6a. 8-Cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-3-(phenylcarbamoyl)-2,3-dihydro-5H[1,3]thiazolo[3,2-a]pyridine-6-carboxylic acid (20) The title compound was prepared by formylation by adaptation of the procedure reported by Sellstedt et al.,28 followed by oxidation with sodium chlorite. A solution of DMF (95 µL, 1.23 mmol) in MeCN (5.3 mL) was cooled to 0 °C. Oxalyl chloride (90 µL, 1.06 mmol) was added cautiously and the mixture stirred for 10 min at rt. The obtained slurry was added to a solution of amide 6a (80 mg, 0.177 mmol) in MeCN (2.4 mL) and the reaction mixture refluxed for 2 h. After cooling to rt, the reaction was diluted with H2O, acidified (pH ≈ 2) with aqueous HCl (1 M), extracted with CH2Cl2 (3×), the organic extracts dried (Na2SO4) and the solvent removed under reduced pressure.

Purification by flash chromatography (SiO2, 0-30% EtOAc in heptane)

afforded the crude aldehyde, which was taken directly to the next step. A solution of NaH2PO4·2H2O (26 mg, 0.166 mmol) dissolved in H2O (0.33 mL) was added dropwise to a solution of the crude aldehyde (40 mg, 0.083 mmol) in DMSO (0.85 mL) at rt. The mixture was then kept on ice and a solution of NaClO2 (30 mg, 0.332 mmol) in H2O (0.3 mL) was added dropwise over 20 min. After stirring the reaction for 1.5 h at rt, the reaction mixture was poured into a separatory funnel containing ice cooled aqueous HCl (1 M). The aqueous phase was extracted with CH2Cl2, and the combined organic extracts washed with H2O (3×), dried (Na2SO4) and the solvent removed under reduced



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pressure. Purification by flash chromatography (SiO2, 0-5% MeOH in CH2Cl2 with 1% AcOH) and freeze-drying (H2O:MeCN; ~3:1) gave the product as a white solid (22 mg, 25%). 1H NMR (400 MHz, DMSO-d6) δ = 0.43-0.55 (m, 2H), 0.61-0.72 (m, 2H), 1.29-1.37 (m, 1H), 3.70 (d, 1H, J = 11.6 Hz, 1H), 3.98-4.04 (m, 1H), 5.03 (d, J = 16.0, 1H)), 5.06 (d, J = 16.0 Hz, 1H), 5.81(d, J = 8.8 Hz, 1H), 6.79 (d, J = 6.8 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 7.32-7.39 (m, 3H), 7.56-7.66 (m, 4H), 7.78 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H), 10.68 (s, 1H), 14.25 (br s, 1H). 13C NMR (100 MHz, DMSO-d6) δ = 7.6, 8.2, 11.9, 32.5, 33.2, 65.7, 115.6, 119.7, 123.8, 124.3, 125.9, 126.3, 126.7, 126.9, 129.0, 129.4, 132.0, 133.7, 135.6, 139.0, 161.4, 165.9, 166.5. HRMS (ESI+) (m/z): [M+Na]+ calcd. for C29H24N2NaO4S, 519.1349; found, 519.1348.

6-Amino-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-N-phenyl-2,3-dihydro-5H[1,3]thiazolo[3,2-a]pyridine-3-carboxamide (21a) The following serves as a representative procedure for the nitration/zinc-reduction preparation of C-6 aminated derivatives. The title compound was prepared by adaptation of the procedure reported by Åberg et al.25 TFA (0.11 ml) was added dropwise to a solution of 6a (60 mg, 0.133 mmol) and NaNO2 (11 mg, 0.159 mmol) in CH2Cl2 (3.25 mL) at rt. The reaction mixture was stirred overnight and quenched by the addition of saturated aqueous NaHCO3. The organic phase was washed with saturated aqueous NaHCO3, dried (Na2SO4) and the solvent removed under reduced pressure. The residue was dissolved in acetic acid (0.8 mL). Freshly activated Zn dust (43 mg, 0.66 mmol) was added in portions and the mixture was allowed to stir for 3 h at rt. After filtration through a pad of Celite®, washing with acetic acid, the solvent was removed under reduced pressure. The residue was diluted with CH2Cl2 and washed with saturated aqueous NaHCO3 (2×). The aqueous layers were extracted with CH2Cl2 (2×) and the combined organic extracts were


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washed with brine, dried (Na2SO4) and the solvent removed under reduced pressure. Purification by flash column chromatography (SiO2, 20-50% EtOAc in heptane), and freeze-drying (H2O:MeCN; ~3:1) afforded the product as a white solid (43 mg, 69%). e.r. = 43:57. 1H NMR (400 MHz, CDCl3) δ = 0.470.58 (m, 2H), 0.64-0.77 (m, 2H), 1.34-1.41 (m, 1H), 3.75 (d, 1H, J = 11.8 Hz, 1H), 3.96-4.07 (m, 1H), 5.05 (d, J = 16.0, 1H)), 5.09 (d, 1H, J = 16.0 Hz), 5.77 (d, J = 8.8 Hz, 1H), 6.98 (d, J = 6.8 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 7.31-7.37 (m, 3H), 7.57-7.67 (m, 4H), 7.78 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 10.40 (s, 1H). 13C NMR (100 MHz, CDCl3) δ = 6.6, 12.1, 29.7, 30.9, 65.0, 116.8, 120.0, 123.1, 123.4, 124.3, 125.7, 126.4, 127.5, 128.9, 129.0, 130.3, 131.9, 132.1, 133.9, 134.1, 138.1, 157.4, 164.9. HRMS (ESI+) (m/z): [M+Na]+ calcd. for C28H25N3NaO2S, 490.1560; found, 490.1560. 6-Amino-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-N-(3-methylphenyl)-5-oxo-2,3-dihydro-5H[1,3]thiazolo[3,2-a]pyridine-3-carboxamide (21b) The title compound was prepared following the general nitration-reduction procedures with 6e (55 mg, 0.118 mmol), NaNO2 (8.5 mg, 0.123 mmol) and TFA (118 µL, 1.54 mmol) in CH2Cl2 (2.2 mL) under an oxygen atmosphere, and subsequently treating the obtained crude with activated zinc dust (42 mg, 0.64 mmol) in acetic acid (1.3 mL). Purification by flash chromatography (SiO2, 0-65% EtOAc in heptane), and freeze-drying (H2O:MeCN; ~3:1) afforded the product as a yellow solid (21 mg, 37%). 1H NMR (400 MHz, CDCl3) δ = 0.44-0.51 (m, 1H), 0.590.79 (m, 3H), 1.53-1.62 (m, 1H), 2.33 (s, 3H), 3.61 (dd, J = 7.7, 11.2 Hz, 1H), 3.85 (br s, 2H), 4.15 (d, J = 11.2 Hz, 1H), 4.46 (d, J = 16.8 Hz, 1H), 4.62 (d, J = 16.7 Hz, 1H), 5.90 (d, J = 7.7 Hz, 1H), 6.89-6.97 (m, 2H), 7.19 (t, J = 7.8 Hz, 1H), 7.30-7.36 (m, 1H), 7.37-7.41 (m, 1H), 7.43-7.45 (m, 1H), 7.54-7.64 (m, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 10.30 (br s, 1H). 13

