Synthesis and Structure-Activity Relationship Studies of C2-Modified Analogs of the Antimycobacterial Natural Product Pyridomycin

Article abstract of DOI:10.1021/acs.jmedchem.9b01457

A series of derivatives of the antimycobacterial natural product pyridomycin have been prepared with the C2 side chain attached to the macrocyclic core structure by a C-C single bond, in place of the synthetically more demanding enol ester double bond found in the natural product. Hydrophobic C2 substituents of sufficient size generally provide for potent anti-Mtb activity of these dihydropyridomycins (minimum inhibitory concentration (MIC) values around 2.5 μM), with several analogs thus approaching the activity of natural pyridomycin. Surprisingly, some of these compounds, in contrast to pyridomycin, are insensitive to overexpression of InhA in Mycobacterium tuberculosis (Mtb). This indicates that their anti-Mtb activity does not critically depend on the inhibition of InhA and that their overall mode of action may differ from that of the original natural product lead.

Full text of DOI:10.1021/acs.jmedchem.9b01457

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Article
Synthesis and Structure-Activity Relationship Studies of C2-Modified
Analogs of the Antimycobacterial Natural Product Pyridomycin
Maryline Kienle, Patrick Eisenring, Barbara Stoessel, Oliver Horlacher, Samuel Hasler, Gwénaëlle
van Colen, Ruben C. Hartkoorn, Anthony Vocat, Stewart Cole, and Karl-Heinz Altmann
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01457 • Publication Date (Web): 06 Jan 2020
Downloaded from pubs.acs.org on January 6, 2020
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Synthesis and Structure-Activity Relationship
Studies of C2-Modified Analogs of the
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Antimycobacterial Natural Product Pyridomycin
†,#
†,#
†,#
†,#
†
Maryline Kienle, Patrick Eisenring, Barbara Stoessel, Oliver P. Horlacher, Samuel Hasler,
†
‡,§
‡
Gwénaëlle van Colen, Ruben C. Hartkoorn, Anthony Vocat,
Stewart T. Cole,‡,% and Karl-Heinz Altmann†*
†ETH Zürich, Department of Chemistry and Applied Biosciences,
Institute of Pharmaceutical Sciences, 8093 Zurich, Switzerland
‡École Polytechnique Fédérale de Lausanne (EPFL), Global Health Institute, 1015 Lausanne,
Switzerland
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ABSTRACT
A series of derivatives of the antimycobacterial natural product pyridomycin have been prepared
with the C2-side chain attached to the macrocyclic core structure by a C-C single bond, in place
of the synthetically more demanding enol ester double bond found in the natural product.
Hydrophobic C2-substituents of sufficient size generally provide for potent anti-Mtb activity of
these dihydropyridomycins (MIC values around 2.5 M), with several analogs thus approaching
the activity of natural pyridomycin. Surprisingly, some of these compounds, in contrast to
pyridomycin, are insensitive to overexpression of InhA in Mtb. This indicates that their anti-Mtb
activity does not critically depend on inhibition of InhA and that their overall mode of action may
differ from that of the original natural product lead.
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INTRODUCTION
Tuberculosis (TB), while often regarded as a disease of the past, as of today is still one of the
major global threats to public health. In 2017, the World Health Organization (WHO) reported an
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estimated 10 million new cases of infection and 1.6 million TB-related deaths. In addition, 1.7
billion individuals globally present an asymptomatic latent tuberculosis infection and have a 5-
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0% lifetime risk of developing active disease. A particular risk for the activation of dormant
bacteria exists for individuals with an impaired (or suppressed) immune system, e. g., as the result
of a human immunodeficiency virus (HIV) infection. However, bacterial activation may also be
triggered by malnutrition, affliction with diabetes or unhealthy lifestyle habits (smoking, alcohol
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abuse). Recently, the emergence of multidrug- and extensively drug-resistant (MDR and XDR,
respectively) strains of the causative pathogen Mycobacterium tuberculosis (Mtb) has become a
serious threat also in industrialized countries.1,2 This development has been exacerbated by the
lack of development of novel anti-TB drugs for several decades; only two new chemical entities
have been approved for TB treatment since the approval of rifampicin in 1968 (Italy; FDA
approval in 1971), namely bedaquiline and delamanid.6,7 Of these, only bedaquiline acts via a
truly novel mechanism of action.4,5 Therefore, in order to eradicate TB and eliminate resistant
strains, the development of improved antimycobacterial agents, ideally equipped with novel
mechanisms of action, is urgently required.
3
4,5
Pyridomycin (1, Figure 1) is a bacterial secondary metabolite that was first isolated in 1953 from
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the Streptomyces strain 6706 (later termed S. pyridomyceticus) by Maeda and co-workers. At the
time of discovery, the compound was shown to exhibit significant antimycobacterial activity and
low systemic toxicity in mice. However, little additional work on the pharmacology of
pyridomycin (1) has been reported for several decades thereafter.
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Figure 1. Structure of pyridomycin (1) and of 2,1’-dihydropyridomycins 2 and 3.
As part of a comprehensive initiative directed towards the discovery of new anti-TB drugs (ERC
FP7 program MM4TB), we recently revisited 1 as a potential lead structure for anti-TB drug
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discovery and we reconfirmed its antimycobacterial activity in vitro (MIC = 0.56 – 1.48 µM). Of
equal importance, we determined the molecular target of pyridomycin (1) as the mycobacterial
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NADH-dependent enoyl-[acyl-carrier-protein] reductase (InhA), which is also the ultimate target
of the clinical TB drug isoniazid (INH). INH acts as a prodrug and requires activation by katG, a
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mycobacterial catalase-peroxidase, to exert its antimycobacterial activity. Resistance to INH
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most often results from mutations in the katG gene rather than in InhA itself. In contrast, we have
shown pyridomycin (1) to be a direct NADH-competitive inhibitor of the reductase; as a
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consequence, most INH-resistant strains remain susceptible to 1, which renders the compound a
promising starting point for TB drug discovery.
The binding mode of 1 to InhA could be determined by X-ray crystallography, which showed
1
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that 1, in contrast to the INH-NADH adduct formed after activation of INH by KatG and other
previously reported direct InhA inhibitors,12–20 blocks both the NADH as well as the lipid binding
2
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site of the enzyme. This binding mode makes pyridomycin unique among InhA inhibitors and
effectively defines a new mode of action.
Only one total synthesis of pyridomycin (1) has been reported in the literature,22,23 with the most
challenging and difficult step being the installment of the exocyclic C2-C1’-double bond of the
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unique enol ester moiety. In order to circumvent this problem, we have investigated analogs of 1
with a single bond between C2 and C1’ which we have termed “dihydropyridomycins”.24
Prototypical examples of this family of pyridomycin analogs are the 2-iso-propyl derivatives 2 and
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(Figure 1), which retain a simple branched C2-side chain. Of these two analogs, the 2R isomer
was only ca. 4-fold less active than 1, while it was 8-fold more potent than its 2S isomer 3 (Figure
). These findings indicated that (a) the enol ester moiety in pyridomycin (1) is not crucial for
antimycobacterial activity, while at the same time (b) highlighting the importance of the
configuration of the newly created stereocenter (in comparison with 1) at C2 for biological activity.
Based on X-ray crystallography, the binding modes of dihydropyridomycin 2 and pyridomycin (1)
to InhA are very similar, with the C2-substituent protruding into a hydrophobic pocket in both
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cases. In light of these findings, we have now prepared a series of new 2R dihydropyridomycin
derivatives with variations in the size and hydrophobicity of the C2-substituent and we have
determined the effects of these variations on antimycobacterial activity. In addition, we have
investigated the importance of the configuration at the C10 and C11 stereocenters of 1 for
biological activity and we have assessed the effect of the replacement of the C12(O)-O1 ester
group of the natural product by an amide moiety (which we thought could lead to improved
metabolic stability).
