Synthesis, structure, and antibiotic activity of aryl-substituted LpxC inhibitors

Article abstract of DOI:10.1021/jm4007774

The zinc-dependent deacetylase LpxC catalyzes the committed step of lipid A biosynthesis in Gram-negative bacteria and is a validated target for the development of novel antibiotics to combat multidrug-resistant Gram-negative infections. Many potent LpxC inhibitors contain an essential threonyl-hydroxamate headgroup for high-affinity interaction with LpxC. We report the synthesis, antibiotic activity, and structural and enzymatic characterization of novel LpxC inhibitors containing an additional aryl group in the threonyl-hydroxamate moiety, which expands the inhibitor-binding surface in LpxC. These compounds display enhanced potency against LpxC in enzymatic assays and superior antibiotic activity against Francisella novicida in cell culture. The comparison of the antibiotic activities of these compounds against a leaky Escherichia coli strain and the wild-type strain reveals the contribution of the formidable outer-membrane permeability barrier that reduces the compounds efficacy in cell culture and emphasizes the importance of maintaining a balanced hydrophobicity and hydrophilicity profile in developing effective LpxC-targeting antibiotics.

Full text of DOI:10.1021/jm4007774

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Article
Synthesis, structure and antibiotic activity of aryl-substituted LpxC inhibitors
Xiaofei Liang, Chul-Jin Lee, Jinshi Zhao, Eric J Toone, and Pei Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4007774 • Publication Date (Web): 05 Aug 2013
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Synthesis, structure and antibiotic activity of aryl-substituted LpxC inhibitors
Xiaofei Liang, Chul-Jin Lee, ‡,§,� Jinshi Zhao, Eric J. Toone, and Pei Zhou
†
,�
‡
†,‡
†,‡,§,*
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†
Department of Chemistry, Duke University, Durham, NC 27708, USA
‡
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
§
Structural Biology & Biophysics Program, Duke University, Durham, NC 27710, USA


These authors contributed equally to this work.
Keywords: LpxC, antibiotic, lipid A biosynthesis, Gram-negative bacteria
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ABSTRACT
The zinc-dependent deacetylase LpxC catalyzes the committed step of lipid A
biosynthesis in Gram-negative bacteria and is a validated target for development of novel
antibiotics to combat multidrug-resistant Gram-negative infections. Many potent LpxC inhibitors
contain an essential threonyl-hydroxamate head group for high-affinity interaction with LpxC.
We report the synthesis, antibiotic activity, and structural and enzymatic characterization of
novel LpxC inhibitors containing an additional aryl-group in the threonyl-hydroxamate moiety,
which expands the inhibitor-binding surface in LpxC. These compounds display enhanced
potency against LpxC in enzymatic assays and superior antibiotic activity against F. novicida in
cell culture. Comparison of the antibiotic activities of these compounds against a leaky E. coli
strain and the wild-type strain reveals the contribution of the formidable outer membrane
permeability barrier that reduces the compound efficacy in cell culture and emphasizes the
importance of maintaining a balanced hydrophobicity and hydrophilicity profile in developing
effective LpxC-targeting antibiotics.
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INTRODUCTION
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Gram-negative bacterial infections remain as a serious risk factor to public health. As the
rate of multidrug-resistant Gram-negative bacterial infections continues to rise, these infections
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2, 3
are becoming more difficult to treat, resulting in increased mortality and morbidity.
Gram-
negative bacteria differ from Gram-positive bacteria in that they possess a unique outer
membrane, with the outer leaflet of the outer membrane enriched with lipid A, the membrane
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anchor of lipopolysaccharide (LPS) and the active component of bacterial endotoxin. Lipid A
shields bacteria from the damage of antibiotics and detergent molecules, and its biosynthesis is
required for the survival of nearly all Gram-negative organisms. Since the essential lipid A
pathway has never been exploited by existing antibiotics, its biosynthetic enzymes, particularly
LpxC that catalyzes the second and committed step of lipid A biosynthesis, have drawn
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increasing attention as promising novel antibiotic targets. LpxC is a zinc-dependent UDP-3-O-
(
acyl)-N-acetylglucosamine deacetylase. Early studies by Merck researchers have led to the
discovery of the first nanomolar LpxC inhibitor 1 (L-161,240) containing a zinc-binding
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hydroxamate group attached to a phenyl oxazoline scaffold (Figure 1A). This compound shows
impressive antibiotic activities against E. coli in vitro and in vivo, but it is inactive against P.
aeruginosa. In the last decade, tremendous progress in medicinal chemistry has resulted in
steady improvement of the potency and spectrum of inhibition for LpxC-targeting antibiotics.
Among well-characterized LpxC inhibitors, the most potent compound reported to date is the
biphenyl diacetylene-based inhibitor 2 (LPC-011), which inhibits E. coli LpxC with a K of
I
~
0.039 nM and exhibits significant antimicrobial activity against a wide range of Gram-negative
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bacteria (Figure 1A).
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Our previous structural and biochemical characterization of the substrate analog 3 (TU-
14) bound to Aquifex aeolicus LpxC has revealed three conserved features of LpxC-inhibitor
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interactions in addition to the essential hydroxamate-zinc interaction, including the acyl-chain
binding hydrophobic passage, a hydrophobic patch consisting of three phenylalanine residues
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adjacent to the passage, and a basic patch located at the opposite side of the active site.
Subsequent studies of the threonyl-hydroxamate-containing biphenyl-acetylene compound 4
CHIR-090) and biphenyl diacetylene compounds 5 (LPC-009) and 2 (Figure 1A) have further
(
validated the important contributions of these three areas for efficient inhibitor interaction with
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LpxC.
In particular, the biphenyl acetylene and biphenyl diacetylene “tail” groups of 4, 5,
and 2 all insert into the hydrophobic passage, whereas their threonyl methyl group forms vdW
contact with the first phenylalanine (F191 of P. aeruginosa LpxC, PaLpxC) of the hydrophobic
patch, and the hydroxyl group forms a hydrogen bond with a catalytically important lysine
residue (K238 of PaLpxC) of the basic patch (Figure 1B). It is interesting to note that in the
1
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PaLpxC/5 complex, the threonyl group can adopt an additional rotameric state (Figure 1B). In
this alternative conformation, the threonyl methyl group points toward the K238, whereas the
hydroxyl group faces up to form a hydrogen bond with the backbone carbonyl group of F191 of
LpxC, leaving the F191-contacting methyl position unoccupied. The observation of two
rotameric states of the compound 5 threonyl head group reveals the existence of additional space
in the LpxC active site that can be further exploited to expand the inhibitor-LpxC interaction
(
Figure 1B).
Here, we describe the synthesis and biochemical and structural characterization of
compound 2 derivatives containing an aryl group in order to enhance the inhibitor interaction
with the hydrophobic patch of LpxC. The best compound of this series 24c is significantly more
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effective than 2 against Francisella novicida, the bacterium closely related with the category A
Gram-negative pathogen Francisella tularensis. Although 24c possesses an overall similar
antibiotic activity as 2 against wild-type E. coli and P. aeruginosa, it displays much enhanced
activity against a leaky E. coli strain, suggesting that the membrane permeability barrier
negatively affects the penetration of 24c and thus its potency. Detailed enzymatic
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characterization reveals a K value of ~0.024 nM of 24c toward E. coli LpxC (EcLpxC), ~1.6-
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fold improvement over 2. This success demonstrates the feasibility to enhance the LpxC-
inhibitor binding by expanding the interaction of the inhibitor head group with the hydrophobic
patch of LpxC.
CHEMISTRY
Synthesis of 8a began with amide coupling between 4-((4-aminophenyl)buta-1,3-diyn-1-
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yl)benzoic acid 6 and L-histidine methyl ester hydrochloride (Scheme 1). Then the methyl ester
was converted to the corresponding hydroxamic acid 8a by treatment with hydroxylamine under
basic conditions. Compounds 8b, 8c and 8d were synthesized by employing the same procedure.
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2, 13
Intermediate serine aldehyde 14 (Scheme 2)
was obtained from Cbz-L-serine 11.
The oxetane tosylate 10 was prepared using standard conditions as a stable crystalline material
with a 72% yield. Subsequent reaction of Cbz-L-serine with the oxetane tosylate 10, in the
presence of 5% tetrabutylammonium iodide and triethylamine in anhydrous DMF afforded the
desired L-serine oxetane ester 12. The formation of the ortho ester 13 from the oxetane ester 12
was performed in DCM with a catalytic amount of BF ꢀEt O (3 mol%). Finally, oxidation of
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ortho ester 13, under Swern conditions, gave the intermediate serine aldehyde 14.