C NMR (100 MHz, CDCl3) δ = 7.2, 8.2, 11.5, 29.9, 36.4, 64.5, 112.6, 115.4, 115.5, 120.3, 123.9,



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125.7, 125.9, 126.5, 127.9, 128.0, 129.1, 132.0, 133.7, 134.1, 140.6, 148.0, 149.4, 151.1, 157.7, 162.4, 165.7. HRMS (ESI+) (m/z): [M+H]+ calcd. for C29H28N3O2S, 482.1897; found, 482.1889.

6-Amino-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-N-(4-methylphenyl)-5-oxo-2,3-dihydro-5H[1,3]thiazolo[3,2-a]pyridine-3-carboxamide (21c) The title compound was prepared following the general nitration-reduction procedures with 6l (60 mg, 0.129 mmol), NaNO2 (9.3 mg, 0.135 mmol) and TFA (121 µL, 1.58 mmol) in CH2Cl2 (2.4 mL) under an oxygen atmosphere, and treating the obtained crude with activated zinc dust (42 mg, 0.64 mmol) in acetic acid (1.3 mL). Purification by flash chromatography (SiO2, 0-60% EtOAc in heptane), and freeze-drying (H2O:MeCN; ~3:1) afforded the product as a yellow solid (26 mg, 42%). 1H NMR (400 MHz, CDCl3) δ = 0.44-0.52 (m, 1H), 0.59-0.79 (m, 3H), 1.53-1.64 (m, 1H), 2.32 (s, 3H), 3.61 (dd, J = 7.8, 11.2 Hz, 1H), 3.85 (br s, 2H), 4.15 (d, J = 11.2 Hz, 1H), 4.46 (d, J = 16.8 Hz, 1H), 4.62 (d, J = 16.7 Hz, 1H), 5.89 (d, J = 7.7 Hz, 1H), 6.95 (d, J = 7.1 Hz, 1H), 7.09-7.13 (m, 2H), 7.30-7.35 (m, 1H), 7.45-7.50 (m, 2H), 7.54-7.65 (m, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 8.19 (d, J = 8.3 Hz, 1H), 10.26 (br s, 1H).

13

C NMR (100 MHz,

CDCl3) δ = 6.7, 7.4, 12.1, 21.0, 29.7, 31.0, 65.1, 116.9, 120.1, 123.2, 123.5, 125.8, 126.1, 126.5, 127.6, 129.1, 129.5, 130.3, 132.0, 132.2, 132.6, 134.05, 134.08, 134.14, 135.6, 157.5, 164.9. HRMS (ESI+) (m/z): [M+Na]+ calcd. for C29H27N3NaO2S, 504.1716; found, 504.1710.

6-Amino-N-(4-carbamoylphenyl)-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-2,3-dihydro-5H[1,3]thiazolo[3,2-a]pyridine-3-carboxamide (21d) The title compound was prepared by adaptation of the procedure reported by Awuah et al.21

The carboxylic acid 24 (65 mg, 0.166 mmol), 4-

aminobenzamide (56 mg, 0.414 mmol), DIPEA (79 µL, 0.455 mmol) and HATU (126 mg, 0.331 mmol) were dissolved in anhydrous DMF (2.125 mL) under an inert atmosphere and heated by MWI at 60 °C for 55 min. The reaction mixture was diluted with CH2Cl2 (30 mL), washed successively with aqueous


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HCl (0.01 M, 2×15 mL), water (3×20 mL), brine (20 mL), dried (Na2SO4) and the solvent removed under reduced pressure. Purification by HPLC (mobile phase: MeCN/H2O with 0.005% formic acid, 35-100% for 45 min; tR = 20.63 min) and subsequent freeze-drying (H2O:MeCN; ~3:1) afforded the product as a white solid (36 mg, 42%). 1H NMR (400 MHz, DMSO-d6) δ = 0.36-0.47 (m, 2H), 0.530.65 (m, 2H), 1.32-1.42 (m, 1H), 3.55 (dd, J = 2.2, 11.9 Hz, 1H), 3.89 (dd, J = 8.9, 11.9 Hz, 1H), 4.414.77 (m, 4H), 5.64 (dd, J = 2.2, 8.8 Hz, 1H), 6.89 (d, J = 6.9 Hz, 1H), 7.27 (br s, 1H), 7.37-7.43 (m, 1H), 7.53-7.70 (m, 4H), 7.78 (d, J = 8.2 Hz, 1H), 7.84-7.98 (m, 4H), 8.30 (d, J = 8.5 Hz, 1H), 10.70 (s, 1H).

13

C NMR (100 MHz, DMSO-d6) δ = 6.7, 6.9, 11.6, 29.8, 31.6, 64.3, 112.7, 118.2, 123.2, 123.7,

125.4, 125.6, 125.7, 126.0, 126.4, 128.5, 129.1, 131.8, 131.9, 132.9, 133.5, 133.9, 141.4, 156.0, 166.6, 167.3. HRMS (ESI+) (m/z): [M+H]+ calcd. for C29H27N4O3S, 511.1798; found, 511.1795. 6-Amino-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2a]pyridine-3-carboxylic acid (24) LiOH (1 M aqueous, 1.24 mL, 1.24 mmol) was added dropwise to a stirred solution of methyl ester 23 (252 mg, 0.620 mmol) in THF (12.5 mL) and stirred at rt overnight. The reaction mixture was quenched with saturated aqueous NH4Cl soln. (15 mL) and extracted with EtOAc (3×30 mL). The aqueous layer was further acidified to circa pH 3 with aqueous HCl (0.5 M), cooled to 0 °C and extracted with EtOAc (6 x 50 mL). The combined organic layers were dried (Na2SO4), and the solvent removed under reduced pressure. The residue was triturated ice-cold Et2O and heptane (2:1; 2×) and freeze-dried (H2O:MeCN; ~3:1) to afford the crude product as an off-white solid which was used without further purification (176 mg, 72%). 1H NMR (400 MHz, DMSO-d6) δ = 0.32-0.43 (m, 2H), 0.51-0.62 (m, 2H), 1.30-1.38 (m, 1H), 3.49 (dd, J = 1.4, 11.6 Hz, 1H), 3.72 (dd, J = 8.6, 11.6 Hz, 1H), 4.43 (d, J = 16.9 Hz, 1H), 4.52 (d, J = 16.9 Hz, 1H), 5.42 (dd, J = 1.3, 8.5 Hz, 1H), 6.88 (d, J = 6.8 Hz, 1H), 7.34-7.40 (m, 1H), 7.53-7.65 (m, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.93-7.97 (m, 1H), 8.29 (d = J = 8.3 Hz, 1H).