RESULTS AND DISCUSSION
Chemistry. As outlined in Scheme 1, the synthesis of all pyridomycin analogs was to follow the
24
same overall strategy that we had developed previously for the synthesis of 2 and 3. Thus, analogs
2
8 and 35 were to be obtained by late-stage amide coupling of 3-hydroxypicolinic acid (27) with
the corresponding macrocyclic amines, which would in turn be formed by simultaneous cleavage
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of the benzyl amino and benzyl ester groups by catalytic hydrogenation of amino acids 25 and 34,
respectively, followed by macrolactamization and cleavage of the BOC-protecting group.
Scheme 1. Retrosynthesis of pyridomycin analogs 28 and 35
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Protected amino acids 25 and 34 would result from an esterification or an amide coupling
reaction of acid 24 with alcohols 8 and amine 33, respectively. Acid 24 can be accessed from
2
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aldehyde 20 by means of an anti-aldol reaction, as described previously, while alcohols 8 and
amine 33 were to be prepared by esterification of acids 5, 11, or 32 with N -BOC-Thr-OBn (6).

Synthesis of alcohols 8. The synthesis of alcohols 8a-c,f departed from the commercially
available hydroxy acids 4a-c,f, which were converted into their TBS-ethers 5a-c,f (via the bis-TBS
2
4
derivative of the hydroxy acid and selective in situ cleavage of the TBS-ester group) (Scheme
25
2
). Yamaguchi esterification of partially protected hydroxy acids 5a-c,f with N

-BOC-L-Thr-
OBn (6) (which is commercially available but was synthesized here in two steps from L-Thr
2
4
following literature procedures ) then delivered intermediates 7a-c,f. Finally, TBS-deprotection
with HF•pyridine furnished the desired free alcohols 8a-c,f.
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Scheme 2. Synthesis of alcohols 8a-c,fa
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aReagents and conditions: (a) i. TBSCl, imidazole, DMF, rt, 24 h; ii. K
0 °C to rt, 4 h, 79% to quant.; (b) 2,4,6-trichlorobenzoyl chloride, Et N, toluene, rt, 5 min, then
alcohol 6, DMAP, rt, 18 h, 12-92%; (c) HF·pyridine, THF, 0 °C to rt, 17 h, 82% to quant.
CO
, MeOH/H O (5:2),
2
3
2
3
Alcohols 8d and 8e, which serve as precursors for dihydropyridomycins with an asymmetric
C2-side chain that are the specific formal hydrogenation products of pyridomycin (1), were
obtained in 6 steps from D-Ile (9e) and D-allo-Ile (9d), respectively (Scheme 3). Diazotation of the
amino acids with aq. H
2
SO
4
and NaNO produced -hydroxy acids 4d,e in excellent yields with
2
retention of configuration.26,27
Scheme 3. Synthesis of alcohols 8d,ea
aReagents and conditions: (a) H
quant.; (b) benzyl bromide, Cs CO
DCM, –78 °C, 30 min, then rt, 15 h, 93% and 77%; (d) H
quant. and 81%; (e) 2,4,6-trichlorobenzoyl chloride, Et N, toluene, rt, 5 min, then alcohol 6,
SO
, DMF, 0 °C to rt, 20 h, 94% and 90%; (c) TBSOTf, lutidine,
(5 bar), Pd/C (10%), EtOAc, rt, 1 h,
1M, NaNO
, H O, 0 °C, 19 h, then rt, 15 min, 97% and
2
4
2
2
2
3
2
3
DMAP, rt, 18 h, 92% and 68%; (f) HF·pyridine, THF, 0 °C to rt, 17 h, quant. The first yield given
is for the respective intermediate leading to 8d, the second for the one leading to 8e.
Applying the conditions previously used for the conversion of 4a-c,f into 5a-c,f (excess
TBSCl/imidazole in DMF, then K
2
CO ) failed to deliver the desired TBS-ethers 5d,e. Hydroxy
3
acids 4d,e were thus transformed into the corresponding benzyl esters with BnBr/Cs
2
CO followed
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by reaction with TBSCl; benzyl ester cleavage by catalytic hydrogenation then furnished acids
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d,e. Finally, Yamaguchi esterification of 5d,e with 6 followed by TBS-deprotection gave free
alcohols 8d,e.
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Compared to all other alcohols 8, the synthesis of alcohol 8i required a different protecting group
regime, due to the need for a protecting group on the prospective C2-hydroxymethyl side chain.
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Starting from methyl ester 10, quantitative ester saponification gave carboxylic acid 11, which
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was esterified with N

-BOC-Thr-OBn (6) under Yamaguchi conditions to give 12 in 71% yield
(Scheme 4). Acetal cleavage with p-TsOH in MeOH followed by selective protection of the
primary hydroxy group as a TBS-ether then furnished alcohol 8i in 41% overall yield from 12.
Scheme 4. Synthesis of alcohol 8ia
aReagents and conditions: (a) KOH (aq. 2M), MeOH, rt, 1 h, 99%; (b) 2,4,6-trichlorobenzoyl
chloride, Et N, THF/DMF (2:3), rt, 5 min, then alcohol 6, DMAP, rt, 18 h, 71%; (c) p-TsOH,
3
MeOH, rt, 20 h, 69%; (d) TBSCl, imidazole, DCM, rt, 18 h, 60%.
The syntheses of the two diastereoisomeric alcohols 8g and 8h were more elaborate than those
of all other building blocks 8, as they required the de novo installation of two chiral centers in
precursor acids 5g and 5h. Both syntheses proceeded through epoxide 14 as a common
intermediate that was obtained by enantioselective Sharpless epoxidation of trans-cinnamyl
alcohol (13) in 74% yield.29,30 Epoxide 14 was opened either in an anti-fashion with MeLi, to
31
32
furnish diol 15 in 74% yield as a single diastereoisomer, or in a syn-fashion with Me Al, to
3
provide 17 in 77% yield as an inseparable mixture of diastereoisomers (d.r. = 3.4:1). The diols 15
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and 17 were doubly TBS-protected and the primary silyl-ether was selectively cleaved using
camphorsulfonic acid (CSA). The resulting alcohols 16 and 18 were oxidized to the corresponding
acids 5g and 5h in good yields (94% and 86%, respectively) following a two-stage Swern-Pinnick
protocol.33–35
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Scheme 5. Synthesis of alcohols 8g,ha
aReagents and conditions: (a) L-(+)-DET, TTIP, t-BuOOH, DCM, –20 °C, 16 h, 74%; (b) for 15,
CuCN, MeLi, Et
2
O, –78 °C to 0 °C, 3 h, 74%; for 17, Me Al, toluene, 0 °C, 2 h, 77% (3.4:1 mixture
3
of diastereoisomers); (c) TBSCl, imidazole, DMF, 0 °C to rt, 12-15 h, 93% and 88% (7.73:1
mixture of diastereoisomers); (d) CSA, DCM/MeOH 1:1, 0 °C, 3-5 h, 60% and 48%; (e) i.
(
COCl)
2
, DMSO, Et
3
N, DCM, –78 °C to rt; ii. NaClO
2
, NaH
2
PO
4
·H
2
O, t-BuOH/THF/H O
2
(4.5:3:1), rt, 2 h, 94% and 86%; (f) 6, 2,4,6-trichlorobenzoyl chloride, Et
3
N, DMAP, toluene, 0 °C
to rt, 2 h, 75% and 60%; (g) HF·pyridine, THF, pyridine, 0 °C to rt, 16 h, 73% and 77%. The first
yield is for the respective intermediate leading to 8g, the second for the one leading to 8h.
