Synthesis, NMR characterization and divergent biological actions of 2′-hydroxy-ceramide/dihydroceramide stereoisomers in MCF7 cells

Bioorganic & Medicinal Chemistry 18 (2010) 7565–7579

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Bioorganic & Medicinal Chemistry

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Synthesis, NMR characterization and divergent biological actions

of 2⁰-hydroxy-ceramide/dihydroceramide stereoisomers in MCF7 cells

Zdzislaw M. Szulc ^a, Aiping Bai ^a, Jacek Bielawski ^a, Nalini Mayroo ^a, Doreen E. Miller ^a,b, Hanna Gracz ^c,

Yusuf A. Hannun ^a, Alicja Bielawska ^a,

⇑

^aLipidomics Shared Resource, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA

^bRoche Carolina, Inc., Florence, SC 29506, USA

^cDepartment of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA

a r t i c l e i n f o

a b s t r a c t

A straightforward method for the simultaneous preparation of (2S,3R,2⁰R)- and (2S,3R,2⁰S)-2⁰-hydroxy-

ceramides (2⁰-OHCer) from (2S,3R)-sphingosine acetonide precursors and racemic mixtures of 2-hydroxy

fatty acids (2-OHFAs) is described. The obtained 2⁰-OH-C4-, -C6-, -C12-, -C16-Cer and 2⁰-OH-C6-dhCer

pairs of diastereoisomers were characterized thoroughly by TLC, MS, NMR, and optical rotation. Dynamic

and multidimensional NMR studies provided evidence that polar interfaces of 2⁰-OHCers are extended

and more rigid than observed for the corresponding non-hydroxylated analogs. Stereospeciﬁc proﬁle

on growth suppression of MCF7 cells was observed for (2⁰R)- and (2⁰S)-2⁰-OH-C6-Cers and their dihydro

Article history:

Received 25 June 2010

Revised 25 August 2010

Accepted 26 August 2010

Available online 28 September 2010

Keywords:

Ceramide

(2⁰R)-2⁰-Hydroxy-ceramide

(2⁰S)-2⁰-Hydroxy-ceramide

Stereospeciﬁcity

Synthesis

LC–MS/MS

NMR

Cytotoxicity

Cellular sphingolipids

analogs. The (2⁰R)-isomers were more active than the (2⁰S)-isomers (IC₅₀ꢀ3

l

M/8

l

M and IC₅₀ꢀ8

lM/

12

l

M, respectively), surpassing activity of the ordinary C6-Cer (IC₅₀ꢀ12

lM) and C6-dhCer (IC₅₀

ꢀ38

l

M). Neither isomer of 2⁰-OH-C6-Cers and 2⁰-OH-C6-dhCers was metabolized to their cellular long

chain 2⁰-OH-homologs. Surprisingly, the most active (2⁰R)-isomers did not inﬂuence the levels of the cel-

lular Cers nor dhCers. Contrary to this, the (2⁰S)-isomers generated cellular Cers and dhCers efﬁciently. In

comparison, the ordinary C6-Cer and C6-dhCer also signiﬁcantly increased the levels of their cellular long

chain homologs. These peculiar anabolic responses and SAR data suggest that (2⁰R)-2⁰-OHCers/dhCers

may interact with some distinct cellular regulatory targets in a speciﬁc and more effective manner than

their non-hydroxylated analogs. Thus, stereoisomers of 2⁰-OHCers can be potentially utilized as novel

molecular tools to study lipid–protein interactions, cell signaling phenomena and to understand the role

of hydroxylated sphingolipids in cancer biology, pathogenesis and therapy.

Published by Elsevier Ltd.

1. Introduction

components of mammalian and plant speciﬁc lipid membranes,

cells, organs, and tissues including brain, epidermis and epithelial

Ceramides (Cers, Scheme 1) have emerged as key modulators of

cancer cell growth and apoptosis.^1–4They are a highly diversiﬁed

structures consisting of different sphingoid base backbones and

an amide linked varied chain fatty acids (FAs; mostly C14–C26 sat-

urated, unsaturated, and hydroxylated FAs).⁵

tissue of the digestive tract.^15–18Despite their prevalence in the

nervous system, epidermis, and kidney as well as suggested and

conﬁrmed contributions to the pathogenesis of many diseases such

as Farber’s disease,^19,20hereditary leukodystrophy,²¹and can-

cer,^22,23their physiological functions remain mostly unknown.¹⁸

2⁰-OH-Sphingomyelin, glucosyl-2⁰-OH-Cer, lactosyl-2⁰-OH-Cer,

and their complex glyco-SPL analogs have been isolated from dif-

ferent natural sources.^18,24–26Galactosylceramide, commonly

called ‘‘cerebroside”, containing cerebronic acid in the N-acyl part

of Cer (2⁰-OH-C24-Cer) is a major glycosphingolipid in brain and

nervous tissues and builds up its complex analogs: sulfatide, gan-

glioside GM4 (sialylgalactosyl-Cer) and Gal2Cer (galabiosyl-

Cer).²⁷Interestingly, the ratio of hydroxy- to non-hydroxy-FA in

cerebrosides increases with the complexity of the central nervous

system.²⁸This cerebroside was also identiﬁed in intestines from

the Japanese quail and Runner and Kidney beans.^29,30

Naturally occurring 2⁰-hydroxy-Cers (2⁰-OHCers), namely

(2S,3R,2⁰R,4E)-2⁰-hydroxy-Cers, Scheme 1), contain an additional

hydroxyl group located in the alpha position to the carbonyl group

in their N-acyl moieties. The formation of an additional chiral cen-

ter in these molecules gives rise to the possibility to create two

stereoisomers: 2⁰R or 2⁰S. Natural 2⁰-OHCers represent (2⁰R)-iso-

mer.^6–14

A large body of recent research showed that sphingolipids

(SPLs) containing 2⁰-OHCers as their building blocks are common

⇑

2⁰-OHCers are present as sub-components of the skin (Cer 4–7,

9 sub-classes).^15,31–33They are thought to have structural roles to

Corresponding author. Tel.: +1 843 792 0273; fax: +1 843 792 6080.

E-mail address: bielawsk@musc.edu (A. Bielawska).

0968-0896/$ - see front matter Published by Elsevier Ltd.

doi:10.1016/j.bmc.2010.08.050

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Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

C6-Cers:

C6-dhCers:

OH

n =12, m = 2

1

2

3

HO

3

HO

n

N

2'

H

m

O

(2S,3R,4E)-Ceramide: Cer

(2S,3R)-dihydroCeramide: dhCer

OH

1

2

3

1

2

3

HO

n

OH

n

OH

N

2'

H

m

O

(2S,3R,2'R)-2'-hydroxy-dihydroCeramide: (2'R)-2'-OHdhCer

(2S,3R,2'R,4E)-2'-hydroxy-Ceramide: (2'R)-2'-OHCer

LCL467: (2'R)-2'-OH-C6-dhCer

LCL366: (2'R)-2'-OH-C6-Cer

OH

1

2

3

HO

n

OH

N

2'

H

m

O

(2S,3R,2'S)-2'-hydroxy-dihydroCeramide: (2'S)-2'-OHdhCer

(2S,3R,2'S,4E)-2'-hydroxy-Ceramide: (2'S)-2'-OHCer

LCL466: (2'S)-2'-OH-C6-dhCer

LCL367: (2'S)-2'-OH-C6-Cer

Scheme 1. Chemical structures of D D

-erythro-Cer/dhCer and (2⁰R)- and (2⁰S)-isomers of -erythro-2⁰-OHCer/dhCers.

facilitate tight packing of lipids and to inﬂuence the conformation

of the head groups via the hydrogen bonds.^33–35This class of Cers

was also identiﬁed in human erythrocytes, the small intestine liver

and kidney of rats and the Harderian gland of guinea pigs.^18,36

Some special 2⁰-OHCers (C3, C16–C26, and C29) were found to

have a remarkable biological effects, including cytotoxicity and

antiproliferative activities related to activation of caspase 3.^37–40

2⁰-OHCers may be produced by the action of fatty acid

2-hydroxylase (FA2H), which has been recently identiﬁed molecu-

larly.^18,41,42Mutation of this enzyme results in inherited human

neurodegenerative disorders.^21,43

Interestingly, deﬁciency in lysosomal CDase causes a lysosomal

storage disease, resulting in the accumulation of 2⁰-OHCers and

Cers in the kidneys, cerebrum, lungs, and stomach of Farber’s dis-

ease patients.^19,20,44,45Studies performed in certain animal cells

showed that 2⁰-OHCers were preferably converted to galactosyl-

ceramides, whereas Cers with normal fatty acids were used for

glucosylceramides formation, but this was not a universal rule.⁴⁶

Elevated cellular toxicity and plant growth regulatory activity of

some natural 2⁰-OHCers, were also reported.^11,14

To date we do not know what factors predominantly control the

structure of 2⁰-OHCer or are responsible for its distinct biological

functions. The following are lines of evidence conﬁrming the roles

of 2⁰-OH group of 2⁰-OHCers: (i) formation of the extensive net-

work of hydrogen bonds between the galactose head group of com-

plex 2⁰-OH-SPL and the polar head of Cer, (ii) promotion of Cers

monolayer condensation to a close-packed arrangement, and (iii)

increased effects on the phase transition temperatures and gel

phases stabilization in comparison to non-hydroxylated

analogs.^35,47,48

Although ceramide has been extensively studied as a mediator

of apoptotic responses, 2⁰-OHCers have never been considered as

ubiquitous signaling molecules. However, our preliminary studies

showed that 2⁰-OHCers were ubiquitously present at a very low le-

vel in numerous cancer cell types (e.g., ꢀ5% of total Cers in MCF7

cells, unpublished) suggesting that these Cers may play a role in

Cer-mediated cell signaling.

Current knowledge on the potential involvement of 2⁰-OHCers

in metabolism and cell signaling phenomena as well as access to

advanced structure–biological activity correlation data is very lim-

ited due to the lack of well-characterized synthetic 2⁰-OHCers to be

potentially used as molecular probes and analytical standards. To

address the above issues and to have non-restricted access to var-

ied chain model 2⁰-OHCers, we developed a simple and efﬁcient

method for the simultaneous preparation of their pure isomers

from easily available precursors and reagents.

In this publication we describe synthesis of (2⁰R)- and (2⁰S)-2⁰-

OH-C4-, C6-, C12-, C16-Cer, and (2⁰R)- and (2⁰S)-2⁰-OH-C6-dhCer

isomers and present their full physiochemical and NMR character-

ization. Basic biological effects of the newly synthesized 2⁰-OH-C6-

Cer and 2⁰-OH-C6-dhCer diastereoisomers were examined in breast

carcinoma MCF7 cells and compared to the activity of C6-Cer and

C6-dhCer. We also studied metabolism and effects of these com-

pounds on cellular SPLs.

2. Results and discussion

2.1. Chemistry

2.1.1. Synthetic strategy and selection of model 2⁰-OHCers

The most important challenge in the synthesis of naturally

occurring 2⁰-OHCer is to construct the amide bond between the

sphingoid base backbone and the appropriate 2-OHFA moiety,

while preserving the stereochemistry of the used building

blocks.^6,7,9,11,39In all reported cases, beside the necessity to make

the appropriate sphingoid base precursor, the synthesis of the

R-isomer of any long chain 2-OHFAs was also required, since these

lipids are not yet commercially available. Generally, all these

methods suffer from many drawbacks, namely tedious synthesis

procedures and multiple puriﬁcation steps, which make them

impractical for a large-scale preparation of varied chains 2⁰-OH-

Cers. Faced with this predicament, we undertook an attempt to

simplify that strategy and develop a practical method of synthesis

of 2⁰-OHCers capable of producing

a chromatographically

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

7567

separable set of their two diastereoisomers simultaneously, when

racemic mixtures of 2-OHFAs are used in the key synthetic step.

The rationale for this approach was based on two encouraging

observations. First, the introduction of the acetonide protective

group into the sphingolipid system facilitated the amide bond for-

mation reaction and secondly, this protecting group was used as a

separation handle for polyol isomers and their analogs.^9,39,49,50

We introduce C4-, C6-, C12-, and C16 homologs of

(2S,3R,2⁰R,4E)-OHCer and their corresponding (2S,3R,2⁰S,4E)-iso-

mers as well as (2S,3R,2⁰R)-2⁰-OH-C6-dhCer and (2S,3R,2⁰S)-2⁰-

OH-C6-dhCer. In order to validate the selected method and conﬁrm

the structures of 2⁰-OHCer and 2⁰-OHdhCer we compared their

properties and spectra to the model (2⁰R)- and (2⁰S)-2⁰-OH-C4-Cer

stereoisomers, which were prepared ﬁrst from enantiomerically

pure 2-hydroxybutyric acids as well as to the commercially avail-

able natural 2⁰-OH-C18-Cer, isolated from cerebroside.

of (R)- and (S)-isomers of 2⁰-OHCers (3b–d, 4b–d, 5b, and 6b),

which were easily separated by normal column chromatography

(D

TLC R_f= 0.18–0.2). Final treatment of the separated 2⁰-OHCer

acetonide isomers with p-TsOH in methanol at room temperature

provided the desired pure isomers of 7a–d, 8a–d, 9b, and 10b in

a satisfactory overall yield (26–33%). All synthesized (2⁰R)-OHCers

gave positive values for their optical rotations, which is in full

agreement with the reported data for these natural isomers.^6–14

2.2. NMR characterization of 2⁰-OHCers

To date, all reported NMR studies of 2⁰-OHCers and their ana-

logs were focused on the structure determination or conﬁrmation

of the isolated and synthesized compounds.^{6–14,38,39,51}Strategi-

cally, most of these experiments focused on the identiﬁcation of

D₂O exchangeable doublet at d 7.20, indicative of the NH proton

of the amide part located at the center of Cer structure. Using this

proton as a starting point and applying COSY, TOCSY, ROESY, and

HSQC experiments, the putative structure of Cer having 2-OHFA

moiety was easily conﬁrmed.^52,53However, there are no reports

dealing with the detailed analysis of 2⁰-OHCer diastereoisomers

spectra and their comparison with the ordinary Cers.