13

C NMR (100 MHz, DMSO-d6) δ = 6.6, 6.8, 11.5, 29.8, 31.8, 63.6,



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112.6, 123.2, 123.7, 124.8, 125.6, 125.6, 126.0, 126.4, 128.5, 131.0, 131.9, 132.9, 133.4, 134.0, 156.0, 169.8. HRMS (ESI-) (m/z): [M-H]- calcd. for C22H19N2O3S, 391.1122; found, 391.1118. 6-Amino-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-N-(pyrimidin-4-yl)-3,5-dihydro-2Hthiazolo[3,2-a]pyridine-3-carboxamide (25)

The title compound was prepared following the

procedure described for 21d with carboxylate 24 (75 mg, 0.191 mmol), 4-aminopyrimidine (45 mg, 0.478 mmol), DIPEA (91 µL, 0.525 mmol) and HATU (145 mg, 0.38 mmol) in anhydrous DMF (2.375 mL) and MWI at 60 °C for 55 min. Purification by HPLC (mobile phase: MeCN/H2O with 0.005% formic acid, 35-100% for 45 min; tR = 22.94 min) and subsequent freeze-drying (H2O:MeCN; ~3:1) afforded the product as a white solid (33 mg, 36%). 1H NMR (400 MHz, CDCl3) δ = 0.44-0.52 (m, 1H), 0.55-0.81 (m, 3H), 1.52-1.62 (m, 1H), 3.65 (dd, J = 7.8, 11.5 Hz, 1H), 4.07 (d, J = 11.5, 1H), 4.44 (d, J = 16.7 Hz, 1H), 4.62 (d, J = 16.8 Hz, 1H), 5.93 (d, J = 7.4 Hz, 1H), 6.91-6.95 (m, 1H), 7.27-7.33 (m, 1H), 7.52-7.65 (m, 2H), 7.75 (d, J = 8.2 Hz, 1H), 7.88-7.93 (m, 1H), 8.10-8.19 (m, 2H), 8.62 (d, J = 5.7 Hz, 1H), 8.90 (d, J = 1.0 Hz, 1H), 11.05 (br s, 1H).

13

C NMR (100 MHz, CDCl3) δ = 6.8, 7.4, 12.2,

29.6, 31.0, 65.1, 110.9, 117.4, 123.2, 123.4, 125.8, 126.1, 126.5, 127.7, 129.1, 130.2, 131.8, 132.2, 133.0, 133.3, 134.1, 157.3, 157.5, 158.5, 158.8, 166.7.

HRMS (ESI+) (m/z): [M+H]+ calcd. for

C26H24N5O2S, 470.1645; found, 470.1646. 5-(2-Chloro-1-hydroxyethylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione

(27)

A

solution

of

chloroacetic acid (2.875 g, 30.42 mmol) and 2,2-dimethyl-1,3-dioxane-4,6-dione (5.262 g, 36.50 mmol) in CH2Cl2 (120 mL) was cooled to 0 °C. DMAP (743 mg, 6.08 mmol) and NEt3 (12.73 mL, 91.33 mmol) were added, followed by dropwise addition of T3P® (50% in EtOAc; 36.20 mL, 60.81 mmol) over 30 min. The reaction was stirred at 0 °C for a further 1.5 h, then allowed to attain rt and stirred for 36 h. The reaction mixture was cooled to 0 °C, quenched cautiously with aqueous KHSO4 solution (3% w/v; 100 mL) and extracted with CH2Cl2 (3×50 mL). The organic extracts were washed with aqueous


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KHSO4 solution (3% w/v; 4×100 mL), brine (100 mL), dried (Na2SO4) and the solvent removed under reduced pressure to afford the acyl Meldrum’s acid as a reddish brown solid, which was used without further purification (6.381 g, 95%). Data was in agreement with published data.28

Methyl (3R)-7-(chloromethyl)-8-cyclopropyl-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-3carboxylate (28) Following the general cyclocondensation procedure with (R)-2 (996 mg, 5.00 mmol) and 27 (3.033 g, 13.75 mmol) in 1,2-dichloroethane (15 mL) with purification by flash chromatography (SiO2, 10-100% EtOAc in heptane) afforded the product as a clear yellow oil (1.444 g, 96%). Data was in agreement with published data.28

Methyl

(3R)-8-cyclopropyl-7-benzyl-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-3-

carboxylate (29a) The following serves as a general procedure for the Suzuki-Miyaura cross-coupling procedure of chloromethyl derivative. The title compound was prepared by adaptation of the procedure reported by Chorell et al.27 The chloromethyl derivative 28 (150 mg, 0.500 mmol), phenylboronic acid (122 mg, 1.00 mmol), Pd(PPh3)2Cl2 (17.5 mg, 0.025 mmol) and KF (58 mg, 1.00 mmol) were dissolved in anhydrous MeOH (4.5 mL) and heated by MWI at 140 °C for 12 min. The reaction mixture was diluted with EtOAc (20 mL), washed successively with saturated aqueous NaHCO3, H2O and brine (20 mL each), dried (Na2SO4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO2; 10-100% EtOAc in heptane) afforded the cross-coupled product as a pale brown solid (119 mg, 70%). [α]D20 = -96 (c = 0.4, CHCl3).

1

H NMR (400 MHz, CDCl3) δ = 0.66-0.69 (m,

2H), 0.82-0.96 (m, 2H), 1.36-1.46 (m, 1H), 3.48 (dd, J = 2.3, 11.7 Hz, 1H), 3.64 (dd, J = 8.6, 11.8 Hz, 1H) , 3.80 (s, 3H), 3.94 (d, J = 15.8 Hz, 1H), 4.01 (d, J = 15.8 Hz, 1H), 5.59 (dd, J = 2.3, 8.5 Hz, 1H), 6.04 (s, 1H), 7.16-7.32 (m, 5H).

