Novel analogs of d-e-MAPP and B13. Part 1: Synthesis and evaluation as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2007.08.033

A series of novel isosteric analogs of the ceramidase inhibitors, (1S,2R)-N-myristoylamino-phenylpropanol-1 (d-e-MAPP) and (1R,2R)-N-myristoylamino-4′-nitro-phenylpropandiol-1,3 (B13), with modified targeting and physicochemical properties were designed, synthesized, and evaluated as potential anticancer agents. When MCF7 cells were treated with the analogs, results indicated that the new analogs were of equal or greater potency compared to the parent compounds. Their activity was predominantly defined by the nature of the modification of the N-acyl hydrophobic interfaces: N-acyl analogs (class A), urea analogs (class B), N-alkyl analogs (class C, lysosomotropic agents), and ω-cationic-N-acyl analogs (class D, mitochondriotropic agents). The most potent compounds belonged to either class D, the aromatic ceramidoids, or to class C, the aromatic N-alkylaminoalcohols. Representative analogs selected from this study were also evaluated by the National Cancer Institute In Vitro Anticancer Drug Discovery Screen. Again, results showed a similar class-dependent activity. In general, the active analogs were non-selectively broad spectrum and had promising activity against all cancer cell lines. However, some active analogs of the d-e-MAPP family were selective against different types of cancer. Compounds LCL85, LCL120, LCL385, LCL284, and LCL204 were identified to be promising lead compounds for therapeutic development.

Full text of DOI:10.1016/j.bmc.2007.08.033

Bioorganic & Medicinal Chemistry 16 (2008) 1015–1031
Novel analogs of D-ef-MAPP and B13. Part 1: Synthesis
and evaluation as potential anticancer agents
Zdzislaw M. Szulc,a Nalini Mayroo,a AiPing Bai,a Jacek Bielawski,a Xiang Liu,b
James S. Norris,b Yusuf A. Hannuna and Alicja Bielawskaa,*
aDepartment of Biochemistry & Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue,
POB 250509, Charleston, SC 29425, USA
bDepartment of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA
Received 8 June 2007; revised 16 August 2007; accepted 20 August 2007
Available online 24 August 2007
Abstract—A series of novel isosteric analogs of the ceramidase inhibitors, (1S,2R)-N-myristoylamino-phenylpropanol-1 (D-e-
MAPP) and (1R,2R)-N-myristoylamino-40-nitro-phenylpropandiol-1,3 (B13), with modified targeting and physicochemical proper-
ties were designed, synthesized, and evaluated as potential anticancer agents. When MCF7 cells were treated with the analogs,
results indicated that the new analogs were of equal or greater potency compared to the parent compounds. Their activity was pre-
dominantly defined by the nature of the modification of the N-acyl hydrophobic interfaces: N-acyl analogs (class A), urea analogs
(class B), N-alkyl analogs (class C, lysosomotropic agents), and x-cationic-N-acyl analogs (class D, mitochondriotropic agents). The
most potent compounds belonged to either class D, the aromatic ceramidoids, or to class C, the aromatic N-alkylaminoalcohols.