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Reaction of serine aldehyde 14 with different Grignard reagents led to the corresponding
protected β-hydroxy amino acids 15a-15c (Scheme 3). The reaction was run at -78 °C in a
mixture of DCM/THF or DCM/Et O, resulting in reasonable yields. The β-hydroxy adducts were
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then oxidized under Swern conditions to afford the corresponding ketones 16a-16c in good
yields. The oxidization products were purified by chromatography on silica gel without
racemization. Reduction of the ketone 16a by LiBH at -78 °C regenerated the β-hydroxy amino
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4, 15
acid 19, but with the opposite configuration at β-carbon.
Grignard reagents afforded the corresponding dialkyl-β-hydroxy α-amino acid derivatives 17a-
7c. Removal of the Cbz group from β-hydroxy amino acids 15a, 15b, 17a, 17b and 19 were
Reaction of ketones 16a-16c with
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accomplished by hydrogenolysis.
Under the standard amide coupling condition (EDC/HOBt/DIPEA), the β-hydroxy amino
acids were reacted with compound 6 to give amide 22 (Scheme 4) in 70-90% yield. Then
exposure of the Oxa-Bicyclic Ortho-ester (OBO ester) 22 to a 1:1:1 mixture of
dioxane/H O/acetic acid allowed for quantitative opening of the OBO ester to give 23, which
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was then converted to the hydroxamic acid 24a, 24b, 24c and 24d, using hydroxylamine in the
presence of KCN.
As shown in Scheme 5, 29 was prepared starting from intermediate 17c, which was
treated with TBAF to remove the TMS group. The terminal alkyne 25 was reacted with
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(azidomethyl)benzene under copper (I)-mediated conditions
to give the triazole 26
regioselectively with concomitant ring-opening of the OBO ester to yield the diol. Amide
coupling of compound 27 with compound 6 under basic conditions afforded the intermediate 28,
which was then converted to the hydroxamic acid 29 as described above.
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RESULTS AND DISCUSSION
Aryl group substitution of the threonyl group
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In our previous study of the LpxC/4 complex, we have noted that the compound 4
threonyl head group and UDP occupy adjacent, but non-overlapping conformational space in the
LpxC active site, with the UDP pyrophosphate group located next to the Cβ atom of the threonyl
head group of 4, suggesting that it is possible to enhance the compound 4-LpxC interaction by
expanding the threonyl head group of 4 through the Cβ-position. Since nearly all LpxC inhibitors
containing the threonyl head group share a similar binding mode, with the methyl group
contacting F191 of the hydrophobic patch and the hydroxyl group forming a hydrogen bond with
K238 of the basic patch, inclusion of an additional substitution would position such a functional
group near the hydrophobic patch of LpxC. We reasoned that introduction of an additional aryl
group at the Cβ position of the threonyl group could augment the inhibitor interaction with the
hydrophobic patch of LpxC without disrupting existing compound-protein interactions, thus
improving the potency of LpxC inhibitors. To search for a suitable aryl group, we first replaced
the threonyl group of 2 with aromatic amino acids such as L-phenylalanine, L-tyrosine, L-
tryptophan and L-histidine and evaluated their effectiveness as antibiotics against wild-type E.
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coli (W3110), E. coli (CMR300)
—a Kdo-deficient strain with enhanced membrane
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permeability, P. aeruginosa (PAO1), and a modified E. coli strain with the native lpxC gene
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replaced by that of P. aeruginosa (W3110PA) (Table 1). In general, replacing the threonyl
group with aromatic side chains reduces the antimicrobial activity. Among tested bacterial
strains, the wild-type E. coli (W3110) is the most tolerant toward bulky aromatic functional
groups, presumably due to a higher degree of conformational flexibility within the EcLpxC
active site. Among the aromatic analogues, substitution of the threonyl group with a tryptophan
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group containing an indole ring at the Cβ position (8c) is most detrimental, resulting a >400-fold
increase of the MIC (minimum inhibitory concentration) value for 8c (12.5 ꢀg/mL) over 2 (0.03
ꢀg/mL) against the wild-type E. coli (W3110). The phenylalanine substituent containing a
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benzene ring at the Cβ position (8b) similarly causes a significantly increase in MIC (6.3
mg/mL). In contrast, tyrosine and histidine analogs are much better tolerated, with MIC values of
0.8 ꢀg/mL for the Tyr analog 8d and 0.4 ꢀg/mL for the His analog 8a. It is interesting to note
that while the MIC values of 8a are identical for the wild-type P. aeruginosa strain and the E.
coli PaLpxC-knock-in strain W3110PA (6.3 ꢀg/mL), 8d is ~3-fold less active against P.
aeruginosa (12.5 mg/mL) than the W3110PA strain (4.7 mg/mL). Since the E. coli W3110PA
strain also expresses PaLpxC instead of EcLpxC, the difference in the MIC values indicates that
8d is less efficient in penetrating the membrane of P. aeruginosa.
In order to determine whether the phenol group and imidazole ring at the Cβ position of
the aromatic substituents occupy the previously noted UDP binding pocket of LpxC and could be
used to augment the inhibitor interaction with the hydrophobic patch of LpxC, we determined the
crystal structure of the PaLpxC/8a complex (Figure 2A). Our structure shows that the imidazole
ring of 8a points toward and cuts across the phenyl ring of F191, forming a favorable π-π
interaction. Additionally, the histidine side chain of 8a forms a hydrogen bond with the backbone
carbonyl of F191and further enhances its interaction with LpxC. Most importantly, the histidine
side chain of 8a occupies a conformational space not utilized by the threonyl group of 2;
although we have not been able to capture a high-resolution structure of the LpxC/8d complex,
the tyrosine analog is expected to adopt a similar side chain romateric position as the side chain
of the histidine analog, suggesting that both the imidazole ring and the phenol group can be
attached to the Cβ position of the threonyl head group to enhance its methyl interaction with the
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hydrophobic patch of LpxC, while maintaining a favorable hydrogen bond between its hydroxyl
group and the catalytically important Lys residue of LpxC (K238 in PaLpxC) from the basic
patch.
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Activity of Cβ-doubly and triply substituted compounds
To test this structure-based prediction, we modified the tyrosine analog 8d and histidine
analog 8a by attaching a hydroxyl group, a methyl group, or both at the Cβ-position. The
attachment of a hydroxyl group in the S-configuration to the tyrosine analog (24b) improves the
antibiotic potency over the parental compound 8d by 5.2- to 12.5-folds against wild-type E. coli,
P. aeruginosa (PAO1) and the E. coli W3110PA strain. The stereochemistry at the Cβ-position
appears crucial for maintaining antibiotic activity of the tyrosine analogues. For example, 24a, of
which the absolute configuration in the Cβ-position is inverted from S to R, displays significantly
diminished activity compared to its S-diastereoisomer 24b (over 500-fold for E. coli). Such a
strict chirality requirement is consistent with the inhibitor binding mode revealed in the crystal
structure of the PaLpxC/24b complex (Figure 2B), where the appended hydroxyl group of 24b
forms a hydrogen bond with the conserved K238, and the inhibitor's phenol ring interacts with
the F191. With the same overall binding mode, but opposite chirality, both of these favorable
interactions are lost, resulting in a large energetic penalty. Inclusion of an additional methyl
attachment to the Cβ-position also requires strict stereochemistry. 24c containing a Cβ-methyl
substitution in the S-configuration displays further improved potency (1.5- to 3-folds) over the
parent compound 24b against the wild-type E. coli, P. aeruginosa and the E. coli W3110PA
strain. In contrast, the R-diastereomer 24d shows reduced antibiotic activity (3- to 5-folds)
compared to the parent 24b.