2
5
Yamaguchi esterification of 5g,h with 6 followed by TBS-removal with HF•pyridine finally
furnished the desired alcohols 8g and 8h in 73% and 77% yield, respectively. Importantly, while
the undesired anti isomer could not be completely removed from the various intermediates en
route from 17 to 8h (ca. 5-10% of the anti product present), 8h was obtained as a single
diastereoisomer.
Synthesis of acids 24 and bis-epi-24. Acid 24 was synthesized from aldehyde 20 and chiral
3
6
24
ester 22 via a Masamune anti-aldol reaction as previously described (Scheme 6). The reaction
gave a separable mixture of diastereomeric aldol products in a 2:1 ratio, from which the desired
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product 23 and bis-epi-23 were isolated as pure isomers in 45% and 18% yield, respectively. The
2
4
configuration of 23 was previously established by X-crystallography of a derived lactam; in this
work, we have also obtained an X-ray crystal structure of the minor diastereoisomer, which
confirmed its structure as bis-epi-23 (see Supporting Information). Finally, base-mediated ester
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hydrolysis with LiOH in MeOH/THF/H
respectively, in high yields.
2
O gave the free carboxylic acids 24 and bis-epi-24,
Scheme 6. Synthesis of acids 24 and bis-epi-24a
aReagents and conditions: (a) c-Hex
LiOH, MeOH/H O/THF (3:2:2), rt, 24 h, 83-96%.
BOTf, Et N, DCM, –78 °C, 45% (23)/18% (bis-epi-23); (b)
2
3
2
Synthesis of analogs 28a-g,i: Analogs 28a-g,i were all synthesized according to the general
2
5
strategy outlined in Scheme 1. Thus, Yamaguchi esterification of acid 24 with alcohols 8a-g,i at
2
4
low temperature (–78 °C to –35 °C) provided 25a-g,i in low to moderate yields (Scheme 7).
Simultaneous removal of all benzyl-protecting groups by catalytic hydrogenation provided the
37
corresponding free amino acids, which were submitted to macrolactamization with HATU and
DIPEA in DCM/DMF (100:1) at high dilution to deliver macrocycles 26a-g,i in yields between
23% and 62%. The exocyclic amino group was then deprotected with TFA (in the case of 26i, this
resulted in concomitant cleavage of the TBS-ether in the C2-substituent) and the free amines were
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coupled to 3-HPA (27) with HATU, furnishing the targeted pyridomycin analogs 28a-g,i in
yields between 30% and 65% over two steps. Importantly, an X-ray crystal structure of 26c could
be obtained and unequivocally confirmed the absolute configuration of the compound (see
Supporting Information).
1
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Scheme 7. Synthesis of C2-modified pyridomycin analogs 28a-g,ia
aReagents and conditions: (a) 2,4,6-trichlorobenzoyl chloride, Et
35 °C, 43 h, then 0 °C, 2 h, 18-72%; (b) H (1 bar), Pd/C, MeOH, rt, 5 h; (c) HATU, DIPEA,
DCM/DMF, rt, 18 h, 23-62% over 2 steps; (d) TFA, DCM, 0 °C, 10 min, then rt, 2 h; (e) 3-HPA
27), HATU, DIPEA, MeCN, rt, 18 h, 30-65% over 2 steps.
N, DMAP, toluene, –78 °C to
3
–
2
(
2
5
Synthesis of analog 28h. Unfortunately, the attempted Yamaguchi esterification of acid 24
with alcohol 8h failed to deliver the desired ester; and the same was true for other esterification
methods investigated. As an alternative to acid 24, we chose to investigate the esterification of
alcohol 8h with acid 29 (Scheme 8), which was obtained in 12 steps from D-glucose following a
3
8
route that had been described previously by Kinoshita and co-workers as part of their total
synthesis of pyridomycin (1) (with some of the steps requiring optimization; see Supporting
Information).
Gratifyingly, acid 29 could be successfully esterified with alcohol 8h under Steglich conditions
3
9
(DCC, DMAP) to deliver ester 30 in high yield (78%). The formyl protecting group was then
removed with KHCO
3
in MeOH, delivering ester 31 in 89% yield. Subsequent catalytic
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hydrogenation led to concomitant benzyl-removal and azide reduction, furnishing the
corresponding amino acid. HATU-mediated macrolactamization followed by BOC-removal with
TFA and HATU-mediated coupling of the ensuing macrocyclic amine with 3-HPA (27) finally
furnished analog 28h.
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Scheme 8. Synthesis of C2-modified pyridomycin analog 28ha
aReagents and conditions: (a) 8h, DCC, DMAP, DCM, 0 °C to rt, 5 h, 78%; (b) KHCO
, MeOH,
3
rt, 1 h, 89%; (c) Pd/C, MeOH, rt, 1.5 h; (d) HATU, DIPEA, DCM, DMF, rt, 16 h, 31% over 2
steps; (e) TFA, DCM, 3 h; (f) 3-HPA (27), HATU, DIPEA, DMF, 16 h, 43% over 2 steps.
Synthesis of analog 35. Instead of protected hydroxy acids 5 or 11, the synthesis of analog 35
 
involved the esterification of N -BOC-Thr-OBn (6) with N -Fmoc-D-Ile (32) (Scheme 9).
Scheme 9. Synthesis of pyridomycin analog 35a
aReagents and conditions: (a) 2,4,6-trichlorobenzoyl chloride, Et
, DMAP, toluene, rt, 1 h, 98%; (b) piperidine, MeCN, rt, 30 min, 64%; (c) 24, HATU, DIPEA,
MeCN/THF (10:1), 0 °C to rt, 48 h, 75%; (d) H (1 atm), Pd/C (10%), MeOH, rt, 2.75 h, 96%
crude); (e) HATU, DIPEA, DCM, DMF (1%), rt, 18 h, 83%; (f) TFA, DCM, rt, 3 h, quant. (crude);
3-HPA (27), HATU, DIPEA, DMF, rt, 18 h, 25%.
N, THF, rt, 5 min, then alcohol
3
6
2
(
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25
The reaction was again conducted under Yamaguchi conditions and furnished the desired ester
in excellent yield (98%). Fmoc-cleavage with piperidine then gave the free amine 33. HATU-
mediated coupling of the latter with acid 24 delivered amide 34 in 75% yield. Global debenzylation
with hydrogen over Pd/C and subsequent macrolactamization with HATU and DIPEA under the
previously established conditions furnished the protected macrocycle in high yield. Cleavage of
the BOC-group and coupling of the free amine with 3-HPA (27) (HATU/DIPEA) finally gave
analog 35 in 20% yield for the four-step sequence from 34 (after RP-HPLC purification).
1
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Synthesis of analogs 39 and 40. Given the availability of significant amounts of acid bis-epi-
2
4 from a number of runs of the aldol reaction between 20 and 22, we also pursued the synthesis
of dihydropyridomicin analogs 39 and 40 with inverted configurations at the C10 and C11
stereocenters (pyridomycin numbering; Scheme 10), in order to gain information on the effect of
these stereochemical changes on antimycobacterial activity. The synthesis of these analogs from
bis-epi-24 and alcohols 8c and 36, respectively, followed the same strategy as for analogs 28a-i
(Scheme 10).