Representative analogs selected from this study were also evaluated by the National Cancer Institute In Vitro Anticancer Drug Dis-
covery Screen. Again, results showed a similar class-dependent activity. In general, the active analogs were non-selectively broad
spectrum and had promising activity against all cancer cell lines. However, some active analogs of the D-e-MAPP family were selec-
tive against different types of cancer. Compounds LCL85, LCL120, LCL385, LCL284, and LCL204 were identified to be promising
lead compounds for therapeutic development.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
mediated apoptosis.5,6 Because of the roles of Cer and
S1P in regulating cell growth and cell death, their meta-
bolic and signaling pathways have emerged as potential
targets for anticancer therapy.7 Many approaches have
been explored to increase endogenous Cer, and a few ap-
pear promising.7,8 These include the application of cell
permeable short-chain Cers,9–11 liposomal formula-
tions,12,13 site-specific cationization,14–17 and induction
of endogenous Cer by modulation of SPL metabolizing
enzymes.18–34
Cancer has proven to be a difficult disease to treat, and
few effective drugs are available. Identification of novel,
efficient, selective, and less toxic anticancer agents re-
mains an important and challenging goal in medicinal
chemistry.1–3
Sphingolipid (SPL) metabolism provides a rich network
of bioactive molecules that play key roles in the regula-
tion of diverse cell functions. Ceramide (Cer) has
emerged as a key modulator of cancer cell growth and
apoptosis.4 Sphingosine 1-phosphate (S1P), generated
from Cer by the action of ceramidases (CDases) to yield
sphingosine (Sph) which is phosphorylated by sphingo-
sine kinase (SK), promotes growth and opposes Cer-
In the course of our investigation of SPLs’ chemistry
and the search for new molecules that mimic the action
of SPLs and/or regulate their metabolism, we have fo-
cused on analogs that affect Cer metabolism, especially
the Cer-Sph-S1P balance, and display particular desired
properties, and we have also attempted to target them to
specific sub-cellular compartments.14–17,27,28,31
Keywords: D-e-MAPP; B13; Ceramide; Lysosomotropic; Mitochond-
riotropic; Ceramidase; Ceramidase inhibitors; Synthesis; Cytotoxicity;
LC–MS; NMR.
Previously, we synthesized a set of lipophilic phenyl-N-
acyl-amino-alcohols (aromatic analogs of Cer, Scheme
1) and evaluated their structure-dependent inhibitory ef-
fects in HL-60 cells.20,21 We found that analog activity
*
Corresponding author. Tel.: +1 843 792 0273; fax: +1 843 792
4322; e-mail: bielawsk@musc.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.033

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Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
Scheme 1. Ceramide, D-e-MAPP and B13 structures and design for aromatic analogs.
depended on stereochemistry and the chain length of the
N-acyl moiety. Analogs with the natural Cer configuration
were not active compounds, whereas their stereoisomers
inhibited cell growth. The active isomers were N-dodec-
anoyl (C12), N-myristoyl (C14), and N-palmitoyl (C16)
homologs, with C14-homologs being the most potent.
Shorter (C2–C10) and longer (C18–C24) homologs were
considerably less active. Pilot modification of some
N-myristoyl analogs (amides) to their N-myristyl-coun-
terpart (N-alkyl-amines) increased their inhibitory activ-
ity in HL-60 cells (A. Bielawska, data not published).
These structural features determine their conforma-
tion(s) and ability to interact effectively with putative
targets, via hydrogen bonding and van der Waals hydro-
phobic interactions. Therefore, the aromatic analogs of
Cer constitute attractive models for the development
of new bioactive lipophilic molecules. The effects of
additional aromatic analogs on SPL metabolism and
SPL intracellular trafficking have been reported, demon-
strating that the phenyl-amino-alcohol foundation is
recognized by many enzymes of SPL metabolism and
serves as a useful structural benchmark for development
of bioactive compounds.18,19,22–26,29,41
Further investigations on the active analogs confirmed
their inhibitory effects on ceramidases (CDases). HL-
60 cell experiments and in vitro studies showed that
the most potent analogs from this group, D-e-MAPP,
stereospecifically inhibited alkaline CDase, whereas its
enantiomer, L-e-MAPP, served as a substrate for this en-
zyme.20 Biological activity of D-e-MAPP was later con-
firmed by several investigators.35–40 Another active
analog: B13 (Scheme 1), which differs from D-e-MAPP
in stereochemistry and functional groups, inhibited acid
CDase.32,34 Treatment with B13 caused the release of
cytochrome C and induced apoptosis.34 Biological activ-
ity of B13 was also demonstrated in malignant mela-
noma, colon, and prostate cancer cells, and in animal
experiments of in vivo cancer growth.32–34
In a previous study, we showed that isosteric replace-
ment of the amide group of Cer by urea or N-alkylamine
units generated inhibitors of neutral CDase, thus illus-
trating the usefulness of this approach.42 Moreover, in
another recent study, we developed the concept of the
fixed positive charge-dependent cellular targeting Cer,
and demonstrated that fixed cationic Cer analogs target
preferentially to the mitochondria.14,16,17
Extending these findings to the aromatic analogs of Cer,
we suspected that introduction of specific structural fea-
tures and other modifications of ꢀCO, X, Y, R, and R1-
functionalities as well as altering the stereochemistry of
D-e-MAPP and B13 structures (Scheme 1) will improve
and modify their physicochemical and targeting proper-
ties to specific compartments.