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In order to obtain direct evidence that inclusion of the phenol group indeed expands the
interaction between the inhibitor and the hydrophobic patch of LpxC, we also determined the
crystal structure of PaLpxC/24c. The complex structure (Figure 2C) reveals that the hydrogen
bond between the inhibitor Cβ-hydroxyl group and K238 of LpxC remains intact, and the
inhibitor Cβ-methyl group similarly makes a favorable vdW contact with the side chain of F191
of LpxC as previously observed in the 2 complex. The additional phenol ring of 24c points
toward F191 with its surface crossing F191 at a nearly perpendicular angle, forming a favorable
T-shaped π-π interaction. Furthermore, the phenolic hydroxyl group picks up an additional OH-π
interaction with adjacent F193 located within the same hydrophobic patch. Indeed, the side chain
of F193 is sufficiently flexible to display two rotameric states in the PaLpxC/8a, PaLpxC/2 and
PaLpxC/24b complexes, whereas it is held in a low energy rotamer by the phenolic hydroxyl
group of 24c. A similar attempt was made to attach the methyl and hydroxyl groups to the Cβ-
position of the 8a. In this case, the imidazole of the 8a was replaced by a triazole ring in order to
facilitate chemical synthesis. This modification (compound 29) also increases the antibiotic
activity over 8a by 6.7- to 12.6-fold against the wild-type E. coli, P. aeruginosa, and E. coli
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(W3110PA). Of note, 29 shares the same stereo center as 24c at the Cβ position, despite an
apparent inversion of nomenclature (S to R) at this position due to the change of IUPAC priority
of the substituted groups (Table 1).
To examine the overall antibiotic profiles of the aryl analog 24c and 29, we measured their
MICs against a panel of Gram-negative bacterial strains, including S. typhimurium, K.
pneumoniae, V. cholerae and F. novicida, a non-virulent subspecies of F. tularensis (Table 2).
While 24c and 29 have an overall antimicrobial potency comparable to that of 2 for many tested
bacterial species, both compounds show enhanced activity (2- to 4-fold) over 2 against F.
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novicida, supporting the notion that expanding the inhibitor interaction with LpxC via the
hydrophobic patch can be utilized to design more potent inhibitors.
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Effect of the LPS membrane barrier on antibiotic activities of LpxC inhibitors
In order to determine whether a lack of improvement in antibiotic activities of 24c and 29
over 2 against E. coli is due to a lack of enhanced inhibition of EcLpxC by these compounds or
due to other factors, such as membrane impermeability, we determined the inhibition constants
of these two inhibitors against EcLpxC using an enzymatic assay. Both 24c and 29 potently
inhibit EcLpxC with a K value of 0.024 ± 0.004 nM, corresponding to ~1.6-fold improvement of
I
potency over 2 (K =0.039 ± 0.003 nM). The more efficient inhibition of EcLpxC by 24c and 29
I
in vitro is consistent with the expanded interaction surface between the additional inhibitor aryl
group and the hydrophobic patch of LpxC, indicating that a lack of improvement in antibiotic
activities of these compounds in cell culture is likely caused by issues related to the membrane
impermeability.
The outer monolayer of the outer membrane of Gram-negative bacteria is enriched with
lipid A-anchored LPS containing O-antigen repeats, which acts as a predominant shield against
hydrophobic molecules. In order to dissect the contribution of the LPS barrier to impeding
membrane penetration of LpxC inhibitors, we tested our compounds against a leaky E. coli strain
(CMR300) that lacks the core saccharides and O-antigen repeats of lipopolyssacharide due to a
1
8
deletion of the kdtA gene. Although all compounds display reduced MIC values against the
leaky E. coli strain, the more hydrophobic compounds experience a greater effect (Table 1),
consistent with the notion that LPS is a major shield against hydrophobic molecules. Since LpxC
contains a hydrophobic substrate passage and a hydrophobic patch in the active site, potent LpxC
inhibitors are typically more hydrophobic compared to antibiotics inhibiting other targets.
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However, our results suggest that despite enhanced potency against LpxC in vitro, a more
hydrophobic compound may not necessarily translate its tighter inhibition in enzymatic assays
into a better antibiotic in cell culture, as the formidable LPS barrier discriminates against the
more hydrophobic compounds. Therefore, a balanced hydrophobic and hydrophilic profile is
most efficient in translating in vitro potency of LpxC inhibitors to antibiotic activities in cell
cultures.
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CONCLUSION
In this study, we have designed and synthesized new series of diacetylene-based
threonyl-hydroxamate derivatives containing an additional aryl head group in order to enhance
the inhibitor interaction with the hydrophobic patch of LpxC, a conserved feature across distinct
LpxC orthologs. Our structure-activity relationship study shows that attaching imidazole- (or
triazole-) and tyrosyl-substituents at the Cβ-position of the threonyl group of 2 introduces
additional interactions with the hydrophobic patch of LpxC and augments the existing threonyl
methyl and hydroxyl group interactions with LpxC. Such an expanded inhibitor-LpxC interaction
has resulted in a further reduction of K values to ~24 pM in E. coli LpxC enzymatic assays and
I
4-fold improved antibiotic activity against F. novicida for the best compound 24c. The newly
identified aryl derivative series show impressive antibiotic activity against a wide range of
Gram-negative bacteria and highlight the Cβ-position of the threonyl group in 2 as an effective
branch point to expand the binding interface of existing LpxC inhibitors. Our study additionally
highlights the importance of balancing the hydrophilicity and hydrophobicity for development of
effective antibiotics targeting Gram-negative bacteria. Such an observation is particularly
important in future efforts to develop in vivo active antibiotics targeting LpxC based on in silico
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screening or structural insights. These compounds can be readily solubilized at concentrations of
10-40 mg/mL in 100 mM β-cyclodextrin, making them compatible with IV formulation for
further evaluations of safety and efficacy in mouse infection models. Our compounds include
both acetylene and hydroxamate moieties, residues that are sometime associated with metabolic
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9, 20
toxicity.
However, both acetylene and hydroxamate groups occur in FDA-approved drugs
21
such as efavirenz, linagliptin, and vorinostat ) for chronic administration, and their presence
22
23
(
in LpxC inhibitors intended for short-term treatment of Gram-negative infections should not
prevent the deployment of this novel class of antibiotics.
EXPERIMENTAL SECTION
MIC tests. The MIC of each compound was tested in triplicate as described previously
using a modified NCCLS protocol, which is adapted to 96-well plates and LB media in presence
7
, 11
of 5% DMSO.
100 ꢀL of various concentrations of compounds are prepared on a standard
9
6-well plate (Corning Costar 3596, flat bottomed with lid, polystyrene wells), in 2-fold dilution
series with range of 0.0005 to 1 ꢀg/mL, 0.0025 to 5 ꢀg/mL or 0.024 to 5 ꢀg/mL. Bacterial cells,
grown to OD600 = 0.6, were diluted 100-fold with fresh LB medium, and 100 ꢀL of the diluted
o
cells was added into each well. The inoculated plates were incubated at 37 C for 22 hours. After
the incubation, 50 ꢀL of 1 mg/mL [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
solution (MTT) was added and incubated at 37 °C for another 3 hr. The MIC was considered the
lowest concentration of compound that showed no visible color change (yellow to black).
LpxC enzymatic inhibition assay. The substrate for the enzymatic assay, UDP-3-O-
3
2
[
(R)-3-hydroxymyristoyl]-N-acetylglucosamine and [α- P] UDP-3-O-[(R)-3-hydroxymyristoyl]-
2
N-acetylglucosamine, were prepared as previously described. The assays were performed in 25
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mM HEPES buffer, pH 7.4, 100 mM KCl, 1 mg/mL BSA, 2 mM DTT, 0.5 µM substrate with
1
0% DMSO or 0.19 pM to 12.5 nM inhibitor in DMSO. The reactions were initiated with
addition of 2 pM of purified E. coli LpxC. Aliquots were taken after 1 to 4 hours of incubation at
0 °C to quantify reaction progression (< 5% conversion of substrate to product). The kinetic
parameters for enzyme activity in the presence of inhibitors were determined as reported
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app
Briefly, KI were determined by plotting fractional activity versus inhibitor
previously.
2
5
concentration and fitting to Morrison’s quadratic equation.
KM and Vmax values were
determined by varying substrate concentration from 0.5 to 50 µM. Finally, the K values were
I
app
calculated based on the relationship between KI and K for a tight binding competitive
I
25
inhibitor. All measurements were done in duplicates.