In our studies NMR experiments were used to: (i) discriminate

between the 2⁰R and 2⁰S-isomers of the synthesized short and long

chain 2⁰-OHCers, (ii) compare their spectra to the natural long

chain 2⁰-OHCers and model non-hydroxylated analogs, and (iii)

analyze the inﬂuence of the additional hydroxy group in the N-acyl

part on the structure of the extended polar head.

2.1.2. Synthesis of 2⁰-OHCer stereoisomers

The synthetic routes of 2⁰-OHCers 7a–d, 8a–d, 9b and 10b are

summarized in Scheme 2. To demonstrate the amenability of our

strategy toward the synthesis of a separable set of varied chain

diastereoisomers of 2⁰-OHCers, we ﬁrst investigated a condensa-

tion of commercially available R- and S-isomers of 2-hydroxybu-

tyric acid with the known acetonide of sphingosine.^9,39Using a

standard procedure for the amide bond formation, that is, dicyclo-

hexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), and

1-hydroxybenzotriazole (HOBt) in ethylene glycol dimethyl ether

(EGDE) solution, ceramides 3a and 4a were obtained in 82% and

95% yields, respectively. Most importantly, these model com-

pounds showed a quite different TLC R_fvalues (0.36 and 0.64,

One and two-dimensional ¹H and ¹³C NMR experiments clearly

exhibited connectivity, chemical shift values and coupling features

expected for 2⁰-OHCers (Fig. 1A–D and data listed in the Section 4).

Also, they showed signiﬁcant differences in the values of d_Hand d_C

for the polar head protons as well as carbons in comparison to the

parent Cers.

respectively) and opposite optical rotations (½a _D²²

+8.9 and ꢂ14.7,

ꢁ

respectively). R_fvalues of 3a and 4a strongly suggested that their

mixtures should be easily separated under normal column chro-

matography conditions. We then explored whether a condensation

of the longer chain 2-OHFA reacemic mixtures with sphingosine

acetonides would follow the same pattern.

Summarizing key ﬁndings of the study: (i) strong deshielding

0

effects for the 2⁰-H protons (

D

d_{2 H}= 1.72–1.78 ppm, Fig. 1A and

0

Indeed, the coupling of the amino component 1 or 2 with the

corresponding racemic 2-OHFAs delivered the expected mixtures

B) and the chiral C₂carbons (

D

d_C2= 35–36 ppm, Fig. 1D) in both

isomers were observed, (ii) the NH protons were signiﬁcantly

OH

O

p-TsOH

C₁₃H₂₇

HO

OH

MeOH

r.t.

N

H

n

O

7a-d: (2S,3R,4E,2'R)

9b: (2S,3R,2'R)

3a-d: (2S,3R,4E,2'R)

5b: (2S,3R,2'R)

HO

n

HO

C₁₃H₂₇

DCC, NHSA

HOBt, EGDE

r.t.

NH₂

1: (2S, 3R, 4E)

2: (2S, 3R)

OH

O

p-TsOH

C₁₃H₂₇

OH

HO

OH

MeOH

r.t.

N

H

n

O

8a-d: (2S,3R,4E,2'S)

10b: (2S,3R,2'S)

4a-d: (2S,3R,4E,2'S)

6b: (2S,3R,2'S)

a: n=1; b: n=3; c: n=9; d: n=13

Scheme 2. Synthetic outline for the preparation of 2⁰-OHCers and 2⁰-OHdhCers.

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Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

Figure 1. 500 MHz NMR spectra of 2⁰-OHCers and Cers. (A) Comparison of the NMR spectra of 2⁰-OH-C6-Cer diastereoisomers with C6-Cer in CD₃OD solution. Signature shift

of the 3-H proton and anisochronism of the polar interface 1Hab/3⁰-Hab protons. (B) Comparison of (2⁰R)-, (2⁰S)-2⁰-OH-C16-Cers and C16-Cer spectra in CDCl₃solution. (C)

¹H–¹H-COSY spectrum of (2⁰R)-2⁰-OH-C6-Cer in CD₃OD solution. (D) ¹H–¹³C-HSQC spectrum of (2⁰R)-2⁰-OH-C6-Cer in CD₃OD solution.

shifted to the lower ﬁeld (

to the corresponding protons in non-hydroxylated Cers, and (iii)

well-separated resonance signals for each methylene 1-Hab and

D

d_NH= 0.9 ppm, Fig. 1B) in comparison

3⁰-Hab protons in both 2⁰-OHCer isomers, in CDCl₃and CD₃OD

solutions, were present (Fig. 1A and B). This is quite a different

behavior than we usually observe in the spectra of ordinary Cers,

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

7569

Fig. 1 (continued)

where the 1-Ha, 1-Hb and the N-acyl chain methylene proton sig-

nals always appeared as one resonance signal (narrow doublet or

multiplet) in CD₃OD solution.^54,55All of the above spectral features

most likely resulted from the extensive intramolecular H-bonding

interactions between the hydroxyl groups and the amide group of

the polar interface of 2⁰-OHCer. As a consequence, elevated rigidity

of the extended polar heads in 2⁰-OHCers is expected.⁵⁴

Generally, all NMR spectra of varied chain 2⁰-OHCer isomers are

similar however, the (2⁰S)-isomers showed higher deshielding

effects for their 3-H protons than (2⁰R)-isomers (

Dd_3-H= 0.06–

0.17 ppm). The observed difference can be the consequence of a

proximal position of the 2⁰-hydroxy amide unit to the 3-H proton

in (2⁰S)-isomer. The juxtaposition of the amide group may poten-

tially cause a deshielding effect of the C@O group on 3-H proton

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Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

(spatial magnetic anisotropy).⁵⁶Also, the 3-OH group of 2⁰-OHCer

have increased opportunity to form more H-bonds between the

remaining proton-donor and acceptor centers than in non-hydrox-

ylated Cer. Nevertheless, to achieve the full capability of interac-

tions, the polar head of Cer must have its all H-donor/acceptor

groups in the gauche spatial disposition (-sc staggered con-

former).⁵⁴In order to fulﬁll this spatial arrangement and launch

effective H-bonding interactions inside the polar interface, the

(2⁰S)- and (2⁰R)-isomers may need to twist their N-acyl parts out

of the line with the sphingosine backbone. The degree of this dis-

tortion may have impact on the overall structure of 2⁰-OHCers. At

this point of the study we do not know exactly which particular

isomer, if not both, may have their backbones in a different spatial

arrangements (i.e., syn or anti) in comparison to the ordinary Cers.

In summary, these structural features can be responsible for the

observed distinct deshielding effect for the 3-H protons of 2⁰-OH-

Cers. Moreover, their NMR resonance signals (d_3H= ꢀ4.30 ppm/

(2⁰S)-isomers vs ꢀ4.13 ppm/(2⁰R)-isomers) can be directly used as

diagnostics to monitor and conﬁrm the level of optical purity of

the prepared 2⁰-OHCer diastereoisomers.

24 h treatments). IC₅₀values for (2⁰R)- and (2⁰S)-isomer of 2⁰-OH-

C6-Cers and 2⁰-OH-C6-dhCers were established as 3/8

M and

8/12 M, respectively. Under identical conditions, the IC₅₀values

for C6-Cer and C6-dhCer were 12 M and 38 M, respectively.

l

Interestingly, all synthesized 2⁰-OH-Cers/dhCer were more potent

than the parent compounds and the naturally occurring (2⁰R)-iso-

mers had signiﬁcantly stronger antiproliferative effects as com-

pared to the (2⁰S)-isomers.

We tested also cytotoxic effect of 2⁰-OH-C4-Cer isomers. Both

isomers showed a very similar inhibitory effects on cell growth

with IC₅₀values corresponding to 7 lM and 9 l

M for the (2⁰R)-

and the (2⁰S)-isomer, respectively. Interestingly, as was previously

reported, the (2⁰S)-2⁰-OH-C3-Cer was much more potent then its

(2⁰R)-isomer in HL60 leukemia cells.³⁹

The potent antiproliferative effect of the (2⁰R)-2⁰-OH-C6-Cer

was also observed with human cervical cancer HeLa cells and hu-

man lung adenocarcinoma A549 cells, indicating that the effect is

not limited to breast cancer cells only (data not shown). A similar

trend, higher cytotoxic activity of 2⁰-OHCers isolated from different

natural sources in comparison to their non-hydroxylated analogs

was previously reported in HL60, K562, A-549, HepG2, Hep3B

and BGC-823 cells lines.^37,38,40

Based on this preliminary exploration, 2⁰-OHCer can be de-

scribed as a distinct lipophilic multidentate ligand possessing a

set of two rigid three-carbon subunits (C1/C2/C3 and C(O)/C⁰2/

C⁰3) in the vicinity of its polar interface. This kind of ligand has

the capability to control its own shape by utilizing electronic and

steric factors: H-bonding interactions to impose conformational

restriction on polar head and ﬁx the spatial arrangement of both

chains. Also, when its environment will change, the multidentate

and rigid polar head of 2⁰-OHCer may interact efﬁciently with its

target protein or other lipids. The proper assessment of 2⁰-OHCer

diastereoisomers⁰conformational preferences in solution will need

more advanced NMR and molecular modeling studies.

In summary, our data show that the introduction of the hydro-

xyl group at position 2⁰in the N-acyl part of Cer not only maintains

but also enhances the activity of 2⁰-OH-C6-dhCer over its normal

and desaturated analogs. These ﬁndings are also of interest when

considering other studies, showing higher cytotoxicity of phyto-

Cers than the corresponding dhCer and Cer analogs in SK-N-

BE(2)C cells.⁵⁹All these observations extend the existing ‘Cer

cytotoxicity paradigm’, which states that the trans double bond

between C4 and C5 atoms in the Cer backbone is essential for its

elevated activity and pro-apoptotic responses.^1,57,60,61Thus, the

cytotoxic activity of Cer seems to be rendered by the presence of

the double bond in the vicinity of its polar head and/or presence

of an additional hydroxyl group at position 2⁰or 4. Our and other

NMR studies pinpoint these electronic features of the Cer structure

as responsible for its polar head enhanced rigidity.⁶¹This may

facilitate the molecule’s molecular recognition and enhance its

binding to the target regulatory proteins.^4,62The formation of that

lipid–protein complex can be responsible for the observed elevated

cytotoxicity of 2⁰-OHCers and 2⁰-OHdhCer.

2.3. Inhibitory effects of 2⁰-OHCers/dhCers on MCF7 breast

carcinoma cells growth

The effects of the newly obtained compounds were examined in

MCF7 cells, using 2⁰-OH-C6-Cer and 2⁰-OH-C6-dhCer stereoiso-

mers, representing cell permeable short chain homologs of 2⁰-OH-

Cers/dhCers. Their activity was compared to the effects of D-e-C6-Cer

and

D-e-C6-dhCer, basic tools commonly used in the sphingo-

lipid research.^55,57,58When added to culture media, these analogs

showed high antiproliferative activities on cell growth (Fig. 2,

2.4. Cellular levels of 2⁰-OHCers/dhCers

Cellular levels of the representative C6-analogs in MCF7 cells

were established by MS analysis. Experimental data from cell treat-

ments at 24 h showed a dose-dependent increase for all tested ana-

logs, however, at different rates (Fig. 3A). (2⁰S)-Isomer (LCL367)

showed the highest level of the C6-Cer series whereas the (2⁰R)-

isomer (LCL366) showed the lowest level and C6-Cer was in the

middle. A similar pattern was observed for the C6-dhCer series.

140.0

C6-Cer

( 2'R) -2'-OH-C6-Cer ( LCL366)

( 2'S) -2'-OH-C6-Cer ( LCL367)

C6-dhCer

( 2'R) -2'-OH-C6-dhCer ( LCL467)

120.0

( 2'S) -2'-OH-C6-dhCer ( LCL466)

100.0

80.0

60.0

40.0

20.0

0.0

Cellular uptakes for 10 lM treatments were fast and at compa-

rable levels up to ꢀ1 h, with the highest levels for LCL366 and C6-

Cer (Fig. 3B). Following a time course, LCL366 was progressively

decreased to the lowest level among all tested analogs at 24 h.

LCL367 was elevated up to 2.5 h followed by a small decrease at

5 h and plateau to 24 h. C6-Cer was progressively increased up to

5 h followed by decrease at 24 h. C6-dhCer followed the curve of

C6-Cer, however at a lower rate. The 2⁰S-isomer of 2⁰-OH-C6-dhCer,

LCL466, was elevated up to 5 h reaching the level of C6-dhCer and

remaining constant up to 24 h. The (2⁰R)-isomer of 2⁰-OH-C6-

dhCer, LCL467, showed the lowest level, remaining in a plateau be-

tween 2.5–24 h.

0

5

10

15

20

25

30

35

40

45

Concentration (µM)

Figure 2. Inhibitory effects of 2⁰-OH-C6-Cer and 2⁰-OHC6-dhCer stereoisomers and

corresponding C6-Cer and C6-dhCer on MCF7 cell growth. Concentration-depen-

dent effect at 24 h.