D-e-MAPP, B13, and Cers are N-acylamino alcohols
with two distinct elements of rigidity: a phenyl ring or
long chain alkenyl unit and the amido group joined to
the chiral backbone of the propane chain (Scheme 1).
These new compounds are shown in Scheme 2. Based on
targeting behavior of alkylamines, we expected that

Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
1017
LCL11-(1S, 2R): X = CH2, R1,2 = H, R3 = CH3
OH
LCL12-(1R ,2S): X = CH2, R1,2 = H, R3 = CH3
LCL13-(1S, 2S): X = CH2, R1,2 = H, R3 = CH3
LCL14-(1R, 2R): X = CH2, R1,2 = H, R3 = CH3
i
A
R1
X
N
LCL15-(1R, 2R): X = NH, R1 = OH,R2 = NO2,R3 = H
LCL16-(1S, 2R): X = NH, R1,2,3 = H
LCL17-(1R, 2S): X = NH, R1,2,3 = H,
R2
R3
ii
8
B
O
LCL81-(1S, 2S): X = NH, R1 = O H,R2 = NO2,R3 =H
LCL18-(1S, 2S): R1,2 = OH , R3,4 = H, n = 8
LCL204-(1R, 2R): R1,2 = OH, R3 = NO2, R4 = H, n = 8
LCL284-(1S, 2R): R1 = OH, R2,3,4 = H, n = 8
LCL286-(1S): R1,3,4 = H, R2 = OH, n = 8
LCL289-(1R, 2S): R1 = OH, R2,3,4 = H, n = 8
LCL343-(1S, 2S): R1,2 = OH, R3 = NO2, R4 = H, n = 8
LCL346-(1R, 2R): R1 = OH, R2,3,4 = H, n = 8
LCL347-(2R): R1,3,4 = H, R2 = OH, n = 8
R1
iii and iv
R2
NH
Cl
C
R3
R4
R1
n
LCL378-(1S, 2R): R1 = OH, R2,3,4 = H, n = 6
LCL381-(1S, 2R): R1 = OH, R2,3 = H2, R4 = CH3, n = 8
LCL385-(1R, 2R): R1,2 = OH, R3,4 = H, n = 8
LCL397- (1S, 2R): R1,3,4 = H, R2 = OH, n = 10
R2
NHR3
R4
OH
1-(1S,2R): R1 = OH,R2,4 = H, R3 = CH3
2-(1R,2S): R1 = OH,R2,4 = H, R3 = CH3
3-(1S,2S): R1 = OH,R2,4 = H, R3 = CH3
4-(1R,2R): R1 = OH,R2,4 = H, R3 = CH3
5-(1S,2R): R1 = OH,R2,3,4 = H
6-(1R,2R): R1 = OH,R2,3,4 = H
7-(1S,2S): R1 = OH,R2,3,4 = H
v and vi
R1
Br
HN
R2
N
LCL82-(1R, 2R): l = 5, k = 12
m
O
LCL85-(1R, 2R): R1 = H, R2 = NO2, m = 13
LCL120-(1S, 2R): R1,2 = H,m = 13
LCL420-(1R, 2S): R1,2 = H, m = 13
D
OH
8-(2S): R2 = OH,R1,3,4 = H
9-(2R): R2 = OH,R1,3,4 = H
10-(1R,2R): R1,2 = OH,R3 = H, R4 = NO2
11-(1S,2S): R1,2 = OH,R3 = H, R4 = NO2
12-(1R,2R): R1,2 = OH,R3,4 = H
13-(1S,2S): R1,2 = OH,R3,4 = H
i, vii, viii and vi
OH
O
N
HN
N
H
Br
l
k
O
Scheme 2. Synthetic outline for the new analogs of D-e-MAPP and B13. Reagents and conditions: (i) Myristoyl chloride, 50% CH3COONa, THF, rt;
(ii) dodecyl isocyanate, C2H5OH, THF, rt; (iii) CH3(CH2)pCHO (p = 10, 12, 14), NaBH3CN, CH3OH, CH3COOH, rt; (iv) 1 M HCl in diethyl ether,
ethyl acetate, rt; (v) 16-bromohexadecanoyl chloride, 50% CH3COONa, THF, rt; (vi) pyridine, toluene, 75–85 ꢁC, 4 h; (vii) Zn, CH3COOH, rt; (viii)
6-bromohexanoyl chloride, 50% CH3COONa, THF, rt.