Crystallization and structure determination. Protein samples were prepared as
1
1
described previously. Purified PaLpxC (1-299, C40S) in a buffer of 25 mM HEPES, pH 7.1,
50 mM NaCl and 2 mM TCEP (tris(2-carboxyethyl)phosphine) was diluted to a final
concentration of 12 mg/mL with the same buffer. A four-fold molar excess of individual
compounds, dissolved in DMSO, was mixed with the diluted protein and then 10 mM zinc
sulfate was added into the complex sample. Crystals of PaLpxC in complex with 8a, 24b or 24c
were obtained using the sitting-drop vapor-diffusion method at 20 ºC in drops containing 1 ꢀL of
the inhibitor-protein complex and 1 ꢀL of well solution consisting of 0.1 M sodium acetate
trihydrate (pH 4.7-5.2) and 2.4-2.7 M ammonium nitrate. The crystals were cryoprotected with
perfluoropolyether (PFO-X175/08) before flash-freezing. Diffraction data were collected in-
house at 100 K using a Rigaku MicroMax-007 HF rotating anode generator and R-Axis IV++
26
detector. The collected diffraction data were processed with HKL2000. Molecular replacement
2
7
using AUTOMR in the PHENIX package
was carried out by using the structure of the
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PaLpxC/5 complex (PDB entry 3P3E) as the search model. The final coordinate was completed
27, 28
by iterative cycles of model building (COOT) and refinement (PHENIX).
The statistics for
the LpxC-inhibitor complexes are shown in Table 3.
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Chemical synthesis. LC/MS analysis was conducted on an Agilent 1200 HPLC with a
quadrupole mass analyzer. LC chromatography used an Agilent XDB-C18 column (4.6×50 mm,
1
.8 ꢀm) with a water/acetonitrile (each with 0.2% (v/v) formic acid) gradient at a flow rate of 0.5
1
13
mL/min. HRMS analyses were performed at the Duke MS Center. H and C spectra were
recorded at 300 (400) and 75 (100) MHz, respectively, on a Varian Spectrometer. Column
chromatography was conducted using either silica gel (Silicycle 40-64 ꢀm) or prepacked
RediSep columns (Teledyne Isco Inc., Lincoln, NE) on an Isco CombiFlash Rf instrument. All
moisture-sensitive reactions were carried out using dry solvents and under a slight pressure of
ultra-pure quality argon. Glassware was dried in an oven at 140 °C for at least 12h prior to use,
and then assembled quickly while hot, sealed with rubber septa, and allowed to cool under a
stream of argon. Reactions were stirred magnetically using Teflon-coated magnetic stirring bars.
Commercially available disposable syringes were used for transferring reagents and solvents. All
reagents were used as received from commercial sources, unless specified otherwise, or prepared
as described in the literature. The purities of all compounds were determined to be > 95% by
NMR and LC-MS.
General Procedure for the amide coupling (Method A). To a stirred mixture of 4-((4-
aminophenyl)buta-1,3-diyn-1-yl)benzoic acid (100 mg, 0.39 mmol, 1.00 equiv) and amino acid
hydrochloride (0.54 mmol, 1.40 equiv) in anhydrous DMF (5 mL) was added N-ethyl-N’-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC hydrochloride) (103 mg, 0.54 mmol,
1
.40 equiv) and 1-hydroxybenzotriazole (HOBt) (73 mg, 0.54 mmol, 1.40 equiv) at room
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temperature. Then the mixture was cooled to 0 °C, and diisopropylethylamine (DIEA) (0.27 mL,
1.53 mmol, 4.00 equiv) was added. The whole reaction mixture was stirred at 0 °C under argon
for 1h, then was allowed to warm to temperature with the stirring continued overnight (18h). The
resulting yellow solution was concentrated to dryness with a rotary evaporator, and the residue
was treated with water (20 mL), extracted with EtOAc (3×50 mL). The combined extracts were
washed with water (20 mL), brine (20 mL), and dried over anhydrous Na SO . Evaporation of
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the solvent afforded the crude products, which was purified by flash chromatography.
General Procedure for the hydroxamic acid (Method B). To an ice-cold solution of ester
(0.23 mmol, 1.00 equiv) dissolved in anhydrous MeOH (1 mL) and THF (1 mL) was added
hydroxylamine hydrochloride (104 mg, 1.50 mmol, 5.00 equiv) followed by 25% sodium
methoxide in methanol solution (0.54 mL, 2.25 mmol, 7.50 equiv). The reaction mixture was
stirred at 0 °C under argon for 2h, then was allowed to warm to ambient temperature with the
stirring continued overnight (16h). The resulting yellow suspension was condensed to dryness
with a rotary evaporator, and the residue was treated water (20 mL), acidified by 0.5 N HCl to
pH 6~7, and extracted with EtOAc (3×50 mL). The combined extracts were washed with water
(20 mL), brine (20 mL), and dried over anhydrous Na SO . Evaporation of the solvent afforded
2
4
the crude product, which was purified by flash chromatography.
General Procedure for the hydroxamic acid (Method C). Ester (0.10 mmol, 1.00 equiv)
was dissolved in a mixture of THF/MeOH (1:1 volume) 2.0 mL. 50% aq hydroxylamine (0.20
mL, 3.0 mmol, 30.00 equiv) was added followed by potassium cyanide (6.5 mg, 0.10 mmol, 1.00
equiv) at room temperature under argon. The reaction mixture was stirred at room temperature
for 14h. Then the yellow solution was concentrated to dryness. The residue was treated with
water (30 mL), acidified by 0.5 N HCl to pH 6~7, extracted with EtOAc (3×50 mL). The
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combined organic layers were washed with water (30 mL), brine (30 mL), and dried (anhydrous
Na SO ). The crude product was purified by flash chromatography.
2
4
General Procedure for Swern Oxdiation (Method D). The alcohol (21.27 mmol, 1.00
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equiv) was dissolved in anhydrous DCM (100 mL) under argon and cooled to -78 °C in flask I.
Oxalyl chloride (17.00 mL, 2.0 M in DCM, 70.19 mmol, 1.60 equiv) was added to anhydrous
DCM (100 mL) in a separate flask II under argon and cooled to -78°C. Anhydrous DMSO (5.36
mL, 70.19 mmol, 3.30 equiv) was added to the oxalyl chloride solution (flask II), and the
mixture was stirred at -78 °C for 15 min. The alcohol solution was transferred by syringe at -78
°C to the flask II over a period of 20 min. The resulting cloudy white mixture was stirred for 1.5h
at -78 °C. DIPEA (18.44 mL, 106.35 mmol, 5.0 equiv) was added, and the reaction mixture was
stirred for 30 min at -78 °C and 10 min at 0 °C. Ice-cold DCM (200 mL) was added, and the
solution was washed with ice-cold 3% NH Cl (3×200 mL), brine (200 mL), dried (anhydrous
4
Na SO ). The crude product was crystallized from DCM/Hexane.
2
4
General Procedure for Grignard Addition (Method E). Aldehyde or ketone (5.00 mmol,
.00 equiv) was dissolved in anhydrous DCM (20 mL) under argon. A solution of RMgBr in
1
THF (20 mL, 20 mmol, 4.00 equiv) was added quickly by syringe at -78 °C or 0 °C and stirred
vigorously. The reaction mixture was stirred at -78 °C for 30 min-2h. Then the reaction mixture
was quenched by saturated NH Cl (300 mL, pH~7) and was stirred for an additional 15 min. The
4
mixture was extracted with DCM (3×80 mL). The combined organic layers were washed with
water (50 mL), brine (50 mL), dried (anhydrous Na SO ). The crude product was purified by
2
4
flash chromatography or recrystallization.
General Procedure for hydrogenolysis (Method F). To a solution of Cbz protected
compound (1.2 mmol) in anhydrous MeOH (10 mL) was added 10% Pd/C (10 w/w%) at room
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temperature under a balloon of hydrogen. The reaction mixture was stirred for 14h. Then the
catalyst was removed by filtration through a pad of celite and washed with MeOH (50 mL). The
filtrate was evaporated under reduced vacuum to obtain the crude product, which was utilized for
further transformation without any purification.
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General Procedure for ring-opening of OBO ester (Method G). OBO ester (0.286 mmol)
was dissolved in dioxane (1.0 mL). The mixture was diluted with acetic acid (1.0 mL) and water
(1.0 mL) at room temperature under argon and the resulting suspension was stirred for 70 min.
Then the yellow clear solution was concentrated to dryness. The residue was treated with
saturated NaHCO (20 mL) to pH~10, extracted with EtOAc (3×40 mL). The combined organic
3
layers were washed with water (2×30 mL), brined (30 mL), and dried (anhydrous Na SO ). The
2
4
crude product was purified by flash chromatography.