13

C NMR (100 MHz, CDCl3) δ = 7.7, 8.1, 11.4, 31.8, 39.4, 53.4, 62.9,



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114.0, 115.8, 126.7, 128.7, 129.3, 138.2, 147.4, 157.1, 161.5, 168.8. HRMS (ESI+) (m/z): [M+H]+ calcd. for C19H20NO3S, 342.1158; found, 342.1155.

Methyl (3R)-8-cyclopropyl-7-(4-chlorobenzyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine3-carboxylate (29c) Following the general Suzuki-Miyaura procedure with 28 (150 mg, 0.500 mmol) and 4-chlorophenylboronic acid (150 mg, 1.00 mmol) and purification by flash chromatography (SiO2, 15-100% EtOAc in heptane) gave the product as an off-white solid (120 mg, 64%). [α]D20 = -26 (c = 1.0, CDCl3). 1H NMR (400 MHz, CDCl3) δ = 0.58-0.68 (m, 2H), 0.81-0.95 (m, 2H), 1.32-1.40 (m, 1H), 3.49 (dd, J = 2.3, 11.8 Hz, 1H), 3.64 (dd, J = 8.6, 11.7 Hz, 1H), 3.80 (s, 3H), 3.90 (d, J = 15.9 Hz, 1H), 3.99 (d, J = 15.9 Hz, 1H), 5.59 (dd, J = 2.3, 8.6 Hz, 1H), 6.01 (s, 1H), 7.09-7.13 (m, 2H), 7.24-7.29 (m, 2H).

13

C NMR (100 MHz, CDCl3) δ = 7.8, 8.1, 11.3, 31.8, 38.8, 53.4, 62.9, 113.8, 115.7, 128.9, 130.6,

132.6, 136.7, 147.8, 156.6, 161.3, 168.7. HRMS (ESI+) (m/z): [M+H]+ calcd. for C19H19ClNO3S, 376.0769; found, 376.0777.

Methyl

(3R)-8-cyclopropyl-7-(2,3-dimethylbenzyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-

a]pyridine-3-carboxylate (29e)

The chloromethyl derivative 28 (150 mg, 0.500 mmol), 2,3-

dimethylphenylboronic acid (150 mg, 1.00 mmol), Pd(PPh3)2Cl2 (26 mg, 0.037 mmol) and KF (58 mg, 1.00 mmol) were dissolved in anhydrous MeOH (5 mL) and heated by MWI at 120 °C for 10 min. Following the work-up as described in the general Suzuki-Miyaura procedure, with purification by flash chromatography (SiO2, 15-100% EtOAc in petroleum ether 60/40), gave the product as an off-white solid (179 mg, 97%). e.r. = 16:84. [α]D20 = -130 (c = 0.35, CHCl3).

1

H NMR (400 MHz, CDCl3) =

0.65-0.73 (m, 2H), 0.87-1.02 (m, 2H), 1.56-1.65 (m, 1H), 2.08 (s, 3H), 2.29 (s, 3H), 3.50 (dd, J = 2.3, 11.7 Hz, 1H), 3.66 (dd, J = 8.5, 11.7 Hz, 1H) , 3.79 (s, 3H), 3.94 (d, J = 17.5 Hz, 1H), 4.01 (d, J = 17.5 Hz, 1H), 5.57 (dd, J = 2.3, 8.5 Hz, 1H), 5.70 (br s, 1H), 6.90-6.93 (m, 1H), 7.01-7.09 (m, 2H).


13

C

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NMR (100 MHz, CDCl3) δ = 7.6, 7.8, 11.2, 15.6, 20.8, 31.9, 37.6, 53.4, 62.8, 113.8, 114.8, 125.9, 128.3, 128.9, 135.3, 136.1, 137.4, 146.7, 157.3, 161.6, 168.9.

HRMS (ESI+) (m/z): [M+H]+ calcd. for

C21H24NO3S, 370.1471; found, 370.1490.

8-Cyclopropyl-7-(4-chlorobenzyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-3-carboxylic acid (30c)

Following the general LiOH hydrolysis procedure with 29c (210 mg, 0.559 mmol),

purification by HPLC (mobile phase: MeCN/H2O with 0.75% formic acid each, 35-100% for 30 min; tR = 17.77 min) and subsequent freeze-drying (H2O:MeCN; ~3:1) gave the product as a white solid (164 mg, 81%). e.r. = 47:52.

1

H NMR (600 MHz, DMSO-d6) δ = 0.48-0.55 (m, 1H), 0.58-0.64 (m, 1H),

0.80-0.92 (m, 2H), 1.33-1.40 (m, 1H), 3.49 (dd, J = 1.3, 11.9 Hz, 1H), 3.78 (dd, J = 9.2, 11.8 Hz, 1H), 3.94 (d, J = 15.6 Hz, 1H), 4.00 (d, J = 15.6 Hz, 1H), 5.38 (dd, J = 1.4, 9.1 Hz, 1H), 5.77 (s, 1H), 7.26 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.3 Hz, 2H), 13.36 (s, 1H).

13

C NMR (150 MHz, DMSO-d6) δ = 7.4, 7.6,

10.8, 31.2, 37.5, 62.4, 111.8, 114.1, 128.4, 130.9, 131.1, 137.7, 148.5, 156.0, 160.0, 169.6. HRMS (ESI+) (m/z): [M+H]+ calcd. for C18H16ClNNaO3S, 384.0432; found, 384.0407.

8-Cyclopropyl-7-(2,3-dimethylbenzyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-3carboxylic acid (30e) The title compound was prepared by a modified version of the general LiOH hydrolysis procedure with 29e (109 mg, 0.295 mmol) and aqueous LiOH (1 M, 0.885 mL, 0.885 mmol). Purification by HPLC (mobile phase: MeCN/H2O with 0.75% formic acid each, 30-100% for 30 min; tR = 18.95 min) and subsequent freeze-drying (H2O:MeCN; ~3:1) gave the product as a white solid (81 mg, 77%). e.r. = 43:57.

1

H NMR (400 MHz, DMSO-d6) δ = 0.52-0.60 (m, 1H), 0.63-0.71 (m, 1H), 0.83-

0.97 (m, 2H), 1.57-1.67 (m, 1H), 2.05 (s, 3H), 2.27 (s, 3H), 3.50 (dd, J = 1.8, 11.9 Hz, 1H), 3.79 (dd, J = 9.1, 11.9 Hz, 1H), 3.94 (d, J = 17.4 Hz, 1H), 4.00 (d, J = 17.4 Hz, 1H), 5.28 (s, 1H), 5.35 (dd, J = 1.7, 9.1 Hz, 1H), 6.94-6.97 (m, 1H), 7.04-7.12 (m, 2H), 13.37 (br s, 1H).