some analogs will locate to lysosomes (e.g., N-alkylami-
no analogs, class C).43–46 In contrast, fixed cations are
expected to act as a mitochondriotropic agents (aro-
matic ceramidoids, class D).17 Finally, neutral analogs
(parent amides, N-methyl amides, class A, and urea ana-
logs, class B) may show no compartmental preferences
as was shown for exogenous Cers.47,48
of the phenyl ring. The stereochemical isomers are:
(1R,2S), (1R,2R), (1S,2R) (1S,2S), (2R), and (2S).
We introduce the following groups of compounds: class
A, N-methyl, N-myristoyl analogs of D-e-MAPP (and
the parent compounds); class B, urea analogs, for which
the N-acyl group is replaced by a nonhydrolyzable urea
group; class C, N-alkylamino analogs for which the
N-acyl moiety was reduced to an N-alkylamino group;
and class D, analogs containing an x-pyridinium salt
unit in the N-acyl group (shown in Scheme 2).
Here we describe the synthesis, full NMR characteriza-
tion, basic physicochemical properties, and growth sup-
pression of breast carcinoma MCF7 cells of new analogs
of D-e-MAPP and B13. Additionally, we present results
from the National Cancer Institute for a full screening
against a 60-human-tumor-cell assay (the NCI’s 60-cell
line assay) performed for the selected analogs from clas-
ses A–D.
2.2. Synthesis
The synthesis of the target compounds was carried out
efficiently according to the procedures presented in
Scheme 2. Four different approaches, based on the pre-
viously reported procedures for the preparation of
Cer,42 their mimics17,19,42 and varied D-e-MAPP, and
B13 analogs,21,22,49 were utilized to synthesize the
amphiphilic compounds LCL11–LCL420. Specifically,
N-methyl amides LCL11–LCL14 were prepared by N-
acylation of the corresponding stereoisomers of amines1–4
with myristoyl chloride under a basic, two-phase system.
The urea analogs of D-e-MAPP and B13, LCL15,
LCL16, LCL17 and LCL81, which have the C12-alkyl
chains mimicking the C13-alkyl chains of the parent amides,
were prepared by condensation of the corresponding
amines 5, 6, 10, and 11 with dodecyl isocyanate. Lipo-
philic amine analogs of D-e-Mapp, L-e-Mapp, and B13
2. Results and discussion
2.1. Chemistry
The compounds designed and synthesized in this study
represent the second generation of analogs that are
based on structural and stereochemical modifications
of D-e-MAPP and B13 (Scheme 1). The selected model
compounds generally contain C14-hydrocarbon chains
in their N-acyl parts. Structural analogs contain a mod-
ified proton donor–acceptor ability of the functional
groups at C1-, C2-, and C3-positions and substituents

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Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
were prepared by the reductive amination of dodecyl,
myristyl, and hexadecyl aldehydes with 1, 5–11, using
NaCNBH3 in a glacial acetic acid-methanol solution,
subsequent to the transformation of the formed di- or
trialkyl amine free bases to their corresponding hydro-
chloride salts LCL18–LCL397. Cationic analogs
LCL85, LCL120, and LCL420, which have a fixed posi-
tive charge located in their N-acyl chains, were prepared
by N-acylation of 5, 6, and 10 with 16-bromo-hexadeca-
noyl chloride after the process of conjugation of the
formed x-bromo-amides,14,15 and 16 with pyridine. Ana-
log LCL82, which has a pyridinium salt moiety attached
at the para-position of the phenyl ring, was prepared in a
three-step synthetic process starting from B13. Thus, the
nitro group of B13 was reduced with Zn in CH3COOH
to afford a 4-amino-derivative.17 This compound was
N-acylated with 6-bromo-hexanoyl chloride to form
the intermediate x-bromo-di-amide,18 which was conju-
gated in the final step with pyridine to give LCL82.