(S)-Methyl
2-(4-((4-aminophenyl)buta-1,3-diyn-1-yl)benzamido)-3-(1H-imidazol-4-
1
yl)propanoate (7a): Method A, (0.97 g, 45% yield) as white solid. H NMR (300 Hz, CDCl ) δ
3
3.20 (s, br, 2H), 3.67 (s, 3H), 4.93 (s, br, 1H), 6.59 (d, J=8.7 Hz, 2H), 7.32 (d, J=11.1Hz, 2H),
1
7.54 (d, J=8.4 Hz, 2H), 7.80-7.83 (m, J=8.4 Hz, 2H), 8.35 (s, br, 1H); C NMR (75 Hz, CDCl )
3
3
δ 29.24, 52.65, 53.38, 72.07, 79.78, 80.10, 83.07, 84.44, 110.45, 114.84, 115.079, 125.99,
+
27.52, 132.49, 132.65, 134.41, 135.47, 148.06, 166.70, 172.17; LCMS: 413.1 [M+H ].
1
(S)-Methyl-2-(4-((4-aminophenyl)buta-1,3-diyn-1-yl)benzamido)-3-phenylpropanoate (7b):
1
Method A, (143 mg, 89% yield) as yellow solid. H NMR (300 Hz, DMSO-d ) δ 3.06-3.19 (m,
6
J=39 Hz, 1H), 3.62 (s, 1H), 4.60-4.68 (m, J=5.846 Hz, 2H), 6.53 (d, J=8.7 Hz, 2H), 7.16-7.27
13
m, J=34.2 Hz, 7H), 7.62 (d, J=8.1 Hz, 2H), 7.78 (d, J=8.4 Hz, 2H), 8.96 (d, J=7.8 Hz, 1H); C
(
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NMR (75 Hz, DMSO-d ) δ 36.86, 52.71, 55.05, 71.77, 77.40, 80.75, 86.45, 105.77, 114.26,
6
124.96, 127.21, 218.43, 128.95, 129.74, 132.70, 134.38, 134.71, 138.30, 144.23, 151.574,
+
66.23, 172.75; LCMS: m/s 424.2 [M+2H] .
1
10
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(S)-methyl-2-(4-((4-aminophenyl)buta-1,3-diyn-1-yl)benzamido)-3-(1H-indol-3-
1
yl)propanoate (7c): Method A, (173 mg, 98% yield) as yellow solid. H NMR (300 Hz,
CD OD) δ 3.29-3.40 (m, J=31.8 Hz, 2H), 3.41 (d, J=5.4 Hz, 1H), 3.70 (s, 3H), 6.60 (d, J=8.4 Hz,
3
2H), 6.99 (t, J=14.4 Hz, 1H), 7.06-7.11 (m, J=15.0 Hz, 2H), 7.23 (d, J=8.1 Hz, 2H), 7.32 (d,
13
J=7.8 Hz, 1H), 7.49 (d, J=8.4 Hz, 2H), 7.55 (d, J=7.8 Hz, 1H), 7.69 (d, J=8.1 Hz, 2H); C NMR
(
75 Hz, CD OD) δ 27.05, 51.63, 54.44, 70.81, 76.61, 79.034, 84.55, 108.33, 109.78, 111.22,
3
114.23, 117.98, 118.73, 121.36, 123.23, 125.89, 127.49, 132.00, 133.83, 136.88, 150.19, 168.07,
+
72.86; LCMS: m/s 462.3 [M+H] .
1
(S)-Methyl-2-(4-((4-aminophenyl)buta-1,3-diyn-1-yl)benzamido)-3-(4-
1
hydroxyphenyl)propanoate (7d): Method A, (148 mg, 90% yield) as yellow solid. H NMR
(300 Hz, CD OD) δ 3.00 (q, J=24.3 Hz, 1H), 3.18 (q, J=19.5 Hz, 1H), 3.70 (s, 3 H), 4.81 (q,
3
J=40.5 Hz, 1H), 6.61 (d, J=8.7 Hz), 6.70 (d, J=8.7 Hz, 2H), 7.06 (d, J=8.7 Hz, 2H), 7.24 (d,
1
3
J=8.7 Hz, 2H), 7.52 (d, J=8.7 Hz, 2H), 7.71 (d, J=8.7 Hz, 2H); C NMR (75 Hz, CD OD) δ
3
36.23, 51.61, 55.07, 70.81, 76.62, 79.05, 84.57, 108.32, 114.23, 115.12, 125.92, 127.48, 127.84,
+
30.06, 132.04, 133.84, 150.20, 156.22, 168.10, 172.56; LCMS: m/s 439.5 [M+H] .
1
(S)-4-((4-aminophenyl)buta-1,3-diyn-1-yl)-N-(1-(hydroxyamino)-3-(1H-imidazol-4-yl)-1-
1
oxopropan-2-yl)benzamide (8a) : Method B, (24 mg, 48% yield) as yellow solid. H NMR (300
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
Hz, DMSO-d ) δ 3.12 (q, 2H, J=20.7 Hz, 4.74 (q, 1H, J=14.4 Hz), 6.62 (d, 2H, J=9.0 Hz), 6.93
6
(s, 1H), 7.22 (d, 2H, J=8.7 Hz), 7.51-7.56 (m, 3H, J=14.1 Hz), 7.68 (d, 1H, J=1.5 Hz), 7.76-7.80
1
3
(
m, 3H, J=12.6 Hz); C NMR (75 Hz, DMSO-d ) δ 29.24, 52.06, 70.70, 76.59, 78.96, 80.01,
6
0
1
2
3
4
5
6
7
8
9
0
1
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8
9
0
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8
9
0
84.56, 108.28, 114.20, 116.74, 125.96, 127.54, 131.85, 131.99, 133.79, 135.09, 150.24, 167.80,
+
169.17; HRMS HRMS: m/z calc'd for C H N O [M] : 413.1488; found: 413.1490.
2
3
19
5
3
(S)-4-((4-aminophenyl)buta-1,3-diyn-1-yl)-N-(1-(hydroxyamino)-1-oxo-3-phenylpropan-2-
1
yl)benzamide (8b): Method B, (77 mg, 77% yield) as yellow solid. H NMR (300 Hz, DMSO-
d6) δ 3.00 (d, J=6.6 Hz 2H), 4.57 (d, J=7.2 Hz, 1H), 5.84 (s, 2H), 6.53 (d, J=8.7 Hz, 2H), 7.15 (d,
1
J=7.2 Hz, 2H), 7.20-7.30 (m, J=30.6, 5H), 7.58 (d, J=8.1 Hz, 2H); C NMR (75 Hz, DMSO-d )
3
6
δ 38.13, 53.66, 71.80, 77.23, 80.84, 86.33, 105.82, 114.26, 124.63, 126.94, 128.50, 129.77,
+
32.52, 134.70, 138.94, 151.54, 165.91, 168.46; HRMS: m/z calc'd for C H N O [M] :
1
2
6
21
3
3
423.1583; found: 423.1585.
(S)-4-((4-aminophenyl)buta-1,3-diyn-1-yl)-N-(1-(hydroxyamino)-3-(1H-indol-3-yl)-1-
1
oxopropan-2-yl)benzamide (8c): Method B, (77 mg, 70% yield) as yellow solid. H NMR (300
Hz, DMSO-d ) δ 3.13 (d, J=6.9 Hz, 2H), 4.63 (q, J=15.6 Hz, 1H), 5.84 (s, 2H), 6.52 (d, J=8.4
6
Hz, 2H), 6.93-7.05 (m, J=35.7 Hz, 2H), 7.18 (s, 1H), 7.22-7.29 (m, J=21.9 Hz, 3H), 7.58 (d,
1
3
J=8.1 Hz, 2H), 8.70 (d, J=8.1 Hz, 1H), 8.88 (s, 1H), 10.76 (s, 1H), 10.86 (s, 1H); C NMR (75
Hz, DMSO-d ) δ 28.31, 52.74, 71.80, 77.25, 80.84, 86.34, 105.81, 110.94, 112.00, 114.26,
6
118.94, 119.18, 121.58, 124.41, 124.64, 127.83, 128.55, 132.51, 134.71, 134.88, 136.72, 151.56,
+
65.92, 169.04; HRMS: m/z calc'd for C H N O [M] : 462.1692; found: 462.1694.