Scheme 10. Synthesis of analogs 39 and 40 with inverted configurations at C10 and C11a
aReagents and conditions: (a) 2,4,6-trichlorobenzoyl chloride, Et
35 °C, 43 h, then 0 °C, 2 h, 51-52%; (b) H (1 bar), Pd/C, MeOH, rt, 5 h; (c) HATU, DIPEA,
DCM/DMF, rt, 18 h, 33-42% over 2 steps; (d) TFA, DCM, 0 °C, 10 min, then rt, 2 h; (e) 3-HPA
N, DMAP, toluene, –78 °C to
3
–
2
(27), HATU, DIPEA, MeCN, rt, 18 h, 37-51% over 2 steps.
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Antimycobacterial Activity and InhA Inhibition. Pyridomycin analogs 28a-i, 35, 39, and 40
were assessed for their antibacterial activity against Mtb strain H37Rv. For those compounds that
displayed significant antimycobacterial activity, their IC50 for inhibition of InhA (S94A) was also
determined with either 2-trans-octenoyl-CoA or 2-trans-dodecenoyl-CoA as the substrate.
As illustrated by the MIC values summarized in Table 1, all dihydropyridomycins with 2-
substituents bulkier than methyl display good antimycobacterial activity, which in several cases
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(28b-h) surpasses the activity of our initial dihydropyridomycin 2 and thus begins to approach the
activity of pyridomycin (1) itself. In contrast, C2-methyl analog 28a is significantly less active
than ethyl derivative 28b, while the presence of a polar side chain as in 28i results in a >150-fold
loss in potency. Although we cannot rule out the possibility that the observed loss of activity for
2
8i results from a decreased ability of the compound to penetrate the mycobacterial cell wall, the
data are in general agreement with the structural data for the complexes between InhA and 1 or 2,
2
1
respectively; in these structures, the C2-substituent protrudes into a hydrophobic pocket. At the
same time, it is still surprising that a relatively small substituent such as ethyl essentially produces
the same activity as the bulkier and more hydrophobic substituents present in 28c-h which would
be expected to engage in more extensive hydrophobic interactions with the hydrophobic protein
cavity. As for asymmetric C2-side chains as in 28d/e and 28g/h, the R diastereoisomer (28d) is
found to be more potent for the 28d/e pair, while no difference is observed between 28g and 28h.
However, even the activity difference between 28d and 28e is small and may not be biologically
significant. Likewise, no meaningful activity difference is observed between C2-benzyl derivative
2
8f and the branched variants 28g/h.
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Table 1. Antimycobacterial and InhA-inhibitory activity of pyridomycin (1) and of
dihydropyridomycins 2, 3, 28a-i, 35, 39 and 40
Relative MIC
MIC/MIC(1))b
IC InhA (S94A)
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0
a
Compound
MIC (M)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
(
(M)
Pyridomycin (1)
0.55 – 3.05
3.2
-
3.43 ± 0.60
32.8 ± 9.20
NMd
2
3
4
26.0
49.9
4.9
32
15
2
NDe
2
8a
8b
28c
8d
8e
28f
8g
8h
28i
2
127 ± 2.31
11.1 ± 0.703
31.5 ± 8.61
87.2 ± 23.4
29.12 ± 1.93
1.3
1.7
2
2
1.5
2
2.9
3.9
2
4.3
NDe
NDe
NDe
NDe
2
2.6
2.3
2.3
167
250
83
148
2
2.6
96.8
184.6
94.6
135.8
3
3
5
9
NDe
40
291 ± 2.42
a
Minimal inhibitory concentrations (MIC) were measured against Mtb strain H37Rv using the
4
0
resazurin method. Data are average values from at least duplicate experiments. MIC’s were
determined in Middlebrook 7H9 broth (Difco) supplemented with 0.2% glycerol, 0.05% Tween 80
and 10% albumin-dextrose-catalase (ADC), which is routinely used in the screening for and testing of
new antimycobacterial agents. The stability of pyridomycin (1) in this medium after 7 days was found
to be >50% (for an initial concentration of 10 g/mL). Some of the analogs 28 were also tested in the
absence of Tween 80, which led to an increase in MIC of 2-4 fold (see Supporting Information). The
exact reasons for these changes are unclear, but it appears conceivable that Tween 80 enhances the
penetration of pyridomycin and pyridomycin analogs into Mtb by disrupting the outer capsule of
41
the bacterial cell envelope. The differences in MIC’s between experiments in the presence or
absence of Tween 80 do not affect the general SAR conclusions derived from the data shown in the
b
Table 1. The relative MIC is defined as the ratio of the MIC’s of the respective analog and the
c
pyridomycin (1) control in the same experiment. Half maximal inhibitory concentrations (IC50
)
were measured against InhA (S94A) with 2-trans-octenoyl-CoA or 2-trans-dodecenoyl-CoA as
9
the substrate. For experimental details cf. ref. Data are average values from experiments carried
d
e
out in triplicates ± SD . NM, no activity detectable up to a concentration of 75 μM. ND, no values
determined.
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Unfortunately, the substitution of an ester for an amide moiety in analog 35 resulted in the
complete loss of antimycobacterial activity. This may be attributed to an intolerable
conformational change of the macrocycle or to unfavorable interactions of the protein with the
more polar amide group. Finally, analogs 39 and 40 did not exhibit any antimycobacterial activity,
which highlights the importance of the natural configurations of the stereocenters at C10 and C11
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(although we cannot exclude that changing only one of these stereocenters, rather than both
simultaneously, would have a less pronounced effect).
The data in Table 1 also show that there is no clear correlation between the antimycobacterial
activity of pyridomycin analogs 28 and the inhibitory activity against their purported target enzyme
InhA in vitro (where determined). There is no straightforward explanation for this discrepancy,
but the data might indicate that at least some of our dihydropyridomycins could exhibit additional
activity(ies) that are unrelated to InhA, perhaps by targeting other short chain enoyl reductase(s)
in Mtb that is (are) different from but still similar to InhA.42,43 To address this question, MIC values
were determined for a group of compounds against the wild type H37Rv strain as well as the
4
4
corresponding InhA-overexpressing H37Rv::pMVinhA strain (Table 2). Pyridomycin (1) and
isoniazid (INH) were used as positive controls, and both compound showed significantly decreased
activity against the InhA-overexpressing strain (ca. 16- and 31-fold reduction in MIC,
respectively). A similar decrease in activity was also observed for analogs 28b,f, thus indicating
that they, too, target InhA. In contrast, and quite intriguingly, 28c,e retained essentially full
antimycobacterial activity against H37Rv::pMVinhA. Thus, while 28c,e show inhibition of InhA
in a biochemical assay, their antibacterial activity appears not to depend critically on inhibition of
bacterial InhA and they act through (an)other mechanism(s) of action that is (are) unaffected by
the overexpression of InhA.
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Table 2. Antimycobacterial activity of selected analogs and INH against wild type H37Rv and
against InhA-overexpressing H37Rv::pMVinhA44
MIC
MIC
Resistance impact
fold MIC increase)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
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0
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Compound
H37Rv::pMV261
H37Rv::pMVinhA
(
a
a
(
M)
0.74
(M)
Pyridomycin (1)
11.56
16
8
2
3.1
4.9
0.7
2.9
4.3
1.2
24.2
>19.1b
0.7
>4
1
2
8b
8c
28e
8f
Isoniazid (INH)
2
2.9
>17.3b
1
2
>4
31
37.3
a
Minimal inhibitory concentrations (MIC) were measured against Mtb strain H37Rv using the
9,40
a
resazurin method
under the conditions detailed in footnote of Table 1. H37Rv::pMVinhA
refers to the Mtb strain H37Rv containing the pMV261::inhA plasmid; H37Rv::pMV261 refers to
4
4 b
H37Rv transfected with the empty plasmid vector pMV261. Highest concentration tested.