2.2.2. Inhibitory effects on MCF7 cell growth. The anti-
proliferative effects of the newly obtained compounds
were examined in MCF7 breast carcinoma cells and
compared to the activity of D-e-MAPP, B13, and their
enantiomers (L-e-MAPP and LCL5). Data are shown
in Figures 2 and 3. IC50 values represent the drug con-
centration that reduces the cell number by 50% during
the drug incubation for 48 h, signifying the growth
inhibitory power of the agents.
D-e-MAPP and B13 were less potent in MCF7 cells com-
pared to HL-60 cells.20,21 D-e-MAPP and B13 inhibited
cell growth in a dose-dependent manner (IC50 at 48 h,
30 lM and 14 lM, respectively), whereas L-e-MAPP
was inactive up to 50 lM (the highest concentration
tested). This compound was also inactive in HL-60
cells.21
The inhibitory profiles for the representative D-e-MAPP
and B13 analogs from classes A–D are shown in Figure
2a and b, respectively. IC50 values for all synthesized ana-
logs were calculated from studies similar to those shown
in Figure 2 as established for the 48-h time point, and
the IC50 for the compounds ranged from 2.3 lM to
ꢁ35.0 lM (Fig. 3). The potency of these analogs de-
pended on the modifications made in the N-acyl- part of
D-e-MAPP or B13. The N-methyl analogs and urea ana-
logs (classes A and B) were active with IC50 > 10 lM,
whereas the N-alkyl-amino analogs and cationic analogs
All synthesized model compounds were characterized by
NMR, MS, and polarimetry, confirming their structural
homogeneity and high optical purity (listed in Section 4).
2.2.1. Cellular levels. The cellular levels of representative
analogs from class A–D in MCF7 cells were established
by high-performance liquid chromatography-mass spec-
trometry (HPLC–MS) analysis. Experimental data from
treating cells with the representative analogs (10 lM)
over the indicated time showed a very fast cellular up-
take for all tested analogs with the highest extent for
analogs from class C (Fig. 1). Concentrations of
LCL284 and LCL204 after 0.5 h of treatment were
established as ꢁ3600 pmols/1 · 106 cells whereas for
the other analogs, the cellular levels were in the range
of 800–1700 pmols/1 · 106 cells with the lowest levels
being the class D analogs. Progressive increases up to
5 h were observed for all analogs. At this time point,
LCL204 and LCL284 were present at concentrations
two times higher than the other analogs. No significant
differences were noted for the representative analogs
from classes B and D compared to D-e-MAPP and B13.
Figure 2. Inhibitory effect on MCF7 cell growth (48 h). (a) Concen-
tration-dependent effect for the representative D-e-MAPP analogs. (b)
Concentration-dependent effect for the representative B13 analogs.
Figure 1. Cellular concentration of selected new analogs in MCF7 cells
(10 lM treatment over time).

Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
1019
Figure 3. IC50 profile for all synthesized compounds in MCF7 cells at 48 h.
(class C, D) were active with IC50 < 10 lM. Interestingly,
all new analogs of inactive L-e-MAPP were also potent
(LCL12, LCL17, LCL289, and LCL420).
whereas the urea analog of B13 (LCL15) and its enan-
tiomer (LCL81) were slightly less potent compared to
the parent compounds (D-e-MAPP, B13 and their enan-
tiomers, respectively). D-e-urea-MAPP, LCL16, was
more potent than its enantiomer, LCL17; however, no
enantiospecificity was found for urea B13 (LCL15 and
LCL81 were equally potent).