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(
S)-4-((4-aminophenyl)buta-1,3-diyn-1-yl)-N-(1-(hydroxyamino)-3-(4-hydroxyphenyl)-1-
1
oxopropan-2-yl)benzamide (8d): Method B, (74 mg, 67% yield) as yellow solid. H NMR (300
Hz, DMSO-d ) δ 2.87 (d, J=7.2 Hz, 2H), 4.47 (d, J=23.4 Hz, 1H), 5.83 (s, 2H), 6.53 (d, J=8.7
6
10
11
12
13
14
15
16
17
18
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57
58
59
60
Hz, 2H), 6.60 (d, J=8.4 Hz, 2H), 7.08 (d, J=8.4 Hz, 2H), 7.24 (d, J=8.4 Hz, 2H), 7.59 (d, J=8.1
Hz, 2H), 7.80 (d, J=8.4 Hz, 2H), 8.68 (d, J=8.4 Hz, 1H), 8.86 (s, 1H), 9.14 (s, 1H), 10.75 (s, 1H);
1
3
C NMR (300 Hz, DMSO-d ) δ 53.96, 71.81, 77.23, 80.85, 86.33, 105.82, 114.26, 115.59,
6
124.63, 128.53, 128.86, 130.74, 132.53, 134.70, 134.93, 151.55, 156.42, 165.96.168.75; HRMS:
+
m/z calc'd for C H N O [M] : 439.1532; found: 439.1533.
2
6
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3
4
(3-methyloxetan-3-yl)methyl 4-methylbenzenesulfonate (10): To a stirred solution of p-
toluenesulfonyl chloride (74.36 g, 0.39 mol, 1.5 equiv) in anhydrous pyridine (300 mL) was
added dropwise 3-methyl-3-oxetane- methanol (26.52 g, 0.26 mol) over 10 min at 0°C under
argon. After 5 min, the reaction mixture was allowed to warm to room temperature with the
stirring continued for an additional 1.5h. The mixture was then slowly added to a vigorously
stirred mixture of milliQ water (800 mL) and crushed ice (800 g) for 30 min. Then the white
precipitate was collected on Whatman filter #1 and washed with cold water (300 mL). The
product was dried under high vacuum to obtain a white power of oxetane tosylate 10 (47.88 g
1
7
2% yield). H NMR (300 MHz, CDCl ) δ 1.28 (s, 3H), 2.43 (s, 3H), 4.08 (s, 2H), 4.29-4.35 (m,
3
1
3
2H), 7.34 (d, J=7.8 Hz, 2H), 7.78 (d, J=7.5 Hz, 2H); C NMR (75 MHz, CDCl ) δ 20.85, 21.86,
3
+
9.45, 74.50, 79.13, 128.15, 130.22, 132.81, 145.34; LCMS: m/s 164.9 [M+H] .
3
(
S)-(3-methyloxetan-3-yl)methyl-2-(((benzyloxy)carbonyl)amino)-3-hydroxypropanoate
12): A solution of Cbz-L-Ser 11 (29.60 g, 0.167 mol), 3-methyl-(3-tosylmethyl oxetane) 10
(
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(
29.95 g, 0.125 mol), tetrabutylammonium iodide (2.30 g, 0.06 mol, 0.05 equiv) and TEA (14.04
g, 0.138 mol, 1.1 equiv) in anhydrous DMF (100 mL) was slowly heated to 70°C for 36h under
argon. The reaction mixture was allowed to cool to room temperature, and the DMF was
removed under reduced pressure. The remaining residue was dissolved in 1.0 L of EtOAc,
0
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7
8
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0
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washed with 1.0 N HCl (2×250 mL), saturated NaHCO (2×250 mL), brine (250 mL), and dried
3
(anhydrous Na SO ). The solvent was removed under reduced pressure, and the remaining
2 4
residue was purified on a CombiFlash system (eluting with 0-50% EtOAc in hexane) to give 12
1
(25.20 g, 64% yield) as pale yellow oil. H NMR (300 MHz, CDCl ) δ 1.26 (s, 3H), 3.34 (br, s,
3
1
H), 3.84-3.87 (m, 1H), 4.02-4.07 (m, 2H), 4.35-4.52 (m, 6H), 5.11 (s, 2H), 5.94 (d, J=7.5 Hz,
1
3
1
H), 7.26-7.33 (m, 5H); C NMR (75 MHz, CDCl ) δ 20.98, 39.70, 56.57, 63.39, 67.34, 69.18,
3
+
79.66, 128.33, 128.44, 128.75, 136.38, 156.51, 170.98; LCMS: m/s 324.1 [M+H] .
(
S)-benzyl-(2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)carbamate
13): Cbz-L-Ser-oxetane ester 12 (11.00 g, 34.00 mmol) was dissolved in anhydrous DCM (100
(
mL) and was cooled to 0°C under argon. BF .Et O (0.13 mL, 0.92 mmol, 0.03 equiv) was diluted
3
2
in DCM (5.0 mL) and added to the reaction flask. The reaction mixture was allowed to warm to
room temperature. After 5h, Et N (1.40 mL, 9.86 mmol, 0.3 equiv) was added with the stirring
3
continued for an additional 30 min. Then the result solution was concentrated to dryness to thick
oil. The crude product was purified on CombiFlash (eluting with 0-5% MeOH in DCM) to afford
1
1
3 (6.90 g, 62.7% yield) as white foam. H NMR (300 MHz, CDCl ) δ 0.78 (s, 3H), 2.64 (q,
3
J=12.6 Hz, 1H), 3.62-3.95 (m, 9H), 5.03-5.14 (m, 2H), 5.37 (d, J=9.0 Hz, 1H), 7.26-7.34 (m,
1
3
5
H); C NMR (75 MHz, CDCl ) δ 14.48, 30.76, 55.60, 62.14, 67.12, 72.90, 108.68, 128.28,
3
+
28.36, 128.69, 136.69, 156.67; LCMS: m/s 324.1 [M+H] .
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(
S)-benzyl-(1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)-2-oxoethyl)carbamate
(14):
1
Method D, (4.3 g, 63% yield) as yellow solid. H NMR (300 MHz, CDCl ) δ 0.81 (s, 3H), 3.93
3
10
11
12
13
14
15
16
17
18
19
20
21
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47
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56
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59
60
(
s, 6H), 4.60 (d, J=8.7 Hz, 1H), 5.12 (s, 2H), 5.36 (d, J=8.7 Hz, 1H), 7.26-7.40 (m, 5H), 9.68 (s,
1
3
1H); C NMR (75 MHz, CDCl ) δ 14.47, 31.09, 63.50, 67.43, 73.12, 107.40, 128.36, 128.70,
3
+
36.40, 156.42, 159.90; LCMS: m/s 307.4 [M+H] .
1
Benzyl((1S,2R)-2-(4-(benzyloxy)phenyl)-2-hydroxy-1-(4-methyl-2,6,7-
trioxabicyclo[2.2.2]octan-1-yl)ethyl)carbamate (15a): Method E, (0.272 g, 54% yield) as foam
1
solid. H NMR (300 MHz, CDCl ) δ 0.83 (s, 3H), 3.36 (s, 1H), 3.98 (s, 6H), 4.07 (d, J=10.5 Hz,
3
1
1H), 4.88-5.08 (m, 5H), 5.48 (d, J=10.5 Hz, 1H), 6.91 (d, J=9.0 Hz, 2H), 7.22-7.46 (m, 12H); C
3
NMR (75 MHz, CDCl ) δ 14.56, 30.93, 58.87, 66.76, 70.16, 70.65, 73.06, 109.04, 114.72,
3
1
27.36, 127.77, 127.99, 128.14, 128.63, 128.80, 132.84, 136.99, 137.414, 156.61, 158.33;
+
LCMS: m/s 506.2 [M+H] .
Benzyl((1S,2R)-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-
1
yl)propyl)carbamate (15b): Method E, (1.52 g, 50% yield) as white solid. H NMR (300 MHz,
CDCl ) δ 0.80 (s, 3H), 1.11 (d, J=6.3 Hz, 3H), 2.87 (s, 3H), 3.75 (d, J=10.5 Hz, 1H), 3.92 (s, 6
3
1
H), 4.35 (q, J=19.5 Hz, 1H), 5.08-5.18 (m, 2H), 5.34 (d, J=10.5 Hz, 1H), 7.26-7.36 (m, 5H); C
3
NMR (75 MHz, CDCl ) δ 14.53, 19.22, 30.84, 57.95, 65.43, 67.06, 72.93, 108.99, 127.77,
3
+
28.19, 128.67, 136.85, 157.19; LCMS: m/s 378.1 [M+H] .
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Benzyl((1S,2R)-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)-4-
(trimethylsilyl)but-3-yn-1-yl)carbamate (15c): Method E, (2.20 g, 70% yield) as white foam.