Experiments aimed at identifying possible additional targets of these compounds are currently
ongoing, but so far have not yielded any conclusive results. Independent of this, the data in Table
2
clearly demonstrate that rather subtle changes in the structure of the C2-substituent in
dihydropyridomycins can completely overcome drug resistance due to overexpression of InhA.
CONCLUSION
In summary, we have achieved the synthesis of 12 new 2,1’-dihydropyridomycins and we have
determined their antimycobacterial activities and, for selected examples, their capacity for
inhibition of InhA. By doing so, we have established the importance of the natural configuration
of the C10 and C11 stereocenters and the significance of the C12(O)-O1 ester linkage for
antibacterial activity. Most importantly, we have identified several new dihydropyridomycins with
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1
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2
2
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2
2
2
2
2
2
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antimycobacterial activities starting to approach the activity of the lead natural product. At the
same time, the (cellular) SAR for lipophilic C2-substituents larger than methyl is surprisingly flat.
It remains to be seen if further modifications of the most potent analogs, for example the
attachment of substituents to the phenyl part of analogs 28f or 28g,h could lead to enhanced
activity. In addition, modifications in the hydroxypicolinic acid part of the
pyridomycin/dihydropyridomycin structure will be required to enhance metabolic stability, in
order to produce analogs suitable for eventual in vivo applications. These questions will be
addressed in a forthcoming publication.
0
1
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EXPERIMENTAL SECTION
General Information. All manipulations were conducted under an argon atmosphere using
flame-dried glassware and standard syringe/septa and Schlenk techniques. Absolute solvents were
purchased from Fluka (absolute over molecular sieves). Commercial chemicals were used without
further purification. Solvents for extractions, flash column chromatography (FC) and thin layer
chromatography (TLC) were purchased as commercial grade and distilled prior to use. TLC was
performed on Merck TLC aluminum sheets (silica gel 60 F254). Spots were visualized with UV
light (λ = 254 nm) or through staining with Ce
2
(SO
4
)
3
/phosphomolybdic acid/H
2
SO (CPS) or
4
KMnO /K CO . FC was performed using Fluka silica gel 60 for preparative column
4
2
3
chromatography (particle size 40-63 μm). NMR spectra were recorded on a Bruker Avance 400
MHz or 500 MHz NMR spectrometer at 300 K. Chemical shifts (δ) are reported in ppm and are
1
referenced to the solvent signal as an internal standard (CDCl
3
δ 7.26 ppm for H and δ 77.00 ppm
1
3
1
13
for C spectra; CD
3
OD δ 3.31 ppm for H and δ 49.00 ppm for C spectra; (CD
3
2
) SO δ 2.50 ppm
1
13
for H and δ 39.43 ppm for C spectra). Data are reported as follows: s = singlet, d = doublet, t =
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triplet, q = quartet, quint = quintet, sext = sextet, m = multiplet, br. = broad signal, J = coupling
constant in Hz. The multiplicity of signals is reported based on appearance (i.e. doublet of doublets
1
3
that are apparent triplets are described as triplets). All C-NMR spectra were measured with
1
13
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1
1
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2
2
2
2
2
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0
complete proton decoupling. H- and C-signals were assigned using two-dimensional correlation
experiments (COSY, HSQC, HMBC). IR spectra were recorded on a Jasco FT/IR-6200 instrument
on thin films. Optical rotations were measured on a Jasco P-1020 polarimeter operating at the
sodium D line (λ = 589 nm) and are reported as follows: [α]
T, concentration (c in g/100 mL) and
D
solvent. Melting points were obtained in open capillary tubes using a Büchi melting point
apparatus B-540 and are uncorrected. Mass spectra were recorded by the MS service of the
Laboratory of Organic Chemistry (LOC) of the ETH Zürich; HR-MS (ESI) spectra were measured
on a Bruker Daltonics maxis (UHR-TOF) and HR-MS (EI) on a Waters Micromass AutoSpec
Ultima instrument. The purity of final products submitted for biological testing was determined
by analytical RP-HPLC to be ≥95%. (For HPLC traces, see the Supporting Information). As the
sole exception, HPLC analysis was not performed on compound 28d; the purity of this compound
1
was estimated to be >90% based on the H-NMR spectrum included in the Supporting Information.
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(
2R,3R)-2-Hydroxy-3-methylpentanoic acid (4d)
0
1
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To a solution of D-Ile (9d) (500 mg, 3.81 mmol, 1.00 eq.) in 1M sulfuric acid (10 mL) was added
a solution of NaNO (2.10 g, 30.5 mmol, 8.00 eq) in water (10 mL) dropwise over 4 h a 0 °C. The
2
reaction mixture was stirred at 0 °C for 15 h, allowed to warm to rt, and stirred for further 15 min.
The reaction was quenched by adding NaCl until saturation of the solution. The aqueous layer was
extracted with EtOAc (3 x 30 mL), and the combined organic layers were washed with water (2 x
1
0 mL) and with brine (2 x 10 mL), dried over MgSO , filtered, and concentrated in vacuo to
4
deliver 4d (489 mg, 97%) as a yellow oil. The crude product was used in the following step without
= 0.17. [α] 20
= +12.6° (c 0.70, CHCl ).
further purification. TLC (SiO
2
, DCM/MeOH 9:1, UV): R
f
D
3
1H-NMR (400 MHz, CDCl
): δ (ppm) = 4.18 (d, J = 3.6 Hz, 1H), 1.94 – 1.85 (m, 1H), 1.49 – 1.39
3
1
3
(
m, 1H), 1.36 – 1.24 (m, 1H), 1.03 (d, J = 6.9 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H). C-NMR (101
-
1
MHz, CDCl ): δ (ppm) = 179.0, 74.8, 39.1, 23.8, 15.5, 11.9. IR (thin film): ʋ (cm ) = 3449, 2965,
3
2937, 2879, 1718, 1646, 1461, 1382, 1273, 1242, 1216, 1169.13, 1135, 1071, 1045, 1017. HR-
+
MS (ESI): Calcd. for C
6
H
13
O
3
[M+H] 133.0859; found 133.0859 m/z.
Benzyl (2R,3R)-2-hydroxy-3-methylpentanoate (4dA)
To a solution of 4d (453 mg, 3.43 mmol, 1.00 eq.) in DMF (20 mL) was added benzyl bromide
(
448 μL, 3.77 mmol, 1.10 eq.) and Cs
2
CO (581 mg, 1.78 mmol, 0.52 eq.) at 0 °C. The white
3
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suspension was vigorously stirred at rt for 20 h. H
2
O (200 mL) was added and the mixture was
extracted with Et
2
O (3 x 200 mL). The combined organic layers were washed with water (2 x
20 mL), dried over MgSO , filtered, and concentrated in vacuo to deliver 4dA (716 mg, 94%) as
4
1
1
1
1
1
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5
5
6
0
1
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7
8
9
0
1
2
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8
9
0
1
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8
9
0
a yellow oil. The crude product was used in the following step without further purification. TLC
= 0.48. [α] 20
= +3.3° (c 0.96, CHCl
). H-NMR (400 MHz,
1
(SiO
2
, hexane/EtOAc 7:3, UV): R
f
D
3
CDCl
3
): δ (ppm) = 7.41 – 7.28 (m, 5H), 5.22 (dd, J = 19.5, 12.1 Hz, 2H), 4.12 (dd, J = 5.9, 3.7 Hz,
1
H), 2.68 (d, J = 6.1 Hz, 1H), 1.88 – 1.74 (m, 1H), 1.38 – 1.27 (m, 1H), 1.27 – 1.16 (m, 1H), 0.97
13
(
d, J = 6.9 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H). C-NMR (101 MHz, CDCl
3
): δ (ppm) = 175.0, 135.3,
-1
1
28.8, 128.7, 128.6, 75.0, 67.4, 39.3, 23.8, 15.6, 11.9. IR (thin film): ʋ (cm ) = 3501, 2964, 2935,
2877, 1729, 1498, 1455, 1379, 1266, 1214, 1135, 1072, 1045, 963, 749, 697. HR-MS (ESI): Calcd.