In summary, we noticed differences in the cellular levels

between (2⁰R)- and (2⁰S)-isomers for 2⁰-OHCers and 2⁰-OHdhCers,

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

7571

250.0

200.0

150.0

100.0

50.0

C24-, C24:1, and C16-Cers are the major components (ꢀ31%, 44%

and 20%, respectively). The remaining C14-, C18-, C18:1-, and

C20-Cers represent almost equal values. Among the dhCer species

C16-dhCer is the major component (ꢀ60%) followed by C24:1-

dhCer (ꢀ12%). The remaining C14-, C20-, and C24-dhCers repre-

sent almost equal values. Among the 2⁰-OHCer species only C24-,

C26-, C16-, C24:1-, and C26:1 (ꢀ24%, 22%, 19%, 18%, and 17%,

respectively) represent components of this class. Similar delin-

quent presence and elevated levels of long chain 2⁰-OHCer species

were found in some mammalian tissues and organs.¹⁸

A

C6-Cer

( 2'R) -2'-OH-C6-Cer ( LCL366)

( 2'S) -2'-OH-C6-Cer ( LCL367)

C6-dhCer

( 2'R) -2'-OH-C6-dhCer ( LCL467)

( 2'S) -2'-OH-C6-dhCer ( LCL466)

2.5.1. Stereospeciﬁc effects of 2⁰-OH-C6-Cers on cellular

2⁰-OHCers, Cers, Sph, and S1P

0.0

0

5

10

15

20

25

30

35

40

45

Concentration (µM)

2.5.1.1. Concentration-dependent effects.

observed for 24 h treatment, both tested isomers of 2⁰-OH-C6-Cers

(2.5–20

M) showed no effect on endogenous 2⁰-OHCer levels

(data not shown). For the maximal concentration used, 10

LCL366 and 20 M LCL367, only a small increase was observed

Surprisingly, as

120.0

100.0

80.0

60.0

40.0

20.0

0.0

l

C6-Cer

B

( 2'R) -2'-OH-C6-Cer ( LCL366)

( 2'S) -2'-OH-C6-Cer ( LCL367)

C6-dhCer

lM

l

( 2'R) -2'-OH C6-dhCer ( LCL467)

(up to 130%; C24- and C24:1-Cers for LCL366; C16-, C24-, and

C24:1-Cers for LCL367). The results suggest that 2⁰-OH-C6-Cers

may not serve as good competitive substrates/inhibitors for en-

zymes of Cer metabolism or they are not efﬁciently delivered to

the cellular compartment where their active metabolic target can

be present. However, it was previously shown that natural 2⁰-OH-

Cer served as a good substrate for neutral ceramidase in vitro.⁶⁷It

was also shown that acidic ceramidase controlled intracellular 2⁰-

OHCer level in vivo.⁴⁴Interestingly, C6-Cer itself was not converted

to the cellular 2⁰-OHCer analogs (data not shown) but an increase

in Sph level was clearly noticed in this case (vide infra, Fig 4B). It

may suggest that the level of 2-OHFA-CoA or FA2H enzyme expres-

sion in cancer cells is low. All these observations are in conﬂict

with results showing formation of 2⁰-OH-C18-Cer from 2-OH-

C18-FA-CoA (racemic mixture) and dhSph in HEK293T cells.⁶⁸

Isomers of 2⁰-OH-C6-Cers did not elevate cellular Cer as effec-

tively as C6-Cer (Fig. 4A). Also, they did not elevate cellular Sph

as was observed for C6-Cer (Fig. 4B). Both, C6-Cer and LCL367

( 2'S) -2'-OH-C6-dhCer ( LCL466)

0

5

10

15

20

25

30

Time (h)

Figure 3. Cellular level of selected analogs in MCF7 cells. (A) Concentration-

dependent levels at 24 h. (B) Time-dependent levels for 10 M treatments.

l

with (2⁰R)-isomers being utilized over time similarly to the parent

compounds, whereas (2⁰S)-isomers being stable.

These results may suggest different metabolism of these com-

pounds and/or their binding to the speciﬁc protein targets.

(2⁰S-isomer), elevated cellular S1P (800% and 600% for 20

lM treat-

ments, respectively), whereas LCL366 (2⁰R-isomer), had no effect

on S1P (data not shown).

2.5. Effects of 2⁰-OHCers/dhCers on bioactive SPLs in MCF7

human breast carcinoma cells

C6-Cer caused a signiﬁcant dose-dependent increase of endog-

enous Cer and Sph (Fig. 3A, up to 900% and 600% for 20

tively). LCL367 followed the pattern of C6-Cer but with a much

lower effect (up to 500%/Cer and 150%/Sph for 20 M). LCL366 only

slightly increased endogenous Cer (up to 130%) and had no effect

lM, respec-

Cellular effects of 2⁰-OHCers on endogenous SPLs and their

comparison to the actions of ordinary Cers have not yet been inves-

tigated. Using the LC–MS/MS method and monitoring changes in

Cer species-Sph-S1P balance we generated a set of preliminary

data pinpointing metabolic junctures and differences between

the actions of model 2⁰-OH-C6-Cer diastereoisomers and their par-

ent analogs.

Studies assessing the roles of Cer in signal transduction phenom-

ena and in regulating tumor cell responses to chemotherapy have

shown that the active agonists, stress factors or cytotoxic drugs, usu-

ally cause an increase of the endogenous Cers, especially C14-, C16-,

and C18-Cers, followed later by cell death.^2,4,63This response was al-

ways observed after cell treatment with exogenous C2- and C6-Cers

at the appropriate concentration.^1,60–64C2- and C6-dhCers are com-

monly used as a negative control for ceramide action.^60,65However,

it is limited only to C2-dhCer as was previously reported by our and

other group.^60,66As shown in Fig. 2, C6-dhCer caused an inhibitory

l

on Sph as noticed for 10

centration used).

lM treatment (the highest possible con-

Next, we examined effects of these compounds on endogenous

Cer species (data not shown). The results showed that C6-Cer ele-

vated all Cer species starting from 5

served for C18-Cer, C14-, and C16-Cers (ꢀ80-, 40-, and 20-fold,

respectively, for 20 M treatment). Increase in the other Cers was

lM. The highest extent was ob-

l

to a much lower extent (C24:1-Cer, ﬁvefold and C24-Cer only

ꢀ0.2-fold). LCL367, the 2⁰S-isomer, followed the pattern of C6-Cer

with a speciﬁc increase in C18-Cer (30-fold), C14-Cer (20-fold),

C16-Cer (sevenfold), C24:1-Cer (fourfold) and C24-Cer (twofold).

LCL366, the 2⁰R-isomer, up to 5

however at 10

C14-Cer (threefold), C16-Cer (1.7-fold) and decrease of C24-Cer

and C24:1-Cer (to 70% and 50%, respectively) was observed. Again,

the (2⁰R)- isomer behaved differently from (2⁰S)-isomer and C6-Cer.

lM had no effect on Cer species,

M a very moderate elevation of C18-Cer (ꢀ5-fold),

l

effect on cell growth at concentrations higher than 10 lM and was

metabolized to C6-Cer and cellular dhCers followed by their desatu-

ration to the cellular Cers (Figs. 6–8).

MCF7 control cells showed the following Cer composition:

ꢀ87% Cers, 8% dhCers and ꢀ5% 2⁰-OHCers. Among the Cers species

2.5.1.2. Time-dependent effects.

Time-dependent effects on

lM treatments (Fig. 5). Both,

cellular Cers were determined for 10

LCL366 (2⁰R) and LCL367 (2⁰S) were not effective in formation of

7572

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

In summary, isomers of 2⁰-OH-C6-Cers inﬂuenced differently

1200.0

A

C6-Cer

bio-transformations of the cellular SPLs as compared to C6-Cer.

(2⁰S)-Isomer showed some similarities to the action of C6-Cer if

used at higher concentration and during longer treatment. The

observed lack of inﬂuence of the (2⁰R)-isomer on basic cellular SPLs

in MCF7 cells and its high cytotoxicity can not be related to its

anabolic transformations, which usually favor hydrolysis and

generation of Sph and long chain Cers.^57,58,60,61Results suggest that

this isomer or its unidentiﬁed yet metabolites may directly target

some key regulatory enzymes (e.g., phosphatases, kinases or casp-

ases).^3,4,69Our preliminary data support this hypothesis, since

some (2⁰R)-OHCers had a higher potency than non-hydroxylated-

Cer for the PPA2 enzyme activation (P. Roddy, D. Perry, and Y. A.

Hannun, unpublished data).

( 2'R) -2'-OH-C6-Cer ( LCL366)

( 2'S) -2'-OH-C6-Cer ( LCL367)

1000.0

800.0

600.0

400.0

200.0

0.0

In conclusion, an uncommon proﬁle of biological action for the

(2⁰R)-isomer, representing natural 2⁰-OHCers, was discovered by

comparison to the effects of C6-Cer and its unnatural (2⁰S)-isomer.

0

5

10

15

20

25

Concentration (µM)

800.0

700.0

600.0

500.0

400.0

300.0

200.0

100.0

0.0

C6-Cer

2.5.2. Stereospeciﬁc effects of 2⁰-OH-C6-dhCers on cellular SPLs

Since a metabolic conversion of C6-dhCer to C6-Cer was ob-

served for 24 h treatment (Fig. 6), we also investigated conversions

of 2⁰-OH-C6-dhCer diastereoisomers, LCL466 (2⁰S-isomer) and

LCL467 (2⁰R-isomer) to their desaturated counterparts as well as

effects on endogenous dhCers, Cers and both sphingoid bases.

In general, 2⁰-OH-C6-dhCers did not serve as a good substrate

for dhCer desaturase since only a small percent of conversion (up

to 12%) of LCL466 to the corresponding LCL367 and LCL467 to

the corresponding LCL366 (up to 5%) was observed as compared

to 42% conversion for C6-dhCer.

B

( 2'R) -2'-OH-C6-Cer ( LCL366)

( 2'S) -2'-OH-C6-Cer ( LCL367)

2.5.2.1. Concentration-dependent effects of 2⁰OH-dhCers on

endogenous SPLs.

0

5

10

15

20

25

2.5.2.1.1. Effect on endogenous dhCers (Fig. 7A). Up to 10 lM of

Concentration (µM)

C6-dhCer and both isomers of 2⁰-OH-C6-dh-Cer did not induce ele-

vation of cellular dhCers. This concentration represented the high-

Figure 4. Concentration-dependent effect of 2⁰-OHC6-Cer isomers on endogenous

bioactive SPLs for 24 h treatment. (A) Effect on total Cer. (B) Effect on Sph.

est concentration tested for (2⁰R)-isomer, since 20

l

M treatment

caused total cell death as shown in Fig. 2. LCL466, (2⁰S)-isomer,

elevated cellular dhCer to the highest extent (ꢀ19-fold/40

treatment). In comparison, C6-dhCer increased cellular dhCer only

M treatment. Regarding dhCer species (results not

l

M

cellular Cer as compared to C6-Cer. Some elevations (ꢀ140%/

LCL366 and ꢀ180%/LCL367) were observed up to 5 h with a plateau

up to 24 h. These effects were different from the action of C6-Cer,

where cellular Cers increased rapidly (500%) up to 5 h and de-

creased (350%) at 24 h. Again, a small increase in cellular Cer by

LCL366 did not correlate with its strong antiproliferative effect

ꢀ7-fold/40

l

shown), both LCL466 and C6-dhCer elevated mostly dhC16-Cer,

the major dhCer for MCF7 control cells (up to 25- and 13-fold,

50.0

(IC₅₀/24 h = 3

l

M, Fig. 2A).

C6-dhCer

45.0

( 2'R) -2'-OH-C6-dhCer ( LCL467)

600.0

( 2'S) -2'-OH-C6-dhCer ( LCL466)

40.0

C6-Cer

( 2'R) -2'-OH-C6-Cer ( LCL366)

( 2'S) -2'-OH-C6-Cer ( LCL367)

35.0

30.0

25.0

20.0

15.0

10.0

5.0

500.0

400.0

300.0

200.0

100.0

0.0

0

10

20

30

40

5

0.0

0

Concentration (µM)

5

10

15

20

25

30

Time (h)

Figure 6. Direct conversion of 2⁰-OHC6-dhCer stereoisomers and C6-dhCer to the

respective unsaturated analogs 2⁰-OHC6-Cer and C6-Cer. Concentration-dependent

effects for 24 h treatments.

Figure 5. Time-dependent effects of 10 l

M 2⁰-OH-C6-Cer stereoisomers and C6-Cer

on cellular Cers.

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

7573

respectively). LCL467 did not elevate C16-dhCer. Interestingly, it

slightly decreased the other dhCers, especially C14-dhCer (to 25%

control).

2.5.2.2. Time-dependent effects.

Time-dependent elevation

of cellular dhCer (Fig. 8A) and Cer (Fig. 8B) were observed for

10 M LCL466, up to 5 h, reaching 200% for both lipids. Ten micro-

l

2.5.2.1.2. Effect on endogenous Cers (Fig. 7B). Effect of these

analogs on endogenous Cer was different from their effects on

dhCers (Fig. 7A). The highest elevation was observed for C6-dhCer

for the full range of the concentration used, reaching 550% for

molar C6-dhCer elevated cellular dhCers to 750% and Cers to 310%.

LCL467, the (2⁰R)-isomer, did not affect cellular dhCers nor Cers for

this period of time; however, at 24 h Cer was slightly elevated

(ꢀ140%) whereas dhCer remained unchanged. At 24 h, dhCer gen-

erated by C6-dhCer decreased to almost control level whereas cel-

lular Cer was only slightly decreased (from 310% to 280%).

Regarding effect of LCL466 at 24 h, both cellular dhCer and Cer

were the same as for 5 h treatment. Among the dhCer species,

LCL466 and C6-dhCer elevated all dhCer species, whereas LCL467

decreased long chain dhCers (ꢀ10–25%) and increased very long

chain dhCers (ꢀ20–30%), data not shown. LCL466 and C6-dhCer

elevated all Cer species, particularly C18-, C14-, and C16-Cers how-

ever to very different extent (data not shown). Cellular dhSph

followed the curves of dhCer whereas Sph was decreased by both

2⁰-OH-dhCers and slightly increased by C6-dhCer (data not shown).