1
H NMR (300 MHz, CDCl ) δ 0.12 (s, 9H), 0.81 (s, 3H), 3.09 (s, 1H), 3.92 (s, 6H), 4.12 (d,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
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7
8
9
0
1
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7
8
9
0
1
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3
4
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7
8
9
0
J=10.2 Hz, 1H), 4.90 (s, 1H), 5.13 (dd, J =21.0 Hz, J =12.3 Hz, 2H), 5.45 (d, J=10.5 Hz, 1H),
2
1
1
3
7.32-7.36 (m, 5H); C NMR (75MHz, CDCl ) δ 0.27, 14.25, 30.65, 57.62, 62.11, 66.91, 72.74,
3
72.89, 89.99, 102.66, 108.31, 127.98, 128.06, 128.43, 136.53. 156.43; LCMS: m/s 420.2
+
M+H] .
[
(S)-benzyl
(2-(4-(benzyloxy)phenyl)-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)-2-
1
oxoethyl)carbamate (16a): Method D, (0. 62 g, 84% yield) as white foam. H NMR (300 MHz,
CDCl ) δ 0.75 (s, 3H), 3.86 (s, 6H), 5.10 (s, 2H), 5.13 (s, 2H), 5.55 (d, J=9.3 Hz, 1H), 5.95 (d,
3
13
J=9.3 Hz, 1H), 7.00 (d, J=9.0 Hz, 2H), 7.27-7.45 (m, 10H), 8.06 (d, J=9.0 Hz, 2H); C NMR (75
MHz, CDCl ) δ 14.48, 30.90, 57.35, 67.23, 70.37, 73.17, 107.60, 114.60, 127.76, 128.30,
3
128.48, 128.67, 128.93, 129.91, 132.120, 136.43, 136.60, 156.16, 163.22, 193.49; LCMS: m/s
+
04.1 [M+H] .
5
(S)-benzyl (1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)-2-oxopropyl)carbamate (16b):
1
Method D, (1.06 g, 82% yield) as white solid. H NMR (300 Hz, CDCl ) δ 0.79 (s, 3H), 2.30 (s,
3
3H), 3.90 (s, 6H), 4.58 (d, J=8.4 Hz, 1H), 5.09 (s, 2H), 5.62 (d, J=8.1 Hz, 1H), 7.26-7.34 (m,
13
5
H); C NMR (300 Hz, CDCl ) δ 14.44, 29.94, 30.87, 63.38, 67.26, 73.17, 107.13, 128.30,
3
+
28.68, 136.50, 156.21, 202.86; LCMS: m/s 356.2 [M+H] .
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(
S)-benzyl(1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)-2-oxo-4-(trimethylsilyl)but-3-
1
yn-1-yl)carbamate (16c): Method D, (0. 62 g, 80% yield) as white foam. H NMR (300 MHz,
CDCl ) δ 0.01 (s, 9H), 0.58 (s, 3H) 3.69 (s, 6H), 4.45 (d, J=9.3 Hz, 1H), 4.89 (s, 2H), 5.40 (d,
3
10
11
12
13
14
15
16
17
18
19
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49
50
51
52
53
54
55
56
57
58
59
60
13
J=9.0 Hz, 1H), 7.04-7.13 (m, 5H); C NMR (75MHz, CDCl ) δ 0.84, 14.21, 30.80, 63.88, 67.17,
3
72.93, 101.23, 102.11, 106.88, 128.12, 128.47, 136.16, 155.72, 181.35; LCMS: m/s 418.1
+
M+H] .
[
Benzyl((1S,2S)-2-(4-(benzyloxy)phenyl)-2-hydroxy-1-(4-methyl-2,6,7-
trioxabicyclo[2.2.2]octan-1-yl)propyl)carbamate(17a): Method E, (0.81 g, 98% yield) as
1
white solid. H NMR (300 MHz, CDCl ) δ 0.73 (s, 3H), 1.50 (s, 3H), 3.73 (s, 3H), 3.71-3.80 (m,
3
6H), 4.22 (d, J=10.2 Hz, 1H), 4.98-5.14 (m, 4H), 5.33 (d, J=10.5 Hz, 1H), 6.90 (d, J=8.7 Hz,
13
2
7
1
H), 7.28-7.43(m, 12H); C NMR (75 MHz, CDCl ) δ 14.45, 27.17, 30.62, 61.09, 66.96, 70.13,
3
2.60, 76.04, 109.50, 113.93, 126.76, 127.11, 127.76, 128.12, 128.68, 128.75, 136.90, 137.51,
+
38.83, 156.60, 157.49; LCMS: m/s 520.2 [M+H] .
Benzyl((1S,2R)-2-(4-(benzyloxy)phenyl)-2-hydroxy-1-(4-methyl-2,6,7-
trioxabicyclo[2.2.2]octan-1-yl)propyl)carbamate(17b): Method E, (0.62 g, 40% yield) as
1
white solid. H NMR (300 MHz, CDCl ) δ 0.82 (s, 3H), 1.66 (s, 3H), 3.48 (s, 1H), 3.87-3.99 (m,
3
1
6H), 4.19 (d, J=10.2 Hz, 1H), 4.87-5.06 (m, 4H), 5.31-5.33 (m, 2H), 7.26-7.46 (m, 12H); C
3
NMR (75 MHz, CDCl ) δ 14.56, 29.71, 30.74, 60.71, 66.50, 70.11, 72.72, 76.03, 109.78, 114.27,
3
126.23, 127.64, 127.77, 127.92, 128.12, 128.55, 128.78, 137.08, 137.45, 138.45, 156.52, 157.60;
+
LCMS: m/s 520.2 [M+H] .
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Benzyl((1S,2S)-2-hydroxy-2-methyl-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)-4-
1
trimethylsilyl)but-3-yn-1-yl)carbamate (17c): Method E, (0.1 g, 74% yield) as white solid. H
(
NMR (300 MHz, CDCl ) δ 0.16 (s, 9H), 0.81 (s, 3H), 1.47 (s, 3H), 3.72 (s, 1H), 3.90 (s, 6H),
3
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
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1
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7
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0
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5
6
7
8
9
0
13
.05 (d, J=10.5 Hz, 1H), 5.13 (s, 2H), 5.27 (d, J=9.6 Hz, 1H), 7.27-7.35 (m, 5H); C NMR
4
(75MHz, CDCl ) δ 0.03, 14.30, 27.38, 30.57, 60.23, 66.92, 68.45, 72.40, 88.53, 107.31, 108.83,
3
+
27.97, 128.02, 128.46, 136.50, 156.55; LCMS: m/s 434.2 [M+H] .
1
4-((1R,2S)-2-amino-1-hydroxy-2-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)phenol
1
(
18): Method F, (0.277 g, 99% yield) as white solid. H NMR (300 MHz, CD OD) δ 0.81 (s,
3
1
3
H), 3.94 (s, 6H), 5.02 (d, J=2.4 Hz, 1H), 6.73 (d, J=8.7 Hz, 2H), 7.17 (d, J=8.7 Hz, 2H); C
3
NMR (75 MHz, CD OD) δ 13.05, 30.36, 60.33, 70.65, 72.57, 108.90, 114.70, 127.11, 133.26,
3
+
56.48; LCMS: m/s 282.2 [M+H] .
1
Benzyl((1S,2S)-2-(4-(benzyloxy)phenyl)-2-hydroxy-1-(4-methyl-2,6,7-
trioxabicyclo[2.2.2]octan-1-yl)ethyl)carbamate (19): Intermediate 18 (0.50 g, 1.00 mmol) was
dissolved in anhydrous DCM (15 mL) and MeOH (15 mL) under argon, and then cooled to -78
°C. Then a solution of 2.0 M LiBH in THF (4.80 mL, 8.00 mmol, 8.0 equiv) was added
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dropwise. The reaction mixture was stirred at -78 °C for 10h, and then slowly warm to room
temperature. The mixture was diluted to with DCM (100 mL) and then quenched with saturated
NH Cl (50 mL). The organic layer was separated. And the aqueous layer was extracted with
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DCM (2×50 mL). The organic layers were combined, washed with saturated NH Cl (50 mL),
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brine (50 mL), and dried (anhydrous Na SO ). The solvent was reduced in vacuum to dryness.