+
for C13
H18NaO
3
[M+Na] 245.1148 m/z; found 245.1154 m/z.
Benzyl (2R,3R)-2-((tert-butyldimethylsilyl)oxy)-3-methylpentanoate (4dB)
A solution of 4dA (700 mg, 3.15 mmol, 1.00 eq.) and 2,6-lutidine (880 µL, 7.56 mmol, 2.40 eq.)
in DCM (32 mL) was cooled to −78 °C. TBSOTf (868 µL, 3.78 mmol, 1.20 eq.) was added
dropwise over 15 min. The mixture was stirred for 30 min at −78 °C, was slowly allowed to warm
to 0 °C and stirred at rt for 15 h. The solution was quenched by the addition of sat. aq. NaHCO
diluted with Et O (15 mL each), and extracted with Et O (3 x 10 mL). The combined organic layers
were dried over MgSO , filtered, and concentrated in vacuo. FC (hexane/EtOAc 9:1 to 6:1 to 4:1)
provided 4dB (987 mg, 93%) as a colorless oil. TLC (SiO , hexane/EtOAc 7:3, UV): R = 0.79.
). H-NMR (400 MHz, CDCl ): δ (ppm) = 7.40 – 7.28 (m, 5H),
3
,
2
2
4
2
f
2
0
1
[α]
D
= +33.6° (c 0.71, CHCl
3
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5
.15 (dd, J = 19.8, 12.2 Hz, 2H), 4.05 (d, J = 5.0 Hz, 1H), 1.82 (m, 1H), 1.50 – 1.40 (m, 1H), 1.24
1.12 (m, 1H), 0.89 (d, J = 8.3 Hz, 3H overlapping s, 9H), 0.85 (t, J = 7.9 Hz, 3H), 0.01 (s, 3H),
–
13
0.00 (s, 3H). C-NMR (101 MHz, CDCl ): δ (ppm) = 173.5, 135.9, 128.6, 128.4, 76.7, 66.4, 39.6,
3
-1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
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7
8
9
0
1
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3
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0
2
1
5.9, 24.1, 18.4, 15.60, 11.6, -4.8, -5.2. IR (thin film): ʋ (cm ) = 3026, 2959, 2930, 2880, 2857,
752, 1731, 1461, 1380, 1252, 1172, 1139, 1069, 1005, 968, 860, 836. HR-MS (ESI): Calcd. for
+
C
19
H32NaO
3
Si [M+Na] 359.2013 m/z; found 359.2020 m/z.
(2R,3S)-2-Hydroxy-3-methylpentanoic acid (4e)
4e was prepared from D-allo-Ile (9e) according to the procedure described for 4d. 4e (568 mg,
quant.) was obtained as a yellow oil. The crude product was used in the following step without
= 0.08. [α] 20
= +7.4 ° (c 0.35, CHCl ).
further purification. TLC (SiO
2
, DCM/MeOH 9:1, UV): R
f
D
3
1H-NMR (400 MHz, CDCl
): δ (ppm) = 4.29 (d, J = 2.0 Hz, 1H), 1.93 – 1.86 (m, 1H), 1.62 – 1.50
3
1
3
(m, 1H), 1.41 – 1.31 (m, 1H), 0.97 (t, J = 7.4 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H). C-NMR (101
-1
MHz, CDCl
3
): δ (ppm) = 180.0, 73.0, 38.5, 26.2, 13.3, 11.9. IR (thin film): ʋ (cm ) = 3521, 2965,
2
924, 2863, 1726, 1464, 1373, 1251, 1201, 1145. HR-MS (ESI): Calcd. for C
6
H11Na
2
3
O [M-
+
H+2Na] 177.0498; found 177.0496 m/z.
Benzyl (2R,3S)-2-hydroxy-3-methylpentanoate (4eA)
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eA was prepared from 4e according to the procedure described for 4dA. 4eA (829 mg, 90%)
was obtained as a yellow oil. The crude product was used in the following step without further
= 0.50. [α] 20
= +4.3° (c 0.81, CHCl
). H-
1
purification. TLC (SiO
2
, hexane/EtOAc 7:3, UV): R
f
D
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR (400 MHz, CDCl
3
): δ (ppm) = 7.40 – 7.34 (m, 5H), 5.22 (dd, J = 17.4, 12.3 Hz, 2H), 4.23
(
dd, J = 5.8, 2.9 Hz, 1H), 2.65 (d, J = 5.8 Hz, 1H), 1.83 (ddd, J = 14.2, 7.0, 2.8 Hz, 1H), 1.61 –
1
3
1
.46 (m, 1H), 1.58 – 1.47 (m, 1H), 0.94 (t, J = 7.4 Hz, 3H), 0.78 (d, J = 6.8 Hz, 3H). C-NMR
(
101 MHz, CDCl
3
): δ (ppm) = 175.5, 135.4, 128.8, 128.7, 128.6, 73.2, 67.5, 38.6, 26.1, 13.3, 12.0.
-1
IR (thin film): ʋ (cm ) = 3537, 3019, 2964, 2935, 2877, 1730, 1456, 1381, 1251, 1214, 1139,
+
1
112, 1048, 753, 668. HR-MS (ESI): Calcd. for C13
H18NaO
3
[M+Na] 245.1148; found 245.1143
m/z.
Benzyl (2R,3S)-2-((tert-butyldimethylsilyl)oxy)-3-methylpentanoate (4eB)
4eB was prepared from 4eA according to the procedure described for 4dB. FC (hexane/EtOAc
9
:1 to 6:1 to 4:1) provided 4eB (956 mg, 77%) as a colorless oil. TLC (SiO
2
, hexane/EtOAc 7:3,
2
0
1
UV): R
f
= 0.80. [α]
D
= +42.9° (c 0.93, CHCl
3
). H-NMR (400 MHz, CDCl ): δ (ppm) = 7.38 –
3
7.30 (m, 5H), 5.15 (dd, J = 14.3, 12.3 Hz, 2H), 4.15 (d, J = 3.7 Hz, 1H), 1.83 – 1.75 (m, 1H), 1.48
1.38 (m, 1H), 1.27 – 1.16 (m, 1H), 0.92 – 0.88 (t, J = 7.4 Hz, 3H overlapping with s, 9H), 0.84
–
1
3
(
d, J = 6.8 Hz, 3H), 0.01 (s, 3H), 0.01 (s, 3H). C-NMR (101 MHz, CDCl
3
): δ (ppm) = 173.9,
1
36.1, 128.7, 128.7, 128.5, 75.3, 66.5, 39.8, 26.3, 26.0, 18.5, 13.9, 12.1, -4.6, -5.2. IR (thin film):
-1
ʋ (cm ) = 2959, 2931, 2881, 2857, 1752, 1461, 1381, 1361, 1252, 1214, 1142, 1072, 836, 752.
+
HR-MS (ESI): Calcd. for C19
H32NaO
3
Si [M+Na] 359.2013 m/z; found 359.2015 m/z.