In conclusion, 2⁰-OH-C6-dhCer diastereoisomers showed differ-

ent effects on cellular dhCers, Cers, dhSph, and Sph. The (2⁰R)-isomer

was not effective, whereas (2⁰S)-isomer acted as a very potent indu-

cer of these lipids being three time more effective than C6-dhCer.

These results suggest that LCL466 may undergo anabolic transfor-

mations and/or may regulate the action of dhCer metabolizing en-

zymes. DhCer/Cer synthases or dhCer desaturase can be

considered as its targets. Most likely, LCL466 may act as an inhibitor

40

the effect of C6-dhCer, however, to a lower extent (ꢀ450% for

40 M treatment). LCL467 as similarly shown for cellular dhCer,

did not effect cellular Cer up to 5 M, however a 10 M treatment

l lM followed

M. LCL 466, the (2⁰S)-isomer, starting from 5

l

increased Cer to ꢀ150%. Regarding Cer species, all compounds ele-

vated mostly C18- and C14-Cers, followed by C24:1-Cer, however

at different rates (results not shown). C16-Cer was elevated up to

ꢀ50 fold by 40

lM LCL466 and C6-dhCer, whereas 1–5 lM

LCL467, caused a rather similar decrease (up to 60%) of this Cer.

2.5.2.1.3. Effect on sphingoid bases. MCF7 control cells (1 ꢃ 10⁶)

contain ꢀ20 pmols Sph and only ꢀ1 pmol of dhSph. Dose-depen-

dent increase of cellular dhSph was caused by LCL466, (2⁰S)-iso-

mer, increasing ꢀ16-fold for 40

did not effect cellular dhSph. C6-dhCer had small effect on dhSph

M) (Fig. 7C). LCL466 also caused elevation

lM treatments, whereas LCL467

elevation (ꢀ5-fold/40

l

of cellular Sph (Fig. 7D), however, to much lower extends in com-

parison to dhSph. LCL467 showed the same small decrease of Sph

(to ꢀ80%), whereas C6-dhCer decreased Sph at a low concentration

(to ꢀ70%), showing some recovery at a higher concentration.

700.0

2500.0

A

B

600.0

C6-dhCer

( 2'R) -2'-OH-C6-dhCer ( LCL467)

( 2'S) -2'-OH-C6-dhCer ( LCL466)

2000.0

C6-dhCer

( 2'R) -2'-OH-C6-dhCer ( LCL467)

( 2'S) -2'-OH-C6-dhCer ( LCL466)

500.0

1500.0

1000.0

500.0

0.0

400.0

300.0

200.0

100.0

0.0

0

5

10

15

20

25

30

35

40

45

0.0

10.0

20.0

30.0

40.0

50.0

Concentration (µM)

1800.0

1600.0

1400.0

1200.0

1000.0

800.0

600.0

400.0

200.0

0.0

C

250.0

200.0

150.0

100.0

50.0

D

dhC6-Cer

( 2'R) -2'-OH-C6-dhCer ( LCL467)

( 2'S) -2'-OH-C6-dhCer ( LCL466)

( 2'R) -2'-OH-C6-dhCer ( LCL467)

( 2'S) -2'-OH-C6-dhCer ( LCL466)

0.0

10.0

20.0

30.0

40.0

50.0

0.0

10.0

20.0

30.0

40.0

50.0

Concentration (µM)

Figure 7. Effect of 2⁰-OH-C6-dhCer stereoisomers and C6-dhCer on cellular SPLs at 24 h. (A). Concentration-dependent effects on cellular dhCers. (B) Concentration-

dependent effects on cellular Cer. (C) Concentration-dependent effects on cellular dhSph. (D) Concentration-dependent effects on cellular Sph.

7574

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

MCF7 cells, and other cell lines, responded differently to the ac-

800.0

A

C6-dhCer

( 2'R) -2'-OH-C6-dhCer ( LCL467)

( 2'S) -2'-OH-C6-dhCer ( LCL466)

tion of these two diastereoisomers in comparison to their non-

hydroxylated analogs. In general, both isomers of 2⁰-OH-C6-Cers

and 2⁰-OH-C6-dhCers were more potent in cell killing than corre-

sponding C6-Cer and C6-dhCer. Moreover, they were not metabo-

lized to their long chain 2⁰-OHCers and showed divergent

stereospeciﬁc effect on cellular Cer. Interestingly, 2⁰-OH-C6-dhCers

did not serve as a good substrate for dhCer desaturase as compared

to C6-dhCer. Data suggest that the observed high cytotoxic effects

of (2⁰R)-isomers are not the result of their anabolic inter-conver-

sions to sphingoid bases and long chain Cers or direct regulatory

actions on the Cer metabolizing enzymes.

In conclusion, the observed similarities in the early cellular

uptakes of (2⁰R)- and (2⁰S)-isomers, differentiated for the longer

treatments, may suggest a higher metabolic rate for (2⁰R)-isomers

or their efﬁcient binding to some important and speciﬁc proteins.

However, taking under consideration the following facts, that the

(2⁰R)-isomers of 2⁰-OH-C6- and 2⁰-OH-dhC6-Cers were not effective

in the generation of cellular Cers, sphingoid bases nor their GluCer

and GalCer analogs (our preliminary observation), we can rather

suggest that their high antiproliferative effects are the result of

their direct actions on the regulatory targets. Finally, the observed

divergences between (2⁰S)-OHCer/dhCer isomers and Cer/dhCer ac-

tions on anabolic transformation of the cellular SPLs pinpoint reg-

ulation of CDase, dhCerS and dhCer desaturase enzymes as the

cause of their elevated cytotoxicity.

700.0

600.0

500.0

400.0

300.0

200.0

100.0

0.0

0

5

10

15

20

25

30

Time (h)

400.0

300.0

200.0

100.0

0.0

B

C6-dhCer

( 2'R) -2'-OH-C6-dhCer ( LCL467)

( 2'S) -2'-OH-C6-dhCer ( LCL466)

Theobservations presentedhere, correlatingsteric andelectronic

inﬂuences on Cer conformation with its cytotoxicity and metabolic

inter-conversions, should prove their usefulness as a guide in defy-

ing key factors which control SPL–protein interactions.

Further studies on effects of 2⁰-OH-C6-Cer/dhCer diastereoiso-

mers on the complex SPLs, search for their direct cellular targets

and mechanism of cell death are pending.

0

5

10

15

20

25

30

Time (h)

Figure 8. Time-dependent effects of 10 l

M 2⁰-OH-C6-dhCer diastereoisomers and

C6-dhCer on cellular SPLs. (A) Effect on cellular dhCers. (B) Effect on cellular Cers.

4. Experimental

4.1. Chemistry

of dhCer synthase or activator of dhCer ceramidase, since elevation

of dhSph was observed. Moreover, LCL466 may additionally act as

an inhibitor of dhCer desaturase, since cellular dhCers generated

by this compound were not so efﬁciently transformed to their oxi-

dized analogs as those generated by the ordinary C6-dhCer (50/25

and 50/13-fold ratio for C16-Cer/C16-dhCer, respectively).

All solvents, general reagents and short chain (2R)- and (2S)-4-

hydroxybutyric acids were purchased from Aldrich–Sigma & Fluka.

D-e-Sph and D-e-dhSph were purchased from Avanti. Long chain

2-hydroxy-FAs and a model sample of naturally occurring (2⁰R)-

2⁰-hydroxy-C18-ceramide were purchased from Matreya. The

homogeneity and optical purity of these lipids were checked by

NMR, MS spectrometry and optical rotation.

3. Summary and conclusion

This work reveals a set of varied chain (2⁰R)- and (2⁰S)-isomers

of 2⁰-OHCers and 2⁰-OHdhCers as novel molecular tools to study

Cer and dhCer biology. Applying these tools and pursuing SAR

and metabolomic studies in MCF7 cells we contribute new insights

into the ‘‘Cer cytotoxicity paradigm”.

4.1.1. Natural (2⁰R)-2⁰-hydroxy-C18-ceramide (from Matreya,

cat. #1323)

TLC R_f= 0.40–42 (CHCl₃–CH₃OH, 45:5, v/v); ¹H NMR (500 MHz,

CDCl₃, d in ppm): 7.08 (d, 1H, J = 8.2, NH), 5.79 (dtd, 1H, J = 15.1,

6.8, 1.0, 5-H), 5.52 (ddt, 1H, J = 15.1, 6.8, 1.1, 4-H), 5.34 (m, ꢀ0.5H,

C⁰H@C⁰H), 4.30 (t, 1H, J = 6.0, 3-H), 4.13 (m, 1H, 2⁰-H_R), 3.92 (m, 2H,

1-Ha and 2-H), 3.74 (m, 1H, 1-Hb), 2.0 (m, ꢀ3H, C(6)H₂and C⁰H₂),

1.82 (m, 1H, 3⁰-Ha), 1.64 (m, 1H, 3⁰Hb), 1.26 (m, ꢀ48H, CH₂), 0.88

(t, 6H, J = 7.1, CH₃); ¹³C NMR (CDCl₃) d 174.4 (C@J), 134.3 (C5),

130.0 (C⁰H@C⁰H), 128.4 (C4), 74.3 (C3), 72.48 (C⁰2), 62.2 (C1), 54.5

(C2), 34.8 (C⁰3), 32.3 (C6), 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2,

29.1, 27.2, 25.0 and 22.7 (C7-C17 and C⁰4-C⁰15), 14.0 (C⁰H₃and

CH₃). ESI-MS (CH₃OH, relative intensity, %) m/z 664.6 ([M₁H]⁺, 32),

628.6 ([M₁HꢂH₂O]⁺, 4), (582.6 ([M₂H]⁺, 40), 564.2 ([M₂HꢂH₂O]⁺,

8), 546.7 (([M₂Hꢂ2H₂O]⁺, 4). Calcd for C₄₂H₈₂NO₄m/z 663.62 [M₁]

and C₃₆H₇₁NO₄m/z 581.54 [M₂].

In summary, we have developed a simple and convenient meth-

od for the simultaneous preparation of (2⁰R)- and (2⁰S)-isomers of

2⁰-OHCers and 2⁰-OH-dhCers starting from sphingoid base precur-

sors and a racemic mixture of 2-OHFA. The developed protocol

may provide an unrestricted access to pure (2⁰R)- and (2⁰S)-isomers

of varied chain 2⁰-OHCers and 2⁰-OHdhCers with enormous possi-

bilities to synthesize sub-classes of skin Cers and other complex

2⁰-OHSPLs for structural and biological studies. The NMR studies

showed that additional presence of the hydroxyl group in the

N-acyl part of Cer increased the hydrogen bonding capacity of

Cer/dhCer polar head and restricted its conformational ﬂexibility.

Thus, the presence or absence of these electronic and steric fea-

tures can be responsible for a different molecular recognition and

binding afﬁnities of 2⁰-OHCers/dhCers and their non-hydroxylated

analogs by metabolic and regulatory enzymes.

1,3-Isopropylidene-(2S,3R,4E)-sphingosine (1) and 1,3-isopro-

pylidene-(2S,3R)-dihydrosphingosine (2) were prepared from the

corresponding sphingoid bases by a slightly modiﬁed procedure

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

7575

described previously.⁹Purity of the synthesized compounds was

conﬁrmed by NMR spectroscopy and the measurement of their

optical rotation values. Reaction progress was monitored by the

analytical normal and reverse phase thin layer chromatography

(NP TLC or RP TLC) using aluminum sheets with 0.25 mm Silica

Gel 60-F₂₅₄(Merck) and 0.150 mm C18-silica gel (Sorbent Technol-

ogies). Detection was done by the PMA reagent (ammonium hepta-

molybdate tetrahydrate cerium sulfate, 5:2, g/g) in 125 mL of 10%

H2SO₄) and the Dragendorff reagent (Fluka) following heating of

the TLC plates at 170 °C or by the UV (254 nm). Flash chromatogra-

phy was performed using EM Silica Gel 60 (230–400 mesh) with

the indicated eluent systems. Melting points were determined in

open capillaries on Electrothermal IA 9200 melting point apparatus

and are reported uncorrected. Optical rotation data were acquired

using a Jasco P-1010 polarimeter. ¹H and ¹³C NMR spectra were re-

corded on Bruker AVANCE 500 MHz spectrometer equipped with

Oxford Narrow Bore Magnet. Chemical shifts are reported in ppm

on the d scale from the internal standard of residual chloroform

(7.26 ppm). Mass spectral data were recorded in a positive ion

electrospray ionization (ESI) mode on Thermo Finnigan TSQ 7000

triple quadrupole mass spectrometer. Samples were infused in

methanol solution with an ESI voltage of 4.5 kV and capillary tem-

perature of 200 °C. Speciﬁc structural features were established by

fragmentation pattern upon electrospray (ESI/MS/MS) conditions,

speciﬁc for each of the compounds studied.^70,71

rosphingosine (2, 106 mg, 0.31 mmol), 2-hydroxy fatty acid

(0.36 mmol), DCC (99%, 75 mg, 0.36 mmol), NHS (97%, 42 mg,

0.36 mmol) and HOBt (ꢀ5% water, 49 mg, 0.36 mmol) in dry EGDE

(5 mL) was stirred at room temperature for 4 h. The reaction mix-

ture was evaporated to dryness under reduced pressure and the

obtained residue was puriﬁed by ﬂash column chromatography

using the suitable solvent system.

4.1.3.1. (2S,3R,2⁰R,4E)-1,3-Isopropylidene-2⁰-hydroxy-C4-cera-

mide (3a).