13

C NMR (100 MHz, DMSO-d6) δ


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= 7.1, 7.5, 10.7, 15.1, 20.3, 32.7, 36.4, 65.2, 111.4, 112.9, 125.5, 127.9, 128.4, 134.9, 136.6, 136.8, 148.5, 155.1, 160.5, 170.7. HRMS (ESI+) (m/z): [M+H]+ calcd. for C20H22NO3S, 356.1315; found, 356.1286.

7-(4-Chlorobenzyl)-8-cyclopropyl-5-oxo-N-phenyl-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-3carboxamide (31c) Following the general LiOH hydrolysis procedure with 30c (34 mg, 0.094 mmol) and aniline (15 µL, 0.165 mmol) afforded after purification by flash chromatography (SiO2, 0-75% EtOAc in heptane) and freeze-drying (H2O:MeCN; ~3:1) the product as a white solid (17 mg, 41%). e.r. = 48:52.

1

H NMR (400 MHz, CDCl3) δ = 0.52-0.60 (m, 1H), 0.65-0.75 (m, 1H), 0.80-0.90 (m, 1H),

0.91-1.00 ( m, 1H), 1.36-1.45 (m, 1H), 3.55 (dd, J = 8.0, 11.2 Hz, 1H), 3.90 (d, J = 16.1 Hz, 1H), 4.00 (d, J = 16.0 Hz, 1H), 4.15 (d, J = 11.2 Hz, 1H), 5.83 (d, J = 7.9 Hz, 1H), 6.06 (s, 1H), 7.04-7.13 (m, 3H), 7.25-7.31 (m, 4H), 7.52-7.58 (m, 2H), 10.32 (s, 1H).

13

C NMR (100 MHz, CDCl3) δ = 7.4, 8.4,

11.5, 29.8, 38.6, 64.8, 115.1, 115.9, 120.0, 124.5, 129.0, 129.1, 130.6, 132.8, 136.3, 138.0149.5, 157.5, 162.5, 164.7. HRMS (ESI+) (m/z): [M+Na]+ calcd. for C24H22ClNaN2O2S, 459.0904; found, 459.0910. 8-Cyclopropyl-7-(2,3-dimethylbenzyl)-5-oxo-N-phenyl-2,3-dihydro-5H-[1,3]thiazolo[3,2a]pyridine-3-carboxamide (31e) Following the general amide-coupling procedure with 30e (47 mg, 0.132 mmol) and aniline (18 µL, 0.199 mmol) and purification by flash chromatography (SiO2, 0-60% EtOAc in heptane) afforded the product as a white solid (30 mg, 53%). e.r. = 46:54.

1

H (400 MHz,

CDCl3) δ = 0.58-0.67 (m, 1H), 0.70-0.82 (m, 1H), 0.85-0.96 (m, 1H), 0.97-1.07 (m, 1H),1.57-1.70 (m, 1H), 2.08 (s, 3H), 2.30 (s, 3H), 3.57 (dd, J = 8.1, 11.1 Hz, 1H), 3.92-4.05 (m, 2H), 4.17 (d, J = 11.2 Hz, 1H), 5.76-5.83 (m, 2H), 6.89-6.94 (m, 1H), 7.03-7.16 (m, 3H), 7.24-7.34 (m, 3H), 7.50-7.69 (m, 2H), 10.34 (br s, 1H).

13

C NMR (100 MHz, CDCl3) δ = 7.1, 8.1,0 11.4, 15.6, 20.8, 29.7, 37.5, 64.7, 114.2,


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115.9, 120.0, 124.4, 126.0, 128.3, 129.0, 129.1, 135.3, 135.7, 137.5, 138.1, 148.4, 158.2, 162.8, 164.8. HRMS (ESI+) (m/z): [M+H]+ calcd. for C26H26N2NaO2S, 453.1607; found, 453.1635.

8-Cyclopropyl-7-(2,3-dimethylbenzyl)-5-oxo-N-(4-methylphenyl)-2,3-dihydro-5H-[1,3]thiazolo[3,2a]pyridine-3-carboxamide (32a) Following the general amide-coupling procedure with 30e (121 mg, 0.340 mmol) and 4-methylaniline (64 mg, 0.596 mmol) afforded after purification by flash chromatography (SiO2, 5-75% EtOAc in heptane) and freeze-drying (H2O:MeCN; ~3:1) the product as a white solid (129 mg, 85%).

1

H NMR (400 MHz, CDCl3) δ = 0.57-0.66 (m, 1H), 0.71-0.80 (m, 1H),

0.85-0.95 (m, 1H), 0.97-1.01 (m, 1H), 1.58-1.68 (m, 1H), 2.07 (s, 3H), 2.28 (s, 3H), 2.30 (s, 3H), 3.55 (dd, J = 8.0, 11.2 Hz, 1H), 3.92-4.04 (m, 2H), 4.17 (d, J = 11.2 Hz, 1H), 5.76-5.80 (m, 2H), 6.89-6.92 (m, 1H), 7.03-7.11 (m, 4H), 7.39-7.44 (m, 2H), 10.23 (s, 1H).

13

C NMR (100 MHz, CDCl3) δ = 7.0,

8.1, 11.4, 15.6, 20.8, 21.0, 29.6, 37.5, 64.6, 114.2, 115.7, 120.0, 125.9, 128.3, 129.1, 129.5, 134.0, 135.3, 135.5, 135.8, 137.5, 148.3, 158.0, 162.8, 164.6.

HRMS (ESI+) (m/z): [M+Na]+ calcd. for

C25H23ClN2NaO2S, 473.1061; found, 473.1068.

7-(4-Chlorobenzyl)-8-cyclopropyl-N-(4-methylphenyl)-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2a]pyridine-3-carboxamide (32b) Following the general amide-coupling procedure with 30c (138 mg, 0.381 mmol) and 4-methylaniline (72 mg, 0.667 mmol) afforded after purification by flash chromatography (SiO2, 5-75% EtOAc in heptane) and freeze-drying (H2O:MeCN; ~3:1) the product as a white solid (139 mg, 81%).