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(
R)-2-((tert-Butyldimethylsilyl)oxy)propanoic acid (5a)
0
1
2
3
4
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0
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To a stirred solution of D-lactic acid (4a) (513 mg, 5.69 mmol, 1.00 eq.) in DMF (6 mL) at 0 °C
were added imidazole (762 mg, 11.2 mmol, 1.97 eq.) and TBSCl (3.30 g, 21.9 mmol, 3.85 eq.).
The white suspension was stirred at rt for 23 h and diluted with hexane/EtOAc (1:1, 90 mL). The
organic layer was washed with aq. 10% citric acid (50 mL) and H
filtered, and concentrated in vacuo. The crude intermediate (2.29 g) was dissolved in MeOH (40
mL), cooled to 0 °C and a solution of K CO (1.52 g, 11.1 mmol, 1.95 eq.) in H O (12.5 mL) was
2
O (90 mL), dried over MgSO ,
4
2
3
2
added. The reaction mixture was stirred at rt for 4 h, was diluted with DCM (150 mL) and was
acidified to pH = 4 using citric acid (aq. 10%). The aqueous layer was extracted with DCM (3 x
1
50 mL). The combined organic layers were dried over MgSO
4
, filtered, and concentrated in vacuo
to deliver 5a (588 mg, crude) as a slightly brown syrup. 5a (crude) was used in the following step
without further purification. TLC (SiO
2
, hexane/EtOAc 1:3 +0.5% CH
3
COOH, KMnO
4
): R =
f
1
0
.18. H-NMR (400 MHz, CDCl
3
) δ (ppm) = 7.61 (br. s, 1H), 4.35 (q, J = 6.7 Hz, 1H), 1.43 (d, J
=
6.8 Hz, 3H), 0.91 (s, 9H), 0.12 (s, 6H).
(R)-2-((tert-Butyldimethylsilyl)oxy)butanoic acid (5b)
5
b was prepared from (R)-hydroxybutyric acid (4b) according to the procedure described for 5a.
5
b (crude) was used in the following step without further purification. TLC (SiO , hexane/EtOAc,
2
1
4
:1 +1% CH
3
COOH, CPS): R
f
= 0.32. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 4.27 (t, J = 5.0 Hz,
1H), 1.93 – 1.66 (m, 2H), 0.99 – 0.94 (m, 3H), 0.93 (s, 9H), 0.13 (s, 6H).
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(
R)-2-((tert-Butyldimethylsilyl)oxy)-2-cyclohexylacetic acid (5c)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
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3
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4
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4
4
4
4
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
c was prepared from (R)-hexahydromandelic acid (4c) according to the procedure described for
5a. FC (hexane/EtOAc 6:1 to 4:1) provided 5c (547 mg, 79% over 2 steps) as a colorless oil. TLC
20
1
(
SiO
CDCl
(s, 9H), 0.11 (s, 3H), 0.09 (s, 3H). C-NMR (101 MHz, CDCl
2
, DCM/MeOH 95:5, UV): R
f
= 0.50. [α]
D
= +15.3° (c 1.41, CHCl ). H-NMR (400 MHz,
3
3
): δ (ppm) = 4.05 (d, J = 4.0 Hz, 1H), 1.85 – 1.54 (m, 6H), 1.19 (d, J = 86.5 Hz, 5H), 0.93
1
3
3
): δ (ppm) = 175.5, 76.6, 42.5,
-1
2
1
9.2, 27.0, 26.1, 26.0, 26.0, 25.7 (3 C), 18.2, -5.0, -5.2. IR (thin film): ʋ (cm ) = 2929, 2856, 1721,
451, 1254, 1179, 1142, 900, 837, 777, 406. HR-MS (ESI): Calcd. for C14
H
28NaO
Si [M+Na]+
3
295.1700 m/z; found 295.1702 m/z.
(2R,3R)-2-((tert-Butyldimethylsilyl)oxy)-3-methylpentanoic acid (5d)
A solution of 4dB (280 mg, 832 μmol, 1.00 eq.) and Pd/C (10%, 89.0 mg, 83.2 μmol, 0.10 eq.)
in EtOAc (16 mL) was stirred under a H -atmosphere (5 bar). After 1 h, the suspension was filtered
through a plug of celite. The filtrate was evaporated to deliver 5d (207 mg, quant.) as a colorless
2
oil. The crude product was used in the following step without further purification. TLC (SiO ,
2
= 0.50. [α] 20
= +5.4° (c 0.94, CHCl
). H-NMR (400 MHz, CDCl
1
): δ
DCM/MeOH 9:1, UV): R
f
D
3
3
(
ppm) = 4.17 (d, J = 3.8 Hz, 1H), 1.83 – 1.77 (m, 1H), 1.55 – 1.46 (m, 1H), 1.35 – 1.24 (m, 1H),
0
.98 – 0.88 (d, J = 8.2 Hz, 3H, overlapping with t, J = 7.5 Hz, 3H), 0.94 (s, 9H), 0.12 (d, J = 5.1
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3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
Hz, 6H). C-NMR (101 MHz, CDCl ): δ (ppm) = 171.4, 76.3, 40.0, 25.8, 24.3, 15.2, 11.9, −4.9,
3
-1
−
5.1. IR (neat, cm ): 2961, 2932, 2859, 1722, 1461, 1254, 1146, 837, 776. HR-MS (ESI): Calcd.
-
for C12
H
25
O
3
Si [M-H] 245.1578 m/z; found 245.1575 m/z.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2R,3S)-2-((tert-Butyldimethylsilyl)oxy)-3-methylpentanoic acid (5e)
5e was prepared from (4eB) according to the procedure described for 5d. 5e (26.7 mg, 81%) was
obtained as a colorless oil. The crude product was used in the following step without further
= 0.61. [α] 20
= +18.3° (c 1.10, EtOH). H-
1
purification. TLC (SiO
NMR (400 MHz, CDCl
2
, DCM/MeOH 9:1, UV): R
f
D
3
): δ (ppm) = 4.20 (d, J = 3.0 Hz, 1H), 1.88 – 1.74 (m, 1H), 1.57 – 1.42
m, 1H), 1.29 – 1.15 (m, 1H), 0.96 – 0.91 (t, J = 7.0 Hz, 3H, overlapping with d, J = 6.8 Hz,
(
13
3
H),0.95 (s, 9H), 0.12 (d, J = 4.3 Hz, 6H). C-NMR (101 MHz, CDCl ): δ (ppm) = 173.3
3
(
extracted from HMBC), 75.8, 39.9, 25.8, 25.8, 13.7, 12.1, -4.7, -5.1. HR-MS (ESI): Calcd. for
+
C
12
H
27
O
3
Si [M+H] 247.1724 m/z; found 247.1730 m/z.
(
R)-2-((tert-Butyldimethylsilyl)oxy)-3-phenylpropanoic acid (5f)
5
f was prepared from D-(+)-3-phenyllactic acid (4f) according to the procedure described for 5a.