Prepared from 1 and (2R)-2-hydroxybutanoic acid

in 82% yield after puriﬁcation by column chromatography (CHCl₃–

CH₃OH, 25:2, v/v). Analytical sample of 3a was prepared by crystal-

lization from ethyl acetate–n-hexane (3:1, v/v) as a white micro-

crystalline powder, mp 75–77 °C. TLC R_f= 0.36 (CHCl₃–CH₃OH,

22

365

25:2, v/v); ½a ²_D²

ꢁ

+8.9 (c 1, CHCl₃); ½

aꢁ

+19.2 (c 1, CHCl₃); ¹H

NMR (500 MHz, CDCl₃) d 6.31 (d, 1H, J = 8.6, NH), 5.74 (dtd, 1H,

J = 15.4, 7.6 and 1.0, 5-H), 5.42 (ddt, 1H, J = 15.4, 7.6 and 1.4, 4-

H), 4.07 (m, 2H, 2⁰-H_Rand 3-H), 3.97 (dd, 1H, J = 5.3 and 11.2, 1-

Ha), 3.86 (m, 1H, 2-H), 3.64 (dd, 1H, J = 9.5 and 11.2, 1-Hb), 2.01

(m, 2H, C(6)H₂), 1.82 (m, 3⁰-Ha, 1H), 1.65 (m, 3⁰-Hb, 1H), 1.49 (s,

3H, CH₃), 1.42 (s, 3H, CH₃), 1.24 (m, 22H, CH₂), 0.93 (t, 3H, J = 7.4,

CH₃), 0.87 (t, 3H, J = 7.1, CH₃). MS (CH₃OH) m/z 873.0 ([2M+Na]⁺,

23), 449.0 ([M+Na]⁺, 100), 368.0 ([MHꢂ(CH₃)₂CO]⁺, 5). Calcd for

C₂₅H₄₇NO₄m/z 425.35 [M].

4.1.3.2.

mide (4a).

(2S,3R,2⁰S,4E)-1,3-Isopropylidene-2⁰-hydroxy-C4-cera-

Prepared from 1 and (2S)-2-hydroxybutanoic acid

4.1.2. Preparation of 1,3-isopropylidene-sphingoid bases

4.1.2.1. 1,3-Isopropylidene-(2S,3R,4E)-sphingosine (1).

A

in 95% yield after puriﬁcation by column chromatography (CHCl₃–

CH₃OH, 25:2, v/v). Analytical sample of 4a was prepared by crystal-

lization from ethyl acetate–n-hexane (3:1, v/v) as a white micro-

mixture of (2S,3R,4E)-sphingosine (300 mg, 1.0 mmol), pyridinium

p-tolulenesulfonate (98%, 252 mg, 1.0 mmol) and 2,2-dimethoxy-

propane (6 mL) in dry toluene (10 mL) was stirred at reﬂux for 4 h.

The reaction mixture was then cooled to room temperature and di-

luted with ethyl acetate (10 mL), washed with saturated sodium

bicarbonate solution and water. The collected organic layer was

dried over anhydrous magnesium sulfate and ﬁltered. The solvent

was evaporated under reduced pressure and the obtained residue

was puriﬁed by ﬂash column chromatography (CHCl₃–CH₃OH–

NH₄OH concd, 15:1:0.025 v/v/v) to give pure 1 (312 mg, 92%) as a

pale yellow oil. This material formed a waxy solid upon storage in

crystalline powder, mp 62–63 °C. TLC R_f= 0.64 (CHCl₃–CH₃OH,

22

365

25:2, v/v); ½a ²_D²

ꢁ

ꢂ14.7 (c 1, CHCl₃₎; ½

a

ꢁ

ꢂ62.3 (c 1, CHCl₃); ¹H

NMR (500 MHz, CDCl₃) d 6.25 (d, 1H, J = 8.2, NH), 5.79 (dtd, 1H,

J = 15.4, 7.6 and 1.0, 5-H), 5.44 (ddt, 1H, J = 15.4, 7.6 and 1.4, 4-

H), 4.16 (dd, 1H, J = 7.8 and 9.4, 3-H), 4.06 (dd, 1H, J = 4.8 and

7.3, 2⁰-H_S), 4.02 (dd, 1H, J = 5.2 and 11.2, 1-Ha), 3.84 (m, 1H, 2-

H), 3.70 (dd, 1H, J = 9.6 and 11.2, 1-Hb), 2.05 (m, 2H, C(6)H₂),

1.87 (m, 3⁰-Ha, 1H), 1.68 (m, 3⁰-Hb, 1H), 1.53 (s, 3H, CH₃), 1.46 (s,

3H, CH₃), 1.24 (m, 22H, CH₂), 0.98 (t, 3H, J = 7.4, CH₃), 0.90 (t, 3H,

J = 7.1, CH₃); MS (CH₃OH) m/z 873.0 ([2M+Na]⁺, 60), 870.0

([2Mꢂ2H+Na]⁺, 100), 449.0 ([M+Na]⁺, 5), 368.0 ([MHꢂ(CH₃)₂CO]⁺,

23). Calcd for C₂₅H₄₇NO₄m/z 425.35 [M].

refrigerator (+4 °C) overnight. TLC R_f= 0.43 (CHCl₃–CH₃OH–NH₄OH

25

65

concd, 15:1:0.025, v/v/v); ½a _D²⁵

ꢁ

+11.0 (c 1, CHCl₃₎; ½

aꢁ

+38.7 (c 1,

CHCl₃); ¹H NMR (500 MHz, CDCl₃) d 5.82 (dtd, 1H, J = 15.2, 7.4 and

1.0, 5-H), 5.37 (ddt, 1H, J = 15.1, 7.6 and 1.4, 4-H), 3.85 (m, 2H, 1-H_a

and 3-H), 3.54 (dd, 1H, J = 10.8 and 11.2, 1-Hb), 2.70 (m, 1H, 2-H),

2.01 (m, 2H, C(6)H₂), 1.49 (s, 3H, CH₃), 1.42 (s, 3H, CH₃), 1.35 (m,

2H, CH₂), 1.24 (m, 22H, CH₂), 0.88 (t, 3H, J = 7.4, CH₃), 0.87 (t, 3H,

J = 7.1, CH₃). ¹³C NMR (500 MHz, CDCl₃) d 137.3, 127.8, 127.7,

100.4, 98.7, 78.4, 65.6, 49.3, 32.6, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5,

29.2, 22.9, 19.8, 19.4, 14.9, 14.4. MS (CH₃OH) m/z 340.8 ([MH]⁺,

100), 282.3 ([MH]⁺ꢂC₃H₆O, 15); Calcd for C₂₁H₄₁NO₂m/z 339.3 [M].

4.1.3.3.

Prepared from 1 and ^DL-2-hydroxyhexanoic acid in

41% yield after puriﬁcation by column chromatography (ethyl ace-

tate–n-hexane, 2:1, v/v). Analytical sample of 3b was prepared by

crystallization from ethyl acetate–n-hexane (1:3, v/v) as a white

microcrystalline powder, mp 77–79 °C. TLC R_f= 0.22 (ethyl ace-

22

365

tate–n-hexane, 1:1, v/v); ½a _D²²

ꢁ

+5.3 (c 0.5, CHCl₃); ½

aꢁ

+16.8 (c

0.5, CHCl₃); ¹H NMR (500 MHz, CDCl₃) d 6.36 (d, 1H, J = 8.8, NH),

5.77 (dtd, 1H, J = 15.1, 7.4, 1.0, 5-H), 5.42 (ddt, 1H, J = 15.1, 7.5,

1.4, 4-H), 4.09 (m, 2H, 2⁰-H_Rand 3-H), 3.97 (dd, 1H, J = 5.6 and

11.2, 1-H_a), 3.88 (m, 1H, 2-H), 3.66 (dd, 1H, J = 9.6 and 11.2, 1-

Hb), 2.38 (d, 1H, J = 4.8, 2⁰-OH), 2.04 (m, 2H, C(6)H_a), 1.87 (m, 2H,

CH₂), 1.81 (m, 1H, 3⁰-Ha), 1.70 (m, 2H, CH₂), 1.60 (m, 1H, 3⁰-Hb),

1.50 (s, 3H, CH₃), 1.43 (s, 3H, CH₃), 1.24 (m, 32H, CH₂), 1.15 (m,

2H, CH₂), 0.88 (m, 6H, 2 ꢃ CH₃). MS (CH₃OH) m/z 929.7

([2M+Na]⁺, 2), 449.5 (100), 414.4 (([MHꢂ(CH₃)₂C], 6) 396.2

([MHꢂ(CH₃)₂CO]⁺, 48). Calcd for C₂₇H₅₁NO₄m/z 453.38 [M].

4.1.2.2. 1,3-Isopropylidene-(2S,3R)-dihydrosphingosine (2).

The same procedure, as described for the synthesis of 1, was per-

formed using (2S,3R)-dihydrosphingosine as a starting material.

The crude product was puriﬁed by ﬂash chromatography (CHCl₃–

CH₃OH, 25:2 v/v) to give pure 2 in 99% yield as a pale yellow oil.

This material solidiﬁed upon storage in refrigerator (+4 °C) over-

night. TLC R_f= 0.58 (CHCl₃–CH₃OH, 25:2 v/v); ½a _D²³

ꢁ

+31.8 (c 1,

23

365

CHCl₃); ½

aꢁ

+97.0 (c 0.5, CHCl₃). Remaining spectral and analyti-

cal data are identical to the literature references.⁹

4.1.3.4.

mide (4b).

(2S,3R,2⁰S,4E)-1,3-Isopropylidene-2⁰-hydroxy-C6-cera-

Prepared from 1 and ^DL-2-hydroxyhexanoic acid in

4.1.3. General procedure for the preparation of 1,3-isopropyl-

idene-2⁰-OH-Cers (3a–d, 4a–d) and 1,3-isopropylidene-2⁰-OH-

C6-dihydroCers (5b, 6b)

A well-stirred mixture of 1,3-ispropylidene-(2S,3R,4E)-sphingo-

sine (1, 105 mg, 0.31 mmol) or 1,3-ispropylidene-(2S,3R)-dihyd-

40% yield after puriﬁcation by column chromatography (ethyl ace-

tate–n-hexane, 2:1, v/v). Analytical sample of 4b was prepared by

crystallization from ethyl acetate–n-hexane (1:3, v/v) as a white

microcrystalline powder, mp 68–69 °C. TLC R_f= 0.41 (ethyl ace-

7576

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

22

365

tate–n-hexane, 1:1, v/v); ½a _D²²

ꢁ

ꢂ11.4 (c 0.5, CHCl₃); ½

aꢁ

ꢂ43.4 (c

4.1.3.8. (2S,3R,2⁰S,4E)-1,3-Isopropylidene-2⁰-hydroxy-C16-cera-

mide (4d). Prepared from 1 and ^DL-2-hydroxyhexadecanoic

acid in 38% yield after puriﬁcation by column chromatography

(ethyl acetate–n-hexane, 2:3, v/v). Analytical sample of 4d was

prepared by crystallization from ethyl acetate–n-hexane (3:1, v/

0.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃) d 6.24 (d,1H, J = 8.4, NH),

5.76 (dtd, 1H, J = 15.1, 7.4, 1.0, 5-H), 5.42 (ddt, 1H, J = 15.1, 7.5,

1.4, 4-H), 4.15 (dd, 1H, J = 8 and 9.6, 3-H), 4.06 (q, 1H, J = 4.0, 2⁰-

H_S), 4.0 (dd, 1H, J = 5.6 and 11.2, 1-H_a), 3.81 (m, 1H, 2-H), 3.66

(dd, 1H, J = 9.6 and 11.2, 1-Hb), 2.26 (d, 1H, J = 4.8, 2⁰-OH), 2.02

(m, 2H, C(6)H_a), 1.80 (m, 1H, 3⁰-Ha), 1.58 (m, 1H, 3⁰-Hb), 1.52 (s,

3H, CH₃), 1.43 (s, 3H, CH₃), 1.24 (m, 32H, CH₂), 1.15 (m, 2H, CH₂),

0.85 (m, 6H, 2 ꢃ CH₃). MS (CH₃OH) m/z 929.7 ([2M+Na]⁺, 2),

449.5 (100), 414.4 ([MHꢂ(CH₃)₂C], 6) 396.2 ([MHꢂ(CH₃)₂CO]⁺,

48). Calcd for C₂₇H₅₁NO₄m/z 453.38 [M].

v) as a white microcrystalline powder, mp 81–83 °C. TLC R_f= 0.44

22

365

(ethyl acetate–n-hexane, 2:3, v/v); ½a _D²²

ꢁ

ꢂ7.6 (c 0.8, CHCl₃); ½ ꢁ

a

ꢂ36.8 (c 0.8, CHCl₃); ¹H NMR (500 MHz, CDCl₃) d 6.20 (d, 1H,

J = 8.2, NH), 5.75 (dtd, 1H, J = 15.3, 6.7, 1.0, 5-H), 5.40 (ddt, 1H,

J = 15.3, 6.6, 1.2, 4-H), 4.13 (dd, 1H, J = 7.7 and 9.6, 3-H), 4.03 (q,

1H, J = 4.1, 2⁰-H_S), 3.98 (dd, 1H, J = 5.2 and 11.2, 1-Ha), 3.80 (m,

1H, 2-H), 3.68 (dd, 1H, J = 9.6 and 11.2, 1-Hb), 2.22 (d, 1H, J = 4.9,

2⁰-OH), 2.02 (m, 2H, C(6)H₂), 1.78 (m, 1H, 3⁰-H_a), 1.58 (m, 1H, 3⁰-

H_b), 1.49 (s, 3H, CH₃), 1.42 (s, 3H, CH₃), 1.24 (m, 46H, CH₂), 0.87

(t, 6H, J = 7.1, CH₃). MS (CH₃OH) m/z 616.6 ([M+Na]⁺, 4), 536.5

([MHꢂ(CH₃)₂CO]⁺, 100), 449.5 (85). Calcd for C₇₃71₃NO₄m/z

593.54 [M].

4.1.3.5. (2S,3R,2⁰R,4E)-1,3-Isopropylidene-2⁰-hydroxy-C12-cera-

mide (3c).