Additionally, activities of representative analogs from
classes C and D were compared to the activity of the
corresponding analogs of Cer: N-myristyl-sphingosine
(C14-ceramine,
LCL342)
and
CCPS
analogs
(LCL30).17,42 The results showed that C14-ceramine
was inactive up to 25 lM (the highest concentration
tested) and activity of LCL30 was comparable to the
activity of analogs from class D (not shown). The
potency of the analogs from class B was compared to
D-e-C6-urea-Cer (LCL25), which represents a cell-per-
meable, short-chain analog of C16-urea-Cer.42 The re-
sults showed that activity of LCL25 (IC50 ꢁ15 lM,
data not shown) was similar to class B analogs.
All N-myristyl-phenylamino-alcohols (class C) ably
inhibited MCF7 cell growth (IC50 3.5–8.0 lM, Fig. 3).
IC50 values for LCL284 and LCL204, analogs of the
parent compounds, were established at 3.5 lM and
4.5 lM, respectively. From available N-myristyl isomers
of D-e-MAPP, the most active was LCL284 bearing the
stereochemistry of D-e-MAPP, followed by its enantio-
mer LCL289 (IC50: 3.5 lM and 5.5 lM, respectively).
LCL346, the threo-isomer, bearing (1R,2R) stereochem-
istry, was less potent than its erythro-diastereoisomer,
LCL284. LCL286 and LCL347, the isomers of
LCL284, which contain the primary instead of the sec-
ondary OH group, were half as potent as LCL284.
LCL381, the N-methyl analog of LCL284, representing
a tertiary lipophilic amine, was almost equally potent as
LCL284. Shorter (C12–, LCL378) and longer (C16–,
LCL379) homologs of LCL284 were only a little less po-
tent than LCL284 (LCL378 IC50 = 9.0 lM; LCL397
IC50: 5.0 lM). Similar chain-length dependency was ob-
served previously for the N-acyl analogs.20,21
As shown in Figure 2a, N-methyl-D-e-MAPP (LCL11)
and D-e-urea-MAPP (LCL16) were twice as effective as
D-e-MAPP (similar IC50 values; however, at a higher
concentration, LCL16 was more effective than LCL11
resulting in 80% and 40% inhibition of growth, respec-
tively). The cationic analog LCL120 and the N-myristyl
analogs (LCL284) were ꢁ10· more potent than D-e-
MAPP. B13 analogs followed the pattern observed for
D-e-MAPP analogs with the exception of urea-B13
(LCL15). The low activity of this compound was also re-
ported for several different cell lines.49
The stereochemical effect of B13 analogs on cell growth
was studied for LCL204/LCL343 and LCL385/LCL18
pairs. These compounds represent (1R,2R) and (1S,2S)
enantiomeric pairs of the threo-isomers only. As shown
in Figure 3, the (1R,2R) isomers were more potent than
the (1S,2S) isomers.
Analogs from class A, with LCLs11–14 representing the
full set of stereoisomers, were more potent than D-e-
MAPP itself (Fig. 3). The least potent was the (1R,2S)
isomer bearing the same stereochemistry of L-e-MAPP
and its (1S,2S) diastereoisomer. The (1S,2R) isomer
bearing stereochemistry of D-e-MAPP and its (1R,2R)
diastereoisomer were more active. In general, isomers
with the threo-configuration were more potent than
their erythro diastereoisomers and the 2R isomers were
more active than 2S counterparts.
The effects of phenyl ring substituents on cell growth
were studied using LCL204 and LCL385 as model com-
pounds. These two analogs have the same stereochemis-
try (1R,2R), contain two hydroxyl groups, and differ
only in the presence or absence of the p-NO2 group in
the phenyl ring. LCL385 was almost equally potent as
LCL204 (Fig. 3).
As shown in Figure 3, urea analogs (class B) of D- and L-
e-MAPP (LCL16 and LCL17) had increased activity,

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Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
In conclusion, all compounds representing class C were
much more potent than the parent compounds, and only
a small influence of the structural changes (hydroxyl
group location and number, chiral center stereochemis-
try, and phenyl ring substituents types) was noticed for
their activity.