5
f (2.24 g, crude) was used in the following step without further purification. TLC (SiO ,
2
1
hexane/EtOAc, 4:1, UV) : R
f
= 0.31. H-NMR (400 MHz, CDCl
3
) δ (ppm) = 7.33 – 7.19 (m, 5H),
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.42 (dd, J = 7.8, 3.8 Hz, 1H), 3.12 (dd, J = 13.7, 3.7 Hz, 1H), 2.95 (dd, J = 13.7, 7.8 Hz, 1H), 0.85
(s, 9H), -0.06 (s, 3H), -0.17 (s, 3H).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2R,3R)-2-((tert-Butyldimethylsilyl)oxy)-3-phenylbutanoic acid (5g)
To a stirred solution of oxalyl chloride (115 mg, 0.91 mmol, 3.00 eq.) in DCM (1.5 mL) was
slowly added DMSO (0.13 mL, 1.82 mmol, 6.00 eq.) at –78 °C. The mixture was stirred for 10 min
and a solution of 16 (85.0 mg, 0.30 mmol, 1.00 eq.) in DCM (1.5 mL) was added at –78 °C. The
reaction was stirred at –78 °C for 20 min and Et
8 °C. The mixture was allowed to warm to rt (over 20 min). The reaction was quenched by the
addition of brine (2 mL) and was extracted with DCM (3 x 10 mL). The combined organic layers
were dried over MgSO , filtered, and concentrated in vacuo. FC (hexane/EtOAc 5:1) provided the
aldehyde (yield considered as quantitative) as a slight yellow oil. To a solution of the aldehyde
303 µmol, 1.00 eq.) in t-BuOH/THF 4.5:3 (3 mL) and 2-methyl-2-butene (1.61 mL, 15.2 mmol,
0.0 eq.) was sequentially added at rt a solution of NaH PO ·2H O (189 mg, 1.21 mmol, 4.00 eq.)
in H O (0.18 mL) and a solution of NaClO (87.7 mg, 0.97 mmol, 3.20 eq.) in H O (0.18 mL). The
mixture was stirred for 2 h at rt. The reaction was quenched with sat. aq. NaHCO solution (3 mL)
and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried over MgSO
3
N (0.34 mL, 2.42 mmol, 8.00 eq.) was added at –
7
4
(
5
2
4
2
2
2
2
3
4
,
filtered, and concentrated in vacuo. FC (hexane/EtOAc 5:1 to 1:1) provided 5g (84.1 mg, 94%) as
20
a white solid. TLC (SiO
2
; hexane/EtOAc 1:1, UV, CPS): R
f
= 0.39. [α]
D
= +29.0° (c 1.00,
1
CHCl
3
). H-NMR (400 MHz, CDCl
3
): δ (ppm) = 7.32 – 7.20 (m, 5H), 4.31 (dd, J = 4.9, 2.2 Hz,
1
3
1H), 3.26 – 3.14 (m, 1H), 1.38 (d, J = 7.3, 1.5 Hz, 3H), 0.90 (s, 9H), 0.03 (s, 6H). C-NMR (101
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1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
MHz, CDCl
3
): δ (ppm) = 177.2, 128.7, 128.4, 127.3, 77.2, 44.00, 25.8, 17.7, -5.3. IR (thin film):
-1
ʋ (cm ) = 2957, 2930, 2886, 2858, 2359, 1721, 1472, 1463, 1455, 1256, 1146, 1115, 1061, 859,
838, 778, 763, 699, 666, 539, 525, 446, 441, 426, 418, 407. HR-MS (ESI): Calcd. for
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
16
H26NaO
3
Si [M+Na] 317.1543 m/z; found 317.1543 m/z.
(2R,3S)-2-((tert-Butyldimethylsilyl)oxy)-3-phenylbutanoic acid (5h)
5
h was prepared from 18 according to the procedure described for 5g. FC (hexane/EtOAc 3:1)
provided 5h (338 mg, 86%) as a colorless oil. TLC (SiO , hexane/EtOAc 1:1, UV, CPS): R
). H-NMR (400 MHz, CDCl ): δ (ppm) = 7.37 – 7.18 (m, 5H),
.33 (d, J = 3.3 Hz, 1H), 3.34 (qd, J = 7.1, 3.3 Hz, 1H), 1.34 (d, J = 7.1 Hz, 3H), 0.86 (s, 9H), -
2
f
=
20
1
0
4
.45. [α]
D
= +8.0° (c 1.00, CHCl
3
3
1
3
0.15 (s, 3H), -0.36 (s, 3H). C-NMR (101 MHz, CDCl
3
): δ (ppm) = 176.1, 142.0, 128.6, 128.5,
-1
1
1
6
28.3, 127.2, 77.3, 43.3, 25.8, 18.2, 13.2, -5.7. IR (thin film): ʋ (cm ) = 2955, 2929, 2885, 2858,
721, 1472, 1463, 1454, 1362, 1256, 1147, 1114, 1099, 1055, 1011, 939, 887, 857, 837, 778, 761,
+
99, 671. HR-MS (ESI): Calcd. for C16
H26NaO
3
Si [M+Na] 317.1543 m/z; found 317.1543 m/z.
Benzyl N-(tert-butoxycarbonyl)-O-((R)-2-((tert-butyldimethylsilyl)oxy)propanoyl)-L-
threoninate (7a)
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To a stirred solution of crude acid 5a (433 mg, 0.873 mmol, 1.00 eq.) in toluene (20 mL) at rt
were added Et N (0.92 mL, 6.62 mmol, 3.13 eq.) and 2,4,6-trichlorobenzoyl chloride (0.42 mL,
3
2.69 mmol, 1.27 eq.). After 5 min, N-Boc-L-Threonine benzyl ester 6 (691 mg, 2.23 mmol, 1.06
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
eq.) and DMAP (559 mg, 4.58 mmol, 2.16 eq.) were added and the suspension was stirred at rt for
1
8 h. The reaction was quenched with sat. aq. NaHCO
3
(20 mL). The aqueous layer was extracted
, filtered, and
with EtOAc (4 x 50 mL). The combined organic layers were dried over MgSO
4
concentrated in vacuo. FC (hexane/EtOAc 9:1 to 4:1) provided 7a (322 mg, 12% over 2 steps) as
20
a colorless oil. TLC (SiO
2
, hexane/EtOAc 4:1, UV): R
f
= 0.52. [α]
D
= +38.1° (c 0.67, CHCl ).
3
1H-NMR (400 MHz, CDCl
): δ (ppm) = 7.40 – 7.29 (m, 5H), 5.46 (qd, J = 6.3, 2.5 Hz, 1H), 5.17
3
(d, J = 10.0 Hz, 1H), 5.13 (d, J = 12.2 Hz, 1H), 5.09 (d, J = 12.1 Hz, 1H), 4.47 (dd, J = 9.7, 2.4
Hz, 1H), 4.19 (q, J = 6.7 Hz, 1H), 1.46 (s, 9H), 1.30 (d, J = 6.4 Hz, 3H), 1.26 (d, J = 6.7 Hz, 3H),
1
3
0
.90 (s, 9H), 0.08 (s, 3H), 0.04 (s, 3H). C-NMR (101 MHz, CDCl
3
): δ (ppm) = 172.9, 170.1,
156.0, 135.1, 128.8, 128.7, 128.6, 80.4, 71.1, 68.2, 67.8, 57.3, 28.4, 25.8, 21.4, 18.4, 17.1, -4.8, -
-
1
5
1
8
.1. IR (thin film): ʋ (cm ) = 3449, 2980, 2955, 2931, 2890, 2858, 1749, 1720, 1499, 1473, 1456,
381, 1367, 1346, 1314, 1251, 1200, 1159, 1143, 1085, 1063, 1035, 975, 939, 902, 894, 867, 830,
13, 778, 748, 697, 667, 601, 582, 540, 534. HR-MS (ESI): Calcd. for C25
H
41NNaO
Si [M+Na]+
7
518.2544 m/z; found 518.2545 m/z.
Benzyl N-(tert-butoxycarbonyl)-O-((R)-2-((tert-butyldimethylsilyl)oxy)butanoyl)-L-
threoninate (7b)
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