1

H NMR (400 MHz, CDCl3) δ = 0.52-0.61 (m, 1H), 0.65-0.74 (m, 1H),

0.79-0.89 (m, 1H), 0.91-1.01 (m, 1H), 1.36-1.45 (m, 1H), 2.29 (s, 3H), 3.55 (dd, J = 8.0, 11.2 Hz, 1H), 3.90 (d, J = 16.0 Hz, 1H), 4.00 (d, J = 16.0 Hz, 1H), 4.16 (d, J = 11.2 Hz, 1H), 5.81 (d, 7.9, 1H), 6.05 (s, 1H), 7.06-7.12 (m, 4H), 7.25-7.30 (m, 2H), 7.40-7.45 (m, 2H), 10.20 (s, 1H).

13

C NMR (100 MHz,

CDCl3) δ = 7.3, 8.4, 11.5, 21.0, 29.6, 38.6, 64.7, 115.3, 115.6, 120.0, 129.0, 129.5, 130.6, 132.8, 134.1,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

135.5, 136.4, 149.3, 157.2, 162.6, 164.5.

Page 44 of 65

HRMS (ESI+) (m/z): [M+H]+ calcd. for C27H29N2O2S,

445.1944; found, 445.1952. 6-Amino-7-(4-chlorobenzyl)-8-cyclopropyl-5-oxo-N-(4-methylphenyl)-2,3-dihydro-5H[1,3]thiazolo[3,2-a]pyridine-3-carboxamide (33a) The title compound was prepared following the general nitration-reduction procedures with 32a (60 mg, 0.11 mmol), NaNO2 (9.6 mg, 0.139 mmol) and TFA (133 µL, 1.74 mmol) in CH2Cl2 (2.66 mL) under an oxygen atmosphere, and subsequently treating the obtained crude with freshly activated zinc dust (44 mg, 0.673 mmol) in acetic acid (2 mL). Purification by flash chromatography (SiO2, 0-90% EtOAc in heptane), and freeze-drying (H2O:MeCN; ~3:1) afforded the product as an off-white solid (47 mg, 76%). 1H NMR (400 MHz, CDCl3) δ = 0.440.53 (m, 1H), 0.61-0.69 (m, 1H), 0.75-0.92 (m, 2H), 1.49-1.60 (m, 1H), 2.29 (s, 3H), 3.56 (dd, J = 7.8, 11.2 Hz, 1H), 3.88 (br s, 2H), 4.00 (d, J = 16.1 Hz, 1H), 4.09-4.20 (m, 2H), 5.83 (d, J = 7.7 Hz, 1H), 7.65-7.12 (m, 4H), 7.23-7.28 (m, 3H), 7.42-7.46 (m, 2H), 10.18 (br s, 1H).

13

C NMR (100 MHz,

CDCl3) δ = 7.1, 7.9, 12.2, 21.0, 29.7, 33.2, 65.1, 116.4, 120.0, 129.2, 129.3, 129.5, 130.0, 132.3, 132.6, 134.1, 134.2, 135.5, 135.9, 157.6, 164.7. HRMS (ESI+) (m/z): [M+H]+ calcd. for C25H25ClN3O2S, 466.1351; found, 466.1348. 6-Amino-8-cyclopropyl-7-(2,3-dimethylbenzyl)-5-oxo-N-(4-methylphenyl)-2,3-dihydro-5H[1,3]thiazolo[3,2-a]pyridine-3-carboxamide (33b) The title compound was prepared following the general nitration-reduction procedures with 32b (61 mg, 0.137 mmol), NaNO2 (9.9 mg, 0.144 mmol) and TFA (137 µL, 1.79 mmol) in CH2Cl2 (2.66 mL) under an oxygen atmosphere, and subsequently treating the obtained crude with freshly activated zinc dust (45 mg, 0.69 mmol) in acetic acid (2 mL). Purification by flash chromatography (SiO2, 0-65% EtOAc in heptane), and freeze-drying (H2O:MeCN; ~3:1) afforded the product as an off-white solid (17 mg, 27%). 1H NMR (400 MHz, CDCl3) δ = 0.410.50 (m, 1H), 0.58-0.67 (m, 1H), 0.69-0.84 (m, 2H), 1.50-1.59 (m, 1H), 2.30 (s, 3H), 2.32 (s, 3H), 2.34


Page 45 of 65

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(s, 3H), 3.59 (dd, J = 7.8, 11.2 Hz, 1H), 3.79 (br s, 1H), 3.94 (d, J = 16.7 Hz, 1H), 4.09-4.16 (m, 2H), 5.86 (d, J = 7.6 Hz, 1H), 6.61 (d, J = 7.4 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 7.3 Hz, 1H), 7.07-7.12 (m, 2H), 7.43-7.48 (m, 2H), 10.25 (s, 1H).

13

C NMR (100 MHz, CDCl3) δ = 6.8, 7.5, 12.1,

15.3, 20.9, 21.0, 29.7, 32.2, 65.1, 116.8, 120.1, 123.9, 126.0, 128.6, 129.5, 131.2, 132.4, 134.0, 134.1, 134.6, 135.2, 135.6, 137.2, 157.5, 164.9.

HRMS (ESI+) (m/z): [M+H]+ calcd. for C27H30N3O2S,

460.2053; found, 460.2055. 8-Cyclopropyl-7-((2-(1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-8-yl)ethyl))-5oxo-N-(3-methylphenyl)-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-3-carboxamide (37) The BODIPY methyl ester 36 (58 mg, 0.109 mmol) was dissolved in MeCN (2% water v/v, 1.15 mL), NEt3 (46 µL, 0.329 mmol) and LiBr (95 mg, 1.094 mmol) added and the reaction mixture heated at 50 °C for 5 h. After cooling to rt, the reaction was diluted with EtOAc (30 mL), washed with aqueous HCl (1 M) and brine (10 mL each), dried (Na2SO4) and the solvent removed under reduced pressure. The crude carboxylic acid was dissolved in anhydrous DMF (1.5 mL), 3-methylaniline (18 µL, 0.165 mmol), DIPEA (43 µL, 0.246 mmol) and HATU (59 mg, 0.155 mmol) added, and the reaction stirred at rt for 17 h. The reaction mixture was diluted with EtOAc (25 mL), washed successively with aqueous HCl (0.5 M), water (3×), brine (10 mL each), dried (Na2SO4) and the solvent removed under reduced pressure. Purification by HPLC (mobile phase: MeCN/H2O with 0.005% formic acid, 40-100% for 25 min; tR = 21.48 min) and subsequent freeze-drying (H2O:MeCN; ~3:1) afforded the product as a bright red solid (14 mg, 21%). 1H NMR (600 MHz, DMSO-d6) δ = 0.46-0.57 (m, 2H), 0.77-0.88 (m, 2H), 1.56-1.62 (m, 1H), 2.25-2.45 (m, 12H), 2.87-3.02 (m, 2H), 3.23-3.37 (m, 2H), 3.52 (d, J = 11.9 Hz, 1H), 3.84-3.89 (m, 1H), 5.51-5.55 (d, J = 9.1 Hz, 1H), 6.19 (s, 1H), 6.27 (s, 2H), 6.88-6.90 (m, 1H), 7.18-7.22 (m, 1H), 7.31-7.35 (m, 1H), 7.45 (s, 1H), 10.36 (s, 1H).