2.2.3. Evaluation of the selected analogs for potential
anticancer activity in the NCI’s in vitro human disease-
oriented tumor cell line screening panel (the NCI’s 60-cell
line assay). Twenty-three compounds selected from the
previous study were evaluated in the in vitro human dis-
ease-oriented tumor cell line screening panel developed
by the NCI in accordance with the protocol of the Drug
Evaluation Branch, National Cancer Institute, Bethes-
da, MD.50–54
The independently synthesized lipophilic amine,
AD2646, which shares the structure of LCL204 (hydro-
chloride of AD2646)—a compound we previously syn-
thesized and studied—was shown to act as a bioactive
molecule in HL-60 cells.22
The panel cell lines used in this test were: leukemia,
non-small cell lung cancer, colon cancer, central ner-
vous system cancer, melanoma, ovarian cancer, renal
cancer, prostate cancer, and breast cancer. The details
of the cell lines used are shown in Tables 2–4. The
cytotoxic and/or growth inhibitory effects were tested
in vitro against the panel of 60 human tumor cell
lines derived from nine neoplastic diseases at 10-fold
dilution of five concentrations ranging from 10ꢀ4 to
10ꢀ8 M. The growth percentage was evaluated spec-
trophotometrically. For each tested compound, three
parameters were measured for each cell line: GI50
(drug concentration that reduces net protein by 50%
during the incubation), TGI (total growth inhibition,
signifying toxicity), and LC50 (drug concentration
resulting in 50% reduction in measured protein at
the end of the drug treatment, compared to the
beginning, signifies net cell loss). The log10 GI50, and
log10 LC50 and log10 TGI were determined and defined
as the mean of the log10 of the individual GI50, TGI,
and LC50 values. The average GI50 values collected
from the 60-cell line assay for the 23 tested com-
pounds are shown in Table 1. Compounds with
log10 GI50 values = ꢀ4 and <ꢀ4 were considered
active.
Analogs from class D, LCL120, LCL85, and LCL420,
were as potent as the analogs from class C (Figs. 2a,
b, and 3). LCL120, LCL85, and LCL420 (analogs of
D-e-MAPP, B13 and L-e-MAPP) had the following
IC50 values: 2.5 lM, 2.3 lM, and 2.4 lM, respectively.
Somewhat surprisingly, again the analog of inactive
L-e-MAPP became active. Additionally, we found that
the location of the pyridinium disk in the molecule
played a significant role on the activity of this class of
compounds. Comparison of LCL85 and LCL82 (con-
tains an x-pyridinium disk in N-acyl part of 40-substitu-
ent of the phenyl ring, Scheme 2) showed that LCL82
had low activity (IC50 = 20 lM, Fig. 3), whereas
LCL85 was one of the most active compounds
(IC50 = 2.3 lM). A similar observation (activity/phenyl
substitution dependency) was noticed when the para-
NO2 group from B13 was replaced by the amino group.
LCL83 was inactive up to 25 lM (the highest concentra-
tion tested, data not shown).
These results indicate that all modifications introduced
into N-acyl part of D-e-MAPP, L-e-MAPP, and B13
structures generated analogs with higher activity than
the parent compounds. The most significant improve-
ments were observed for L-e-MAPP analogs.
Full profiles (60 cell lines) of the GI50, TGI, and LC50
values for the selected analogs LCL284, LCL289,
LCL286, LCL204, LCL385, LCL120, LCL420, and
LCL85 are shown in Tables 2–4, respectively.
From our previous work,21 we have learned that stereo-
chemistry of phenyl-N-acyl-amino alcohols plays an
important role on their activity. Analogs with the con-
figuration of natural Cer were not active as was shown
for L-e-MAPP, whereas their stereoisomers inhibited cell
growth with D-MAPP, the enantiomer of L-e-MAPP,
being the most potent. This effect may be related to
the fact that L-e-MAPP was metabolized on its N-amido
group, whereas D-e-MAPP inhibited this process sug-
gesting involvement of amidase/ceramidase enzymes.20
However, this enantiospecific effect was not observed
for the new analogs. Forced localization of LCL120
and LCL420 (enantiomeric pair of aromatic ceramid-
oids) to mitochondria demonstrated almost equal po-
tency suggesting an effect on some non-enantiospecific
enzymes. Enantiomeric pairs of class B and C analogs,
where the N-amido group was replaced by urea or N-al-
kyl groups also showed a similar activity suggesting that
these analogs can be recognized by some enzymes
(ceramidases) but do not serve as their substrates.