Prepared from 1 and DL-2-hydroxydodecanoic acid

in 33% yield after puriﬁcation by column chromatography (ethyl

acetate–n-hexane, 2:3, v/v). Analytical sample of 3c was prepared

by crystallization from ethyl acetate–n-hexane (3: 1, v/v) as a

white microcrystalline powder, mp 85–87 °C. TLC R_f= 0.22 (ethyl

23

acetate–n-hexane, 2:3, v/v); ½a _D²³

ꢁ

+5.60 (c 0.5, CHCl₃); ½

a

ꢁ

+11.0

4.1.3.9. (2S,3R,2⁰R)-1,3-Isopropylidene-2⁰-hydroxy-C6-dihydro-

365

(c 0.5, CHCl₃); ¹H NMR (500 MHz, CDCl₃) d 6.32 (d, 1H, J = 8.8,

NH), 5.74 (dtd, 1H, J = 15.1, 7.4, 1.0, 5-H), 5.40 (ddt, 1H, J = 15.1,

7.5, 1.4, 4-H), 4.04 (m, 2H, 2⁰-H_Rand 3-H), 3.96 (dd, 1H, J = 5.2

and 11.2, 1-H_a), 3.86 (m, 1H, 2-H), 3.64 (dd, 1H, J = 9.6 and 11.2,

1-Hb), 2.24 (d, 1H, J = 4.4, 2⁰-OH), 2.01 (m, 2H, C(6)H_a), 1.92 (m,

2H, CH₂), 1.78 (m, 1H, 3⁰-Ha), 1.64 (m, 2H, CH₂), 1.59 (m, 1H, 3⁰-

Hb), 1.48 (s, 3H, CH₃), 1.41 (s, 3H, CH₃), 1.24 (m, 32H, CH₂), 1.05

(m, 2H, CH₂), 0.88 (t, 6H, J = 7.1, CH₃). MS (CH₃OH) m/z 538.6

([MH]⁺, 2), 498.5.6 ([MHꢂ2H₂O]⁺, 10), 488.4 ([MH-(CH₃)₂CO]⁺,

100), 449.5 (10). Calcd for C₃₃H₆₃NO₄m/z 537.48 [M].

ceramide (5b).

Prepared from 2 and ^DL-2-hydroxyhexanoic

acid in 32% yield after puriﬁcation by column chromatography

(ethyl acetate–n-hexane, 1:1, v/v). Analytical sample of 5b was

prepared by crystallization from ethyl acetate–n-hexane (3:1, v/

v) as

a white microcrystalline powder, mp 61–62 °C. TLC

R_f= 0.26 (ethyl acetate–n-hexane, 1:1, v/v); ½a _D²³

ꢁ

+29.4 (c 0.5,

23

365

CH₃OH); ½

aꢁ

+93.4 (c 0.5, CH₃OH); ¹H NMR (500 MHz, CDCl₃)

d 6.43 (d, 1H, J = 8.4, NH), 4.14 (q, 1H, J = 4.2, 2⁰-H_R), 3.86 (m,

2H, 1-Ha and 3-H), 3.53 (m, 2H, 2-H and 1-Hb), 1.82 (m, 3⁰-Ha,

1H), 1.63 (m, 3⁰-Hb, 1H), 1.23–1.52 (m, 36H, CH₂and 2 ꢃ CH₃),

0.85 (m, 6H, 2 ꢃ CH₃); MS (CH₃OH) m/z 456.4 ([MH]⁺, 100),

398.4 ([MHꢂ(CH₃)₂CO]⁺, 30). Calcd for C₂₇H₃₁NO₄m/z 455.71

[M].

4.1.3.6. (2S,3R,2⁰S,4E)-1,3-Isopropylidene-2⁰-hydroxy-C12-cera-

mide (4c).

Prepared from 1 and ^DL-2-hydroxydodecanoic acid

in 37% yield after puriﬁcation by column chromatography (ethyl

acetate–n-hexane, 2:3, v/v). Analytical sample of 4c was prepared

by crystallization from ethyl acetate–n-hexane (3:1, v/v) as a white

4.1.3.10. (2S,3R,2⁰S)-1,3-Isopropylidene-2⁰-hydroxy-C6-dihydro-

ceramide (6b).

Prepared from 2 and ^DL-2-hydroxyhexanoic

microcrystalline powder, mp 80–82 °C. TLC R_f= 0.40 (ethyl ace-

acid in 35% yield after puriﬁcation by column chromatography

(ethyl acetate–n-hexane, 1:1, v/v). Analytical sample of 6b was

prepared by crystallization from ethyl acetate–n-hexane (3:1, v/

v) as a white microcrystalline powder, mp 83–84 °C. TLC R_f= 0.46

23

tate–n-hexane, 2:3, v/v); ½a _D²³

ꢁ

ꢂ6.84 (c 0.5, CHCl₃); ½

aꢁ

ꢂ34.2 (c

365

0.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃) d 6.21 (d,1H, J = 8.4, NH),

5.74 (dtd, 1H, J = 15.0, 7.5, 1.0, 5-H), 5.40 (ddt, 1H, J = 15.1, 7.5,

1.4, 4-H), 4.12, (dd, 1H, J = 7.6 and 9.2, 3-H), 4.04 (q, 1H, J = 4.2,

2⁰-H_S), 3.97 (dd, 1H, J = 5.2 and 11.2, 1-H_a), 3.79 (m, 1H, 2-H),

3.64 (dd, 1H, J = 9.6 and 11.2, 1-Hb), 2.24 (d, 1H, J = 4.4, 2⁰-OH),

2.0 (m, 2H, C(6)H_a), 1.92 (m, 0.5H, CH₂), 1.78 (m, 1H, 3⁰-Ha), 1.67

(m, 0.5H, CH₂), 1.58 (m, 1H, 3⁰-Hb), 1.48 (s, 3H, CH₃), 1.42 (s, 3H,

CH₃), 1.24 (m, 36.5H, CH₂), 1.05 (m, 0.5H, CH₂), 0.88 (t, 6H,

J = 7.1, CH₃). MS (CH₃OH) m/z 538.6 ([MH]⁺, 2), 498.5.6

([MHꢂ2H₂O]⁺, 10), 488.4 ([MHꢂ(CH₃)₂CO]⁺, 100), 449.5 (10). Calcd

for C₃₃H₆₃NO₄m/z 537.48 [M].

(ethyl acetate–n-hexane, 1:1, v/v); ½a _D²³

ꢁ

+11.0 (c 0.5, CH₃OH);

23

365

½

aꢁ

+23.4 (c 0.5, CH₃OH); ¹H NMR (500 MHz, CDCl₃) d 6.35 (d,

1H, J = 8.5, NH), 4.12 (q, 1H, J = 4.1, 2⁰-H_S), 3.90 (m, 2H, 1-Ha and

3-H), 3.55 (m, 2H, 2-H and 1-Hb), 1.82 (m, 3⁰-Ha, 1H), 1.60 (m,

3⁰-Hb, 1H), 1.20–1.52 (m, 36H, CH₂and 2 ꢃ CH₃), 0.90 (m, 6H,

2 ꢃ CH₃). MS (CH₃OH) m/z 456.4 ([MH]⁺, 100), 398.4

([MHꢂ(CH₃)₂CO]⁺, 30). Calcd for C₂₇H₃₁NO₄m/z 455.71 [M].

4.1.4. General procedure for thepreparation of 2⁰-OHCers (7b–d,

8b–d) and 2⁰-OH-C6-dhCers (9b, 10b)

4.1.3.7. (2S,3R,2⁰R,4E)-1,3-isopropylidene-2⁰-hydroxy-C16-cera-

A

mixture

of

1,3-isopropylidene-2⁰-hydroxy-ceramide

mide (3d).

Prepared from 1 and ^DL-2-hydroxyhexadecanoic

(0.07 mmol) and p-toluenesulfonic acid monohydrate (19 mg,

0.10 mmol) in dry methanol (2 mL) was stirred at room tempera-

ture for 4 hrs. The reaction mixture was evaporated under reduced

pressure to dryness. The obtained residue was puriﬁed by ﬂash col-

umn chromatography using the suitable solvent system.

acid in 40% yield after puriﬁcation by column chromatography

(ethyl acetate–n-hexane, 2:3, v/v). Analytical sample of 3d was

prepared by crystallization from ethyl acetate–n-hexane (3:1, v/

v) as a white microcrystalline powder, mp 100–101 °C. TLC

R_f= 0.25 (ethyl acetate–n-hexane, 2:3, v/v); ½a _D²²

ꢁ

+8.6 (c 0.8, CHCl₃);

22

365

½

aꢁ

+17.5 (c 0.8, CHCl₃); ¹H NMR (500 MHz, CDCl₃) d 6.29 (d, 1H,

4.1.4.1. (2S,3R,2⁰R,4E)-2⁰-Hydroxy-C4-ceramide (7a).

Pre-

J = 8.6, NH), 5.74 (dtd, 1H, J = 15.4, 7.6, 1.0, 5-H), 5.42 (ddt, 1H,

J = 15.4, 7.7, 1.4, 4-H), 4.07 (m, 2H, 2⁰-H_Rand 3-H), 3.97 (dd, 1H,

J = 4.4 and 11.4, 1-Ha), 3.86 (m, 1H, 2-H), 3.64 (dd, 1H, J = 9.6 and

11.4, 1-Hb), 2.01 (m, 2H, C(6)H_a), 1.92 (m, 0.5H, CH₂), 1.78 (m,

1H, 3⁰-Ha), 1.68 (m, 0.5H, CH₂), 1.57 (m, 1H, 3⁰-Hb), 1.49 (s, 3H,

CH₃), 1.42 (s, 3H, CH₃), 1.24 (m, 44.5H, CH₂), 1.1 (m, 0.5H, CH₂),

0.88 (t, 6H, J = 7.1, CH₃). MS (CH₃OH) m/z 616.6 ([M+Na]⁺, 4),

536.5 ([MH-(CH₃)₂CO]⁺, 100), 449.5 (85). Calcd for C₇₃71₃NO₄m/z

593.54 [M].

pared from 3a in 80% yield after puriﬁcation by column chroma-

tography (CHCl₃–CH₃OH, 45:5, v/v). Analytical sample of 7a was

prepared by crystallization from acetone as a white microcrystal-

line powder, mp 94–96 °C. TLC R_f= 0.32 (CHCl₃–CH₃OH, 45:5, v/

22

365

v); ½a ²_D²

ꢁ

+18.4 (c 0.25, CHCl₃); ½

aꢁ

+43.1 (c 0.25, CHCl₃); ¹H

NMR (500 MHz, CDCl₃) d 7.12 (d, 1H, J = 7.7, NH), 5.78 (dtd, 1H,

J = 15.1, 6.8, 1.0, 5-H), 5.52 (ddt, 1H, J = 15.1, 6.8, 1.1, 4-H), 4.31

(m, 1H, 3-H), 4.12 (dd, 1H, J = 4.1 and 7.2, 2⁰-H_R), 3.93 (m, 2H, 1-

Ha and 2-H), 3.74 (dd, 1H, J = 3.1 and 11.0, 1-Hb), 2.04 (q, 2H,

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

7577

J = 7.0, C(6)H₂), 1.87 (m, 1H, 3⁰-Ha), 1.70 (m, 1H, 3⁰-Hb), 1.39 (m,

4.1.4.4.

(2S,3R,2⁰S,4E)-2⁰-Hydroxy-C6-ceramide

(8b,

2H, C(7)H₂), 1.23 (m, 20H, CH₂), 0.99 (t, 3H, J = 7.1, C⁰H₃), 0.87 (t,

3H, J = 7.1, CH₃); ¹³C NMR (CDCl₃) d 174.7 (C@J), 134.6 (C5),

128.3 (C4), 74.3 (C3), 73.2 (C⁰2), 62.1 (C1), 54.3 (C2), 32.3 (C⁰3),

31.9 (C6), 29.6, 29.4, 29.3, 29.2, 29.0, 27.6 and 22.6 (C7–C17),

14.1 (C⁰H₃), 9.15 (CH₃). (CD₃OD) d 176.8 0 (C@J), 134.78 (C5),

130.80 (C4), 73.83 (C3), 73.21 (C⁰2), 61.79 (C1), 55.97 (C2), 33.23

(C⁰3), 32.94 (C6), 30.66, 30.58, 30.52, 30.34, 30.29, 30.11, 28.59

and 23.61 (C7–C17), 14.35 (C⁰H₃), 9.68 (CH₃) MS (CH₃OH) m/z

368.1 ([MHꢂH₂O]⁺, 14), 386.0 ([MH]⁺, 10), 793.1 ([2M+Na]⁺, 100).

Calcd for C₂₂H₄₉NO₄m/z 385.32 [M].

LCL367).