13

C NMR (150 MHz, DMSO-d6) δ = 7.3, 7.5, 10.5, 14.1,

15.7, 21.1, 25.0, 31.6, 32.3, 63.9, 110.7, 111.4, 116.2, 119.6, 121.9, 124.2, 128.7, 130.8, 138.1, 138.7,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

141.0, 145.4, 148.9, 153.6, 155.2, 160.2, 166.0.

Page 46 of 65

HRMS (ESI+) (m/z): [M+Na]+ calcd. for

C33H35BF2N4NaO2S, 623.2434; found, 623.2446.


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Supporting Information: Synthetic procedures and characterization for all other compounds, chiral HPLC chromatographs. Corresponding Author Information *S.B: email: [email protected]; phone: +46 90 785 6726. *F.A: email: [email protected]; phone: +46 90 786 6925. Present Addresses K Syam Krishnan: Department of Chemistry, Mannam Memorial NSS College, Kottiyam, Kollam, Kerala, 691571, India. P. Engström: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA. Conflict of interest Sven Bergström and Fredrik Almqvist are cofounders of Quretech Bio AB. Acknowledgements We thank Dr. Emma Andersson, Umeå University for assistance with cytotoxicity assays and Ingela Nilsson, Umeå University for assistance with biological evaluation. We thank Dr Sofia Essén, Lund University for assistance with HRMS measurements. We acknowledge funding from the Swedish Research Council (F.A. and S.B.), the Knut and Alice Wallenberg Foundation (F.A. and S.B.), the Göran Gustafsson Foundation (F.A.), the Swedish Foundation for Strategic Research (F.A.), the Swedish Government Fund for Clinical Research ALF (ÅG) and the Scandinavian Society for Antimicrobial Chemotherapy Foundation (Å.G.). K.S.K. thanks the JC Kempe Foundation for funding.



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Page 48 of 65

PE is currently funded by a postdoctoral fellowship from the Swedish Society of Medical Research (SSMF). Abbreviations: C. albicans, Candida albicans; C. trachomatis, Chlamydia trachomatis; comp. Log P, computed Log P; DAPI, (4′,6-diamidino-2-phenylindole), DIPEA, N,N-Diisopropylethylamine; DMEM, Dulbecco's modified eagle medium; EB, elementary body; e.r., enantiomeric ratio; Escherichia coli, E. coli; FBS, fetal bovine serum; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b]pyridinium 3-oxid hexafluorophosphate); HBSS, Hanks balanced salt solution; hpi, hours post infection; IFU, inclusion forming unit; G6-P, glucose 6-phosphate; MOI, multiplicity of infection; MOMP, Chlamydial major outer membrane protein; MWI, microwave irradiation; OD, optical density; RB, reticulate body; rt, room temperature; PBS, phosphate buffered saline; propylphosphonic anhydride, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane

2,4,6-trioxide

solution;

SAR,

structure-activity

relationship; STI, sexually transmitted infection; T3P®, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide solution; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate. References 1.

World Health Organisation. Global incidence and prevalence of selected curable sexually

transmitted infections - 2008; Geneva, Switzerland, 2012; p 20. 2.

Burton, M. J.; Mabey, D. C. The global burden of trachoma: a review. PLoS Negl. Trop. Dis.

2009, 3, e460. 3.

Haggerty, C. L.; Gottlieb, S. L.; Taylor, B. D.; Low, N.; Xu, F.; Ness, R. B. Risk of sequelae

after Chlamydia trachomatis genital infection in women. J. Infect. Dis. 2010, 201, S134-S155. 4.

Ceovic, R.; Gulin, S. J. Lymphogranuloma venereum: diagnostic and treatment challenges.

Infect. Drug. Resist. 2015, 8, 39-47.


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5.


AbdelRahman, Y. M.; Belland, R. J. The chlamydial developmental cycle. FEMS Microbiol.

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Beeckman, D. S.; De Puysseleyr, L.; De Puysseleyr, K.; Vanrompay, D. Chlamydial biology and

its associated virulence blockers. Crit. Rev. Microbiol. 2014, 40, 313-328. 7.

Hammerschlag, M. R.; Kohlhoff, S. A. Treatment of chlamydial infections. Expert Opin.

Pharmacother. 2012, 13, 545-552. 8.

Dethlefsen, L.; Relman, D. A. Incomplete recovery and individualized responses of the human

distal gut microbiota to repeated antibiotic perturbation. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 Suppl 1, 4554-61. 9.

Jernberg, C.; Löfmark, S.; Edlund, C.; Jansson, J. K. Long-term impacts of antibiotic exposure

on the human intestinal microbiota. Microbiology 2010, 156, 3216-23. 10.

Vincent, C.; Manges, A. R. Antimicrobial use, human gut microbiota and Clostridium difficile

colonization and infection. Antibiotics (Basel, Switz.) 2015, 4, 230-253. 11.

Engström, P.; Krishnan, K. S.; Ngyuen, B. D.; Chorell, E.; Normark, J.; Silver, J.; Bastidas, R. J.;

Welch, M. D.; Hultgren, S. J.; Wolf-Watz, H.; Valdivia, R. H.; Almqvist, F.; Bergström, S. A 2pyridone-amide inhibitor targets the glucose metabolism pathway of Chlamydia trachomatis. mBio 2015, 6, e02304-14. 12.

Engström, P.; Bergström, M.; Alfaro, A. C.; Krishnan, K. S.; Bahnan, W.; Almqvist, F.;

Bergström, S. Expansion of the Chlamydia trachomatis inclusion does not require bacterial replication. Int. J. Med. Microbiol. 2015, 305, 378-382. 13.

Schwöppe, C.; Winkler, H. H.; Neuhaus, H. E. Properties of the glucose-6-phosphate transporter

from Chlamydia pneumoniae (HPTcp) and the glucose-6-phosphate sensor from Escherichia coli (UhpC). J. Bacteriol. 2002, 184, 2108-15.



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14.