From Table 1, we can conclude that all tested com-
pounds were active in this test showing MGMID Log10
-
GI50 values from ꢀ4.02 to ꢀ5.77 (1.71–95.0 lM); d,
0.11–3.03; range, 0.3–3.30.
D-e-MAPP and B13 had a broad spectrum of activity
with the GI50 values ꢁꢀ4.0 (ꢁ100 lM); d < 1; and range
<1, indicating no cell specificity. Other analogs from
class A were more potent (GI50 ꢁꢀ4.9, ꢁ11 lM) but
the d values were also <1. Interestingly, LCL12, the ana-
log of L-e-MAPP, had a high GI50 (ꢀ4.96) with a d and
range >3. This compound was specifically active in
SNB-57 and MDA-MS-435 cancer cell lines (not
shown).
Class B analogs had GI50 values ꢁꢀ4.8 (ꢁ15 lM).
LCL16 and LCL15, urea analogs of the parent com-
pounds, had d values <1 and range values <1. However,
LCL17 and LCL81, the enantiomers of LCL16 and
LCL15 had d and range values >1, showing cancer cell
specificity to HOP-92 and BT-549 by LCL17 and BT-
549 by LCL81 (data not shown).
Considering cellular levels of the analogs from classes C
and D (Fig. 1), the most active analogs were the cationic
analogs: LCL85, LCL120, and LCL420.

Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
1021
Table 1. Average GI50 values collected from the NCI’s 60-cell line
assay
instead of the secondary OH group present in
LCL284, was very potent in BT-549 breast cancer cells
(ꢀ8.00, ꢁ10 nM). LCL120 was specifically very potent
in CCRF-CEM leukemia cells (<ꢀ8.00, <10 nM) and
was selective for breast cancer cells NCI-ADR-RES ver-
sus BT-549 (v4.71, 19.5 lM and ꢀ7.16, 68 nM, respec-
tively). Similar selectivity was detected for LCL420,
the enantiomer of LCL120 (>4.00 vs ꢀ8.00). B13 ana-
logs LCL385, LCL204, and LCL85 were also very active
(GI50 ꢁꢀ5.5, 2.8 lM) with broad-spectrum activity and
low specificity, except for LCL85.
MG MID Log10 GI50
Delta
Range
Class A
D-Mapp
B13
ꢀ4.03
ꢀ4.02
ꢀ4.90
ꢀ4.84
ꢀ4.96
0.32
0.47
0.75
0.37
3.03
0.35
0.49
1.48
0.60
3.30
LCL5
LCL11
LCLI2
Class B
LCL16
LCLI7
LCL15
LCL81
ꢀ4.76
ꢀ4.84
ꢀ4.84
ꢀ4.83
0.15
2.33
0.54
1.20
0.30
2.57
0.72
1.36
A full screen on the in vitro total growth inhibition
(TGI, M) by these selected analogs is shown in Table
3. Data indicate that all tested analogs were cytostatic
with TGI values between ꢀ4.84 and ꢀ5.5 (14.3–
2.8 lM). LCL120 and LCL420 showed some safety
specificity to NCI-ADR-RES breast cancer cells and
HCT-15 colon cancer cells (ꢁꢀ4, ꢁ100 lM).
Class C
LCL18
ꢀ5.73
ꢀ5.58
ꢀ5.70
ꢀ5.67
ꢀ5.75
ꢀ5.71
ꢀ5.77
ꢀ5.74
ꢀ5.77
ꢀ5.68
0.15
0.32
0.24
0.38
1.31
0.11
1.46
2.26
2.23
0.15
1.01
1.06
1.20
1.17
2.09
0.94
2.42
3.16
3.27
1.01
LCL204
LCL385
LCL343
LCL284
LCL289
LCL346
LCL286
LCL347
LCL381
A full screen on the in vitro tumor 50% lethal concentra-
tion (LC50, M) of these analogs is shown in Table 4.
Data indicate that all tested analogs were cytotoxic with
LC50 values between >ꢀ4.00 and ꢀ5.00 (10–100 lM). In
most cases, cytotoxic effects of the class D compounds
were