Prepared from 4b in 88% yield after puriﬁcation by

column chromatography (CHCl₃–CH₃OH, 45:5, v/v). Analytical

sample of 8b was prepared by crystallization from acetone as a

white microcrystalline powder, mp 80–82 °C. TLC R_f= 0.40

(CHCl₃–CH₃OH, 45:5, v/v); ½a _D²²

ꢁ

ꢂ35.4 (c 0.25, CHCl₃); ½ ꢁ

a

22

365

ꢂ124.8 (c 0.25, CHCl₃); ¹H NMR (500 MHz, CDCl₃) d 7.13 (d, 1H,

J = 7.7, NH), 5.80 (dtd, 1H, J = 15.1, 6.8, 1.0, 5-H), 5.52 (ddt, 1H,

J = 15.1, 6.8, 1.1, 4-H), 4.35 (t, 1H, J = 5.2, 3-H), 4.14 (dd, 1H,

J = 3.9 and 8.0, 2⁰-H_R), 3.97 (dd, 1H, J = 4.0 and 11.4, 1-Ha), 3.90

(m, 1H, 2-H), 3.72 (dd, 1H, J = 3.5 and 11.4, 1-Hb), 2.05 (q, 2H,

J = 7.1, C(6)H₂), 1.83 (m, 1H, 3⁰-Ha), 1.65 (m, 1H, 3-⁰Hb), 1.37 (m,

4H, CH₂), 1.26 (m, 22H, CH₂), 0.92 (t, 3H, J = 7.1, C⁰H₃), 0.88 (t,

3H, J = 7.1, CH₃); (CD₃OD) d 7.56 (d, 0.5H, J = 9.0, NH), 5.70 (dtd,

1H, J = 15.1, 7.0, 1.0, 5-H), 5.47 (ddt, 1H, J = 15.1, 7.0, 1.1, 4-H),

4.15 (t, 1H, J = 6.7, 3-H), 3.97 (dd, 1H, J = 3.8 and 8.1, 2⁰-H_S), 3.84

(m, 1H, 2-H), 3.75 (dd, 1H, J = 5.4 and 11.1, 1-Ha), 3.63 (dd, 1H,

J = 4.4 and 11.1, 1-Hb), 2.03 (q, 2H, J = 7.1, C(6)H₂), 1.74 (m, 1H,

3⁰-Ha), 1.57 (m, 1H, 3-⁰Hb), 1.4 (m, 4H, CH₂), 1.28 (m, 22H, CH₂),

0.91 (t, 3H, C⁰H₃), 0.89 (t, 3H, J = 7.1, CH₃); ¹³C NMR (CDCl₃) d

175.2 (C@J), 134.5 (C5), 127.9 (C4), 73.7 (C3), 72.3 (C⁰2), 62.2

(C1), 54.5 (C2), 34.3 (C⁰3), 32.3 (C6), 31.9, 29.6, 29.4, 29.3, 29.2,

29.1, 27.2, 22.6 and 22.4 (C7-C17, C⁰4 and C⁰5), 14.1 (C⁰H₃), 13.9

(CH₃); (CD₃OD) d 177.0 (C@J), 134.6 (C5), 131.7 (C4), 73.1 (C3),

72.8 (C⁰2), 61.9 (C1), 56.2 (C2), 35.4 (C⁰3), 33.3 (C6), 33.0, 30.8,

30.7, 30.6, 30.4, 30.3, 28.3, 23.7 and 23.6 (C7-C17, C⁰4 and C⁰5),

14.4 (C⁰H₃), 14.3 (CH₃); MS (MeOH) m/z 436.4 ([M+Na]⁺, 20),

414.4 ([MH]⁺, 100), 396.4 ([MHꢂH₂O]⁺, 10). Calcd for C₂₄H₄₇NO₄

m/z 413.35 [M].

4.1.4.2.

(2S,3R,2⁰S,4E)-2⁰-Hydroxy-C4-ceramide (8a).

Pre-

pared from 4a in 81% yield after puriﬁcation by column chroma-

tography (CHCl₃–CH₃OH, 45:5, v/v). Analytical sample of 8a was

prepared by crystallization from acetone as a white microcrystal-

line powder, mp 80–82 °C. TLC R_f= 0.34 (CHCl₃–CH₃OH, 45:5, v/

22

365

v); ½a ²_D²

ꢁ

ꢂ25.1 (c 0.25, CHCl₃), ½

a

ꢁ

ꢂ95.8 (c 0.25, CHCl₃); ¹H

NMR (500 MHz, CDCl₃) d 7.13 (d, 1H, J = 7.4, NH), 5.82 (dtd, 1H,

J = 15.1, 7.0, 1.0, 5-H), 5.54 (ddt, 1H, J = 15.1, 7.0, 1.1, 4-H), 4.38

(t, 1H, J = 5.4, 3-H), 4.15 (dd, 1H, J = 4.1 and 7.1, 2⁰-H_s), 4.01 (dd,

1H, J = 4.0 and 11.4, 1-Ha), 3.84 (m, 1H, 2-H), 3.75 (dd, 1H, J = 4.1

and 11.4, 1-Hb), 2.08 (q, 2H, J = 7.0, C(6)H₂), 1.91 (m, 1H, 3⁰-Ha),

1.74 (m, 1H, 3⁰-Hb), 1.41 (m, 2H, C(7)H₂), 1.23 (m, 20H, CH₂),

1.03 (t, 3H, J = 7.1, C⁰H₃), 0.92 (t, 3H, J = 7.1, CH₃); (CD₃OD) d 5.70

(dtd, 1H, J = 15.4, 7.0, 1.0, 5-H), 5.49 (ddt, 1H, J = 15.4, 7.0, 1.0, 4-

H), 4.15 (t, 1H, J = 7.0, 3-H), 3.94 (dd, 1H, J = 5.6 and 7.0, 2⁰-H_S),

3.84 (m, 1H, 2-H), 3.75 (dd, J = 4.9 and 11.2, 1-Ha), 3.63 (dd, 1H,

J = 4.2 and 11.2, 1-Hb), 2.04 (q, 2H, J = 7.0, C(6)H₂), 1.77 (m, 1H,

3⁰-Ha), 1.62 (m, 1H, 3⁰Hb), 1.37 (m, 2H, CH₂) 1.28 (m, 42H, CH₂),

0.94 (t, 6H, J = 7.0, CH₃), 0.89 (t, 6H, J = 7.0, CH₃); (CD₃OD) d

177.01 (C@J), 134.86 (C5), 130.92 (C4), 74.03 (C3), 73.36 (C⁰2),

62.09 (C1), 56.44 (C2), 33.55 (C⁰3), 33.23 (C6), 30.97, 30.96,

30.95, 30.92, 30.81, 30.49, 30.47, 28.80 and 23.89 (C7-C17), 14.58

(C⁰H₃), 9.77 (CH₃). MS (CH₃OH) m/z 385.9 ([MH]⁺, 28), 368.1

([MHꢂH₂O]⁺, 25), 793.1 ([2M+Na]⁺, 100). Calcd for C₂₂H₄₉NO₄m/

z 385.32 [M].

4.1.4.5. (2S,3R,2⁰R,4E)-2⁰-Hydroxy-C12-ceramide (7c).

Pre-

pared from 4c in 75% yield after column chromatography puriﬁca-

tion (Silica Gel 60; CHCl₃–MeOH, 45:5, v/v). Analytical sample of

7c was prepared by crystallization from acetone as a white micro-

crystalline powder. TLC R_f= 0.37 (CHCl₃–MeOH, 45:5, v/v); ½a _D²³

ꢁ

23

365

+8.4 (c 0.25, CHCl₃); ½

aꢁ

+19.0 (c 0.25, CHCl₃); ¹H NMR (500 MHz,

CD₃OD–CDCl₃, 3:1, v/v)/ d 5.63 (dtd, 1H, J = 15.1, 6.8, 1.0, 5-H), 5.35

(ddt, 1H, J = 15.1, 6.8, 1.1, 4-H), 4.0 (t, 1H, J = 5.6, 3-H), 3.95 (m, H₂O

and 2⁰-H_R), 3.65 (m, 2H, 1-Ha and 2-H), 3.57 (dd, 1H, J = 3.6 and

11.2, 1-Hb), 1.95 (q, 2H, J = 7.1, C(6)H₂), 1.65 (m, 1H, 3⁰-Ha), 1.44

(m, 1H, 3⁰-Hb), 1.15 (m, 38H, CH₂), 0.76 (t, 6H, J = 7.1, CH₃); ESI-MS

(CH₃OH, relative intensity, %) m/z 498.5 ([MH]⁺, 100), 480.4

([MHꢂH₂O]⁺, 15). Calcd for C₃₀H₅₉NO₄m/z 497.44 [M].

4.1.4.3.

(2S,3R,2⁰R,4E)-2⁰-Hydroxy-C6-ceramide (7b, LCL366).

Prepared from 3b in 78% yield after puriﬁcation by column chro-

matography (CHCl₃–CH₃OH, 45:5, v/v). Analytical sample of 7b

was prepared by crystallization from acetone as a white microcrys-

talline powder, mp 85–86 °C. TLC R_f= 0.38 (CHCl₃–CH₃OH, 45:5, v/

22

365

v); ½a ²_D²

ꢁ

+7.7 (c 0.25, CHCl₃); ½

aꢁ

+28.6 (c 0.25, CHCl₃); ¹H NMR

4.1.4.6. (2S,3R,2⁰S,4E)-2⁰-Hydroxy-C12-ceramides (8c).

Pre-

(500 MHz, CDCl₃) d 7.21 (d, 1H, J = 7.0, NH), 5.77 (dtd, 1H,

J = 15.1, 6.8, 1.0, 5-H), 5.52 (ddt, 1H, J = 15.1, 6.8, 1.1, 4-H), 4.29

(t, 1H, J = 5.0, 3-H), 4.13 (dd, 1H, J = 3.7 and 8.2, 2⁰-H_R), 3.92 (m,

2H, 1-Ha and 2-H), 3.75 (dd, 1H, J = 5.7 and 12.0, 1-Hb), 2.05 (q,

2H, J = 7.1, C(6)H₂), 1.82 (m, 1H, 3⁰-Ha), 1.62 (m, 1H, 3-⁰Hb), 1.37

(m, 4H, CH₂), 1.26 (m, 22H, CH₂), 0.91 (t, 3H, C⁰H₃), 0.88 (t, 3H,

J = 7.1, CH₃); (CD₃OD) d 5.73 (dtd, 1H, J = 15.1, 7.0, 1.0, 5-H), 5.47

(ddt, 1H, J = 15.1, 7.0, 1.1, 4-H), 4.09 (t, 1H, J = 7.0, 3-H), 3.97 (dd,

1H, J = 3.8 and 8.1, 2⁰-H_R), 3.83 (m, 1H, 2-H), 3.77 (dd, 1H, J = 5.4

and 11.2, 1-Ha), 3.66 (dd, 1H, J = 4.1 and 11.2, 1-Hb), 2.03 (q, 2H,

J = 7.1, C(6)H₂), 1.74 (m, 1H, 3⁰-Ha), 1.53 (m, 1H, 3-⁰Hb), 1.4 (m,

4H, CH₂), 1.28 (m, 22H, CH₂), 0.91 (t, 3H, J = 7.1, C⁰H₃), 0.89 (t,

3H, J = 7.1, CH₃); ¹³C NMR (CDCl₃) d 175.5 (C@J), 134.6 (C5),

128.3 (C4), 74.0 (C3), 72.4 (C⁰2), 62.0 (C1), 54.4 (C2), 34.2 (C⁰3),

32.3 (C6), 32.0, 29.7, 29.5, 29.3, 29.2, 29.1, 27.2, 22.7 and 22.5

(C7-C17, C⁰4 and C⁰5), 14.1 (C⁰H₃), 13.9 (CH₃); (CD₃OD) d 177.1

(C@J), 134.8 (C5), 131.0 (C4), 73.3 (C3), 72.9 (C⁰2), 61.8 (C1),

56.0 (C2), 35.5 (C⁰3), 33.4 (C6), 33.0, 30.7, 30.6, 30.4, 30.3, 30.2,

28.4, 23.7 and 23.6 (C7–C17, C⁰4 and C⁰5), 14.4 (C⁰H₃), 14.3 (CH₃);

MS (CH₃OH) m/z 436.4 ([M+Na]⁺, 20), 414.4 ([MH]⁺, 100), 396.4

([MHꢂH₂O]⁺, 10). Calcd for C₂₄H₄₇NO₄m/z 413.35 [M].

pared from 4c in 70% yield after column chromatography puriﬁca-

tion (Silica Gel 60; CHCl₃–CH₃OH, 45:5, v/v). Analytical sample of

8c was prepared by crystallization from acetone as a white micro-

crystalline powder. TLC R_f= 0.46 (CHCl₃–CH₃OH, 45:5, v/v); ½a _D²³

ꢁ

23

365

ꢂ26.9 (c 0.25, CHCl₃); ½

aꢁ

ꢂ99.0 (c 0.25, CHCl₃); ¹H NMR

(500 MHz, CDCl₃) d 7.30 (d,1H, J = 8.4, NH), 5.70 (dtd, 1H, J = 15.1,

6.8, 1.0, 5-H), 5.40 (ddt, 1H, J = 15.1, 6.8, 1.1, 4-H), 4.17 (m, 1H,

3-H), 3.93 (dd, 1H, J = 4.0 and 8.4, 2⁰-H_S), 3.72 (m, 2H, 1-Ha and

2-H), 3.53 (dd, 1H, J = 5.6 and 13.2, 1-Hb), 1.96 (q, 2H, J = 7.0,

C(6)H₂), 1.72 (m, 1H, 3⁰-Ha), 1.48 (m, 1H, 3⁰-Hb), 1.20 (m, 38H,

CH₂), 0.79 (t, 6H, J = 7.1, CH₃). ESI-MS (CH₃OH, relative intensity,

%) m/z. 498.5 ([MH]⁺, 100), 480.4 ([MHꢂH₂O]⁺, 15). Calcd for

C₃₀H₅₉NO₄m/z 497.44 [M].

4.1.4.7. (2S,3R,2⁰R,4E)-2⁰-Hydroxy-C16-ceramide (7d).