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Stephens, R. S.; Kalman, S.; Lammel, C.; Fan, J.; Marathe, R.; Aravind, L.; Mitchell, W.;

Olinger, L.; Tatusov, R. L.; Zhao, Q.; Koonin, E. V.; Davis, R. W. Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 1998, 282, 754-759. 15.

Iliffe-Lee, E. R.; McClarty, G. Regulation of carbon metabolism in Chlamydia trachomatis. Mol.

Microbiol. 2000, 38, 20-30. 16.

Nicholson, T. L.; Chiu, K.; Stephens, R. S. Chlamydia trachomatis lacks an adaptive response to

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Figure Legends Figure 1: Structure of compound 1 and schematic of SAR questions addressed in this study. Figure 2: The fluorescent amide 37 localizes to the bacterial inclusion. Compound 37 (top row) or a BODIPY control 38 (bottom row) were added to infected HeLa cells at the beginning of the experiment (1 µM each) and were not removed until 48 hpi when the cells were washed with PBS (3×), fixed and prepared for microscopic imaging. The red channel in this image shows the major outer membrane



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protein (MOMP) signal which delineates the bacterial cell wall, the green channel shows the BODIPY fluorophore and the merge of both channels with a bright-field image of the same field which highlights the cellular outline and subcellular localization of the signals. The data shown in this figure are confocal micrographs generated using identical imaging and image modification parameters and thus the image intensities are directly comparable across experimental conditions. The data shown here are representative of at least 2 independent experiments.

Figure 3: Co-treatment with compounds 6e or 21a reduces the accumulation of the fluorescent analogue 37 within bacterial inclusions. a) Infected HeLa cells were incubated with 1 µM 37 alone or with 37 alongside equimolar concentrations of 6e or 21a. White arrows point to the Chlamydial inclusions. The cells were then fixed and imaged by confocal microscopy. The images shown were generated using identical imaging and image analysis parameters for all samples, and thus the image intensities are directly comparable across experimental conditions. b) The fluorescent intensity per inclusion was analyzed using ImageJ software and divided by the area of the inclusion. Every data point on the graph indicates the fluorescent intensity per one single inclusion. The fluorescence intensity of 37 was measured in more than 40 inclusions and the data shown in this graph is representative of the results seen in at least 2 independent experiments. * indicates p < 0.05 and thus, statistical significance using a 2 tailed student t-test.

Scheme Legends Scheme 1: General route for the preparation of new amides 6-19. Reagents and conditions: (a) 1 eq. TFA, DCE, MWI at 120 °C, 3 min, 84%; (b) LiOH (1 M aq.), THF, rt, 48 h, 86%; (c) for 6a-f, 8-9, 1119: HNR1R2, propylphosphonic anhydride (50% in EtOAc), pyridine, MeCN/EtOAc (1:1), -10 °C to rt,


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16-48 h; (d) for 7: 2-aminopyridine, TBTU, DIPEA, CH2Cl2, rt, 18 h, 65%; (e) for 10: 4aminopyrimidine, HATU, DIPEA, DMF, MWI at 60 °C , 1 h, 43%. Scheme 2: Functionalization of the C-6 position. Reagents and conditions: (a) oxalyl choride, DMF, MeCN, 0 °C to rt, 10 min; (b) MeCN, reflux, 2 h; (c) aq. NaH2P2O4, DMSO, rt; (d) aq. NaClO2, 0 °C to rt, 2 h, 25% from 6a; (e) NaNO2, TFA, CH2Cl2, O2 atmosphere, rt, 16 h; (f) activated Zn dust, AcOH, rt, 3 h; 21a: 69% from 6a; 21b: 37% from 6e; 21c: 42% from 6l. Scheme 3: Synthesis of C-6 amine analogues. Reagents and conditions: (a) NaNO2, TFA, CH2Cl2, O2 atmosphere, rt, 15 h, 74%; (b) activated Zn dust, AcOH, rt, 20 h, 82%; (c) LiOH (1 M aq.), THF, rt, 17 h, 72%; (d) HNR1R2, HATU, DIPEA, DMF, MWI 60 °C, 1 h. (21d: 42% 25: 36%). Scheme 4: General route for the synthesis of C-7 substituted derivatives via Suzuki-Miyaura cross-coupling. Reagents and conditions: (a) Meldrum’s acid, propylphosphonic anhydride (50% in EtOAc), NEt3, CH2Cl2, rt, 48 h, 95%; (b) (R)-2, TFA, DCE, MWI at 120 °C, 3 min, 96%; (c) R1PhB(OH)2, cat. Pd(PPh3)2Cl2, KF, MeOH, MWI 140 °C, 12 min; (d) LiOH (1 M aq.), THF, rt, 16-48 h; (e) R2-aniline, propylphosphonic anhydride (50% in EtOAc), pyridine, MeCN/EtOAc (1:1),-10 °C to rt, 16-48 h; (f) NaNO2, TFA, CH2Cl2, O2 atmosphere, rt, 16 h; (g) activated Zn dust, AcOH, rt, 3 h; 33a: 76% from 32a; 33b: 27% from 32b. Scheme 5: Enantioselective synthesis of amides (R)-6 and (S)-6.

Reagents and conditions: (a)

Meldrum’s acid 3, 0.2 eq. TFA, DCE, MWI at 120 °C, 2 min 20 sec; (b) LiBr, NEt3, MeCN (2% water v/v) rt, 3 h; (c) TMS-diazomethane, benzene, 30 min, rt; (d) aniline, propylphosphonic anhydride (50% in EtOAc), pyridine, EtOAc, -15 °C, 16 h; (e) aniline, HATU, DIPEA, CH2Cl2, rt, 6 h. Scheme 6: Synthesis of fluorescent analogue 37. Reagents and conditions: (a) LiBr, NEt3, MeCN (2% water v/v) 50 °C, 3 h; (b) 3-methylaniline, HATU, DIPEA, DMF, rt, 16 h, 21% over 2 steps.



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Figures

7

8

S

Lead optimization

2

N

6

O

3

O

1 KSK 120

NH

S C-3 Stereocenter?

N

R O

O

C-6 & C-7 Substituents?

Figure 1

Figure 2


NH C-3 Amide?


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Figure 3


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Schemes

Scheme 1



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S

S

a,b,c,d

N 1

O

6

O

N H

R

HO

N O

e,f

O

O

NH

20

S N

H2N O

O

21a-c

Scheme 2


N H

R1

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Scheme 3



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Scheme 4


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Scheme 5



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Scheme 6


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Table of Contents Graphic


Thiazolino 2-Pyridone Amide Inhibitors of Chlamydia trachomatis

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