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4
The crude product was purified on CombiFlash (eluting with 0-60% EtOAc in hexane) to obtain
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9 (0.44g, 88% yield) as white solid. H NMR (300 MHz, CD Cl) δ 0.83 (s, 3H), 3.96 (s, 6H),
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.11 (d, J=1.5 Hz, 1H), 4.14-4.21 (m, 1H), 4.78-4.87 (m, 3H), 5.00 (s, 1H), 5.02 (s, 2H), 6.91 (d,
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J=8.4 Hz, 2H), 7.11 (d, J=6.6 Hz, 2H), 7.25-7.45 (m, 10H); C NMR (75 MHz, CD Cl) δ 14.51,
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10
11
12
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0.92, 58.94, 66.72, 70.10, 73.03, 74.02, 108.96, 114.51, 127.71, 127.83, 128.11, 128.60, 128.78,
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129.12, 132.95, 136.83, 137.38, 156.04, 158.62; LCMS: m/s 505.2 [M+H] .
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-((1S,2S)-2-amino-1-hydroxy-2-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)phenol
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(20): Method F, (0.280 g, 96% yield) as white solid. H NMR (300 MHz, CD OD) δ 0.82 (s,
3
3H), 3.02 (d, J=7.2 Hz, 1H), 3.95 (s, 6H), 4.72 (d, J=7.2 Hz, 1H), 6.75 (d, J=8.4 Hz, 2H), 7.19 (d,
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J=8.7 HZ, 2H); C NMR (75 MHz, CD OD) δ 12.97, 30.32, 59.78, 72.52, 73.42, 108.66,
3
+
114.71, 128.85, 157.18; LCMS: m/s 282.2 [M+H] .
4-((1S,2S)-1-amino-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)propan-2-
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yl)phenol (21a): Method F, (0.343 g, 97% yield) as white solid. H NMR (300 MHz, CD OD) δ
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0.77 (s, 3H), 1.60 (s, 3H), 2.96 (s, 1H), 3.86 (s, 6H), 6.73 (d, J=8.7 Hz, 2H), 7.26 (d, J=8.7 Hz,
1
3
2H); C NMR (75 MHz, CD OD) δ 12.98, 24.07, 30.11, 62.99, 72.29, 75.52, 109.59, 114.24,
3
+
27.21, 136.78, 16.25; LCMS: m/s 296.1 [M+H] .
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4-((1S,2R)-1-amino-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)propan-2-
1
yl)phenol (21b): Method F, (0.339 g, 99 % yield) as white solid. H NMR (300 MHz, CDCl ) δ
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0.82 (s, 3H), 1.57 (s, 3H), 3.93 (s, 6H), 4.06 (br, s, 2H), 5.29 (s, 1H), 6.54 (d, J=8.1 Hz, 2H), 7.13
1
3
(d, J=7.8 Hz, 2H); C NMR (75 MHz, CDCl ) δ 14.51,29.91, 30.67, 61.76, 72.74, 74.88,
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+
09.85, 115.72, 125.63, 137.17, 155.57; LCMS: m/s 296.1 [M+H] .
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-((4-aminophenyl)buta-1,3-diyn-1-yl)-N-((1S,2S)-2-hydroxy-2-(4-hydroxyphenyl)-1-(4-
methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)benzamide (22a): Method A, (165 mg, 87%
0
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9
0
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yield) as yellow solid. H NMR (300 MHz, CD OD) δ 0.82 (s, 3H), 3.98 (s, 6H), 4.54 (d, J=8.4
3
Hz, 1H), 4.95 (d, J=8.4 Hz, 1H), 6.61 (d, J=8.7 Hz, 2H), 6.68 (d, J=8.7 Hz, 2H), 7.21 (d, J=6.6
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Hz, 2H), 7.23 (d, J=6.6 Hz, 2H), 7.43 (s, 4H); C NMR (75 MHz, CD OD) δ 12.99, 30.460,
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5
7.49, 70.77, 72.67, 73.07, 76.29, 79.06, 84.38, 108.35, 108.66, 114.21, 114.47, 125.41, 127.26,
+
128.85, 131.68, 131.79, 133.79, 134.74, 150.19, 156.97, 167.68; LCMS: m/s 525.1 [M+H] .
4-((4-aminophenyl)buta-1,3-diyn-1-yl)-N-((1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-(4-
methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)benzamide (22b): Method A, (337 mg, 98%
1
yield) as yellow solid. H NMR (300 MHz, CD OD) δ 0.79 (s, 3H), 3.96 (s, 6H), 4.41 (d, J=1.5
3
Hz, 1H), 5.27 (s, 1H), 6.61 (d, J=9.0 Hz, 2H), 6.67 (d, J=8.7 Hz, 2H), 7.17 (d, J=8.4 Hz, 2H),
13
7.25 (d, J=8.7 HZ, 2H), 7.52 (d, J=8.7 Hz, 2H), 7.70 (d, J=8.7 Hz, 2H); C NMR (75 MHz,
CD OD) δ 13.08, 30.46, 58.04, 70.50, 7.91, 72.68, 76.58, 79.16, 84.58, 108.38, 108.51, 114.28,
3
114.68, 125.72, 127.08, 127.36, 132.07, 132.68, 133.87, 134.41, 150.159, 156.47, 167.73;
+
LCMS: m/s 525.2 [M+H] .
4-((4-aminophenyl)buta-1,3-diyn-1-yl)-N-((1S,2S)-2-hydroxy-2-(4-hydroxyphenyl)-1-(4-
methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)propyl)benzamide (22c): Method A, (350 mg,
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7
6
2
8% yield) as yellow solid. H NMR (300 MHz, DMSO-d ) δ 0.70 (s, 3H), 1.50 (s, 3H), 3.78 (s,
6
H), 4.42 (d, J=8.7 Hz, 1H), 4.53 (s, 1H), 5.82 (s, 1H), 6.53 (d, J=8.4 Hz, 4H), 7.18 (d, J=8.7 Hz,
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H), 7.24 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.4 Hz, 2H), 7.68-7.73 (m, 3H), 9.00 (s, 1H); C NMR
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75 MHz, DMSO-d ) δ 14.29, 27.15, 30.57, 49.29, 60.06, 71.80, 72.32, 75.56, 77.08, 80.89,
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6.26, 105.86, 109.45, 114.27, 124.39, 127.54, 132.44, 134.68, 135.48, 137.60, 151.54, 155.96,
+
65.50; LCMS: m/s 539.4 [M+H] .
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4-((4-aminophenyl)buta-1,3-diyn-1-yl)-N-((1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-(4-
methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)propyl)benzamide (22d): Method A, (353 mg,
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7
9.1% yield) as yellow solid. H NMR (300 MHz, DMSO-d ) δ 0.74 (s, 3H), 1.56 (s, 3H), 3.84
6
(s, 6H), 4.47 (s, 1H), 5.82 (s, 2H), 6.51 (d, J=2.7 Hz, 2H), 6.54 (d, J=2.7 Hz, 2H), 7.16 (d, J=8.7
13
Hz, 2H), 7.23 (d, J=8.4 Hz, 2H), 7.49-7.57 (m, 4H), 9.00 (s, 1H); C NMR (75 MHz, DMSO-d )
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δ 14.38, 29.55, 30.62, 59.47, 71.79, 72.33, 75.58, 77.08, 80.81, 86.25, 105.85, 109.40, 114.26,
114.65, 124.35, 126.62, 128.19, 132.53, 134.68, 135.42, 138.53, 151.53, 155.90, 165.04; LCMS:
+
m/s 539.2 [M+H] .
(2S,3S)-3-hydroxy-2-(hydroxymethyl)-2-methylpropyl 2-(4-((4-aminophenyl)buta-1,3-diyn-
1-yl)benzamido)-3-hydroxy-3-(4-hydroxyphenyl)propanoate (23a): Method G, (126 mg, 88%
1
yield) as yellow solid. H NMR (300 MHz, CD OD) δ 0.86 (s, 3H), 3.45-3.43 (m, 4H), 4.09 (q,
3
J=32,4 Hz, 2H), 4.89 (d, J=7.5 Hz, 1H), 5.05 (d, J=7.5 Hz, 1H), 6.60 (d, J=8.7 Hz, 2H), 6.78 (d,
J=8.4 Hz, 2H), 7.24 (d, J=8.7 Hz, 2H), 7.26 (d, J=8.7 Hz, 2H), 7.48 (d, J=8.4 Hz, 2H), 7.60 (d,
1
3
J=8.7 Hz, 2H); C NMR (75 MHz, CD OD) δ 15.75, 40.83, 59.21, 64.58, 67.31, 70.92, 73.33,
3
76.75, 79.10, 84.65, 108.44, 114.32, 115.03, 125.98, 127.42, 127.96, 131.62, 132.07, 133.76,
+
33.88, 150.10, 157.23, 167.84, 171.21; LCMS: m/s 543.2 [M+H] .
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