Pre-

pared from 3d in 88% yield after column chromatography

puriﬁcation as a white microcrystalline powder (Silica Gel 60;

CHCl₃–CH₃OH, 45:5, v/v). Analytical sample of 7d was prepared

by crystallization from acetone as a white microcrystalline powder,

mp 101–103 °C. TLC R_f= 0.38 (CHCl₃–CH₃OH, 45:5, v/v); ½a _D²³

ꢁ

+5.2

7578

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

23

365

(c 0.25, MeOH–CHCl₃, 2:8, v/v); ½

a

ꢁ

+32.2 (c 0.25, CH₃OH–CHCl₃,

Gel 60; CHCl₃–CH₃OH, 10:1, v/v). Analytical sample of 9b was pre-

pared by crystallization from acetone as a white microcrystalline

2: 8, v/v); ¹H NMR (500 MHz, CD₃OD–CDCl₃, 1:3, v/v) d 5.76 (dtd,

1H, J = 15.1, 6.8, 1.0, 5-H), 5.47 (ddt, 1H, J = 15.1, 6.8, 1.1, 4-H),

4.12 (t, 1H, J = 6.0, 3-H), 4.01 (dd, 1H, J = 3.7 and 8.2, 2⁰-H_R), 3.85

(m, 1H, 2-H), 3.80 (dd, J = 8.6 and 11.3, 1-Ha), 3.68 (dd, 1H, J = 3.7

and 11.3, 1-Hb), 2.05 (q, 2H, J = 7.1, C(6)H₂), 1.78 (m, 1H, 3⁰-Ha),

1.58 (m, 1H, 3⁰Hb), 1.26 (m, 46H, CH₂), 0.88 (t, 6H, J = 7.1, CH₃);

(CDCl₃) d 7.11 (d, 1H, J = 8.2, NH) 5.78 (dtd, 1H, J = 15.1, 6.8, 1.0,

5-H), 5.52 (ddt, 1H, J = 15.1, 6.8, 1.1, 4-H), 4.30 (t, 1H, J = 6.0, 3-

H), 4.13 (dd, 1H, J = 3.5 and 8.0, 2⁰-H_R), 3.93 (m, 2H, 1-Ha and 2-

H), 3.74 (dd, 1H, J = 5.7 and 13.0, 1-Hb), 2.05 (q, 2H, J = 7.1,

C(6)H₂), 1.82 (m, 1H, 3⁰-Ha), 1.64 (m, 1H, 3⁰-Hb), 1.26 (m, 46H,

CH₂), 0.88 (t, 6H, J = 7.1, CH₃); (CD₃OD, 30 °C, 700 MHz) d 5.72

(dtd, 1H, J = 15.3, 7.0, 0.9, 5-H), 5.47 (ddt, 1H, J = 15.3, 7.3, 0.9, 4-

H), 4.08 (t, 1H, J = 7.4, 3-H), 4.12 (dd, 1H, J = 3.8 and 7.9, 2⁰-H_R),

3.84 (m, 1H, 2-H), 3.76 (dd, J = 4.9 and 11.2, 1-Ha), 3.64 (dd, 1H,

J = 5.0 and 11.2, 1-Hb), 2.05 (q, 2H, J = 7.0, C(6)H₂), 1.76 (m, 1H,

3⁰-Ha), 1.57 (m, 1H, 3⁰Hb), 1.40 (m, 4H, CH₂) 1.29 (m, 42H, CH₂),

0.90 (t, 6H, J = 7.1, CH₃); ¹³C NMR (CDCl₃) d 174.7 (C@J), 134.5

(C5), 128.7 (C4), 74.4 (C3), 72.46 (C⁰2), 62.3 (C1), 54.6 (C2), 34.9

(C⁰3), 32.3 (C6), 31.9, 29.7, 29.5, 29.4, 29.3, 29.2, 29.1, 25.0 and

22.7 (C7–C17 and C⁰4–C⁰15), 14.0 (C⁰H₃and CH₃); (CD₃OD) d

177.3 (C@J), 135.0 (C5), 131.2 (C4), 73.4 (C3), 73.2 (C⁰2), 62.1

(C1), 56.1 (C2), 36.0 (C⁰3) , 33.6 (C6), 33.2, 30.98, 30.96, 30.94,

30.92, 30.9, 30.88, 30.82, 30.7, 30.62, 30.61, 30.49), 26.3 and 23.8

(C7-C17 and C⁰4 -C⁰15), 14.5 (C⁰H₃and CH₃). ESI-MS (CH₃OH, rela-

tive intensity, %) m/z 1130.5 and 1129.4 ([2M+Na]⁺and

([2MꢂH+Na]⁺, 70 and 100), 1107.0 ([2M+H]⁺, 35), 554.1 (MH⁺,

16), 536.3 ([MHꢂH₂O]⁺, 27). Calcd for C₃₄H₆₇NO₄m/z 553.51 [M].

powder, mp 112–114 °C. TLC R_f= 0.24 (CHCl₃–CH₃OH, 45:5, v/v);

23

½

a ²_D³

ꢁ

+38.20 (c 0.125, MeOH); ½

aꢁ

+124.91 (c 0.125, CH₃OH); ¹H

365

NMR (500 MHz, CDCl₃) d 7.28 (d, 1H, J = 8.4, NH), 4.12 (q, 1H,

J = 3.6, 2⁰-H_S), 3.95 (dd, 1H, J = 3.6 and 11.2, 1-Ha), 3.82 (m, 2H,

2-H and 3-H), 3.78 (m, 1H, J = 3.1 and 11.2, 1Hb), 1.82 (m, 1H, 3⁰-

Ha), 1.61 (m, 1H, 3⁰-Hb), 1.51 (m, 2H, C(4)H₂), 1.25 (m, 36H,

CH₂), 0.90 (t, 3H, J = 7.1, C⁰H₃), 0.87 (t, 3H, J = 7.1, CH₃). MS (CH₃OH)

m

/z 416.4 ([MH]⁺, 100), 498.3 ([MHꢂH₂O]⁺, 4). Calcd for C₂₄H₄₉NO₄

m/z 415.37 [M].

4.1.4.10.

LCL446).

(2S,3R,2⁰S)-2⁰-Hydroxy-C6-dihydroceramide

Prepared from 6b in 95% yield after column chroma-

(10b,

tography puriﬁcation as a white microcrystalline powder (Silica

Gel 60; CHCl₃–CH₃OH, 10:1, v/v). Analytical sample of 10b was

prepared by crystallization from acetone as a white microcrystal-

line powder, mp 90–91 °C. TLC R_f= 0.33 (CHCl₃–CH₃OH, 45:5, v/

23

365

v); ½a ²_D³

ꢁ

ꢂ17.20 (c 0.125, CH₃OH); ½

a

ꢁ

ꢂ62.03 (c 0.125, MeOH);

¹H NMR (500 MHz, CDCl₃) d 7.28 (d, 1H, J = 8.4, NH), 4.14 (q, 1H,

J = 3.8, 2⁰-H_S), 4.02 (dd, 1H, J = 3.5 and 11.4, 1-Ha), 3.82 (m, 2H,

2-H and 3-H), 3.78 (m, 1H, J = 3.1 and 11.4, 1Hb), 1.86 (m, 3⁰-Ha,

1H), 1.68 (m, 3⁰-Hb, 1H), 1.54 (m, 2H, C(4)H₂), 1.25 (m, 36H,

CH₂), 0.90 (t, 3H, J = 7.1, C⁰H₃), 0.86 (t, 3H, J = 7.1, CH₃). MS (CH₃OH)

m

/z 416.4 ([MH]⁺, 100), 498.3 ([MHꢂH₂O]⁺, 4). Calcd for C₂₄H₄₉NO₄

m/z 415.37 [M].

4.2. Biology

4.2.1. Cell Culture

4.1.4.8. (2S,3R,2⁰S,4E)-2⁰-Hydroxy-C16-ceramides (8d).

Pre-

MCF7 cells (breast adenocarcinoma, pleural effusion) were pur-

chased from American type Culture Collection (ATCC, Rockville,

MD, USA) and grown in RPMI 1640 media (Life Technologies, Inc)

supplemented with 10% fetal calf serum (FCS, Summit Biotechnol-

ogy, CO, USA) and maintained under standard incubator conditions

(humidiﬁed atmosphere 95% air, 5% CO₂, 37 °C). A parallel set of

cells was used to determine cell proliferation and to prepare lipid

extracts for MS analysis.

pared from 4d in 81% yield after column chromatography

puriﬁcation as a white microcrystalline powder (Silica Gel 60;

CHCl₃–MeOH, 45:5, v/v). Analytical sample of 8d was prepared by

crystallization from acetone as a white microcrystalline powder,

mp 100–102 °C. TLC R_f= 0.39 (CHCl₃–CH₃OH, 45:5, v/v); ½a _D²³

ꢁ

23

365

ꢂ16.1 (c 0.25, MeOH–CHCl₃, 2:8, v/v); ½

aꢁ

ꢂ50.7 (c 0.25, CH₃OH–

CHCl₃, 2: 8, v/v); ¹H NMR (500 MHz, CD₃OD–CDCl₃, 1: 3, v/v) d 7.42

(d, ꢀ0.5H, J = 8.2, NH), 5.76 (dtd, 1H, J = 15.1, 6.8, 1.0, 5-H), 5.47

(ddt, 1H, J = 15.1, 6.8, 1.1, 4-H), 4.22 (t, 1H, J = 6.3, 3-H), 4.01 (dd,

1H, J = 3.8 and 8.3, 2⁰-H_S), 3.85 (m, 1H, 2-H), 3.79 (dd, 1H, J = 8.2

and 11.2, 1-Ha), 3.62 (dd, 1H, J = 5.6 and 11.2, 1-Hb), 2.04 (q, 2H,

J = 7.0, C(6)H₂), 1.78 (m, 1H, 3⁰-Ha), 1.58 (m, 1H, 3⁰-Hb), 1.27 (m,

46H, CH₂), 0.88 (t, 6H, J = 7.1, CH₃); (CDCl₃) d 7.10 (d, 1H, J = 8.2,

NH) 5.80 (dtd, 1H, J = 15.1, 6.8, 1.0, 5-H), 5.53 (ddt, 1H, J = 15.1, 6.8,

1.1, 4-H), 4.34 (t, 1H, J = 5.0, 3-H), 4.12 (dd, 1H, J = 4.0 and 8.0, 2⁰-

H_R), 3.95 (dd, J = 4.0 and 11.5, 1-Ha), 3.89 (m, 1H, 2-H), 3.72 (dd,

1H, J = 3.5 and 11.5, 1-Hb), 2.06 (q, 2H, J = 7.0, C(6)H₂), 1.82 (m, 1H,

3⁰-Ha), 1.64 (m, 1H, 3⁰Hb), 1.26 (m, 46H, CH₂), 0.88 (t, 6H, J = 7.1,

CH₃); (CD₃OD, 30 °C, 700 MHz) d 5.73 (dtd, 1H, J = 15.3, 7.0, 1.0, 5-

H), 5.49 (ddt, 1H, J = 15.3, 7.0, 1.0, 4-H), 4.16 (t, 1H, J = 6.3, 3-H),

4.12 (q, 1H, J = 5.6, 2⁰-H_S), 3.84 (m, 1H, 2-H), 3.79 (dd, J = 5.1 and

11.2, 1-Ha), 3.64 (dd, 1H, J = 4.0 and 11.2, 1-Hb), 2.04 (q, 2H, J = 7.0,

C(6)H₂), 1.71 (m, 1H, 3⁰-Ha), 1.55 (m, 1H, 3⁰Hb), 1.37 (m, 4H, CH₂)

1.29 (m, 42H, CH₂), 0.90 (t, 6H, J = 7.1, CH₃); ¹³C NMR (CDCl₃) d

174.6 (C@J), 134.5 (C5), 128.5 (C4), 74.2 (C3), 72.38 (C⁰2), 62.4

(C1), 54.7 (C2), 35.0 (C⁰3), 32.9 (C6), 31.9, 29.7, 29.6, 29.54 29.5,

29.4, 29.3, 29.2, 29.0, 25.0 and 22.6 (C7–C17 and C⁰4–C⁰15), 14.0

(C⁰H₃and CH₃); (CD₃OD) d 177.2 (C@J), 134.9 (C5), 130.9 (C4),

73.3 (C3), 73.03 (C⁰2), 62.1 (C1), 56.5 (C2), 30.98, 30.96, 30.94,

30.92, 30.9, 30.88, 30.82, 30.7, 30.62, 30.61, 30.49, 26.2 and 23.9

(C7–C17 and C⁰4–C⁰15), 14.5 (C⁰H₃and CH₃).

4.2.2. Cell proliferation

MCF7 cells were seeded at a density of ꢀ50% (corresponding to

1 ꢃ 10⁶cells) in 10 ml of 10% FCS and, after an overnight incuba-

tion, were treated with the compounds at (0–50 lM) in ethanol

(<0.1%) and the changes in cell numbers after 24 h were deter-

mined and expressed as a percentage of the untreated controls.

Brieﬂy, media were removed, cells were washed twice with PBS,

detached using 1% Trypsin and centrifuged at 800 rpm. Cell pellets

were resuspended in PBS and Trypan blue (Sigma Chemicals, St.

Louis, MO, USA) was added (1:1 dilution). Under a light micro-

scope, the percentage of unstained and stained cells was assessed.

The IC₅₀represents the drug concentration resulting in 50% growth

inhibition compared to control cells.

4.2.3. LC–MS/MS analysis of endogenous SPLs and cellular levels

of exogenously added compounds

Advanced analyses were performed on a Thermo Finnigan TSQ

7000, triple-stage quadrupole mass spectrometer operating in a

Multiple Reaction Monitoring (MRM) positive ionization mode as

described.^70,71Quantitative analysis of the cellular level of 2⁰-OH-

C6-Cers/dhCers and C6-Cer/dhCer was based on the calibration

curves generated by spiking an artiﬁcial matrix with known

amounts of target standards and an equal amount of the internal

standard (IS). The target analyte to IS peak area ratios from the

samples were similarly normalized to their respective IS and com-

pared to the calibration curves using a linear regression model. Fi-

4.1.4.9.

LCL447).

(2S,3R,2⁰R)-2⁰-Hydroxy-C6-dihydroceramide

Prepared from 5b in 93% yield after column chroma-

(9b,

tography puriﬁcation as a white microcrystalline powder (Silica

Z. M. Szulc et al. / Bioorg. Med. Chem. 18 (2010) 7565–7579

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Article Doi

Synthesis, NMR characterization and divergent biological actions of 2′-hydroxy-ceramide/dihydroceramide stereoisomers in MCF7 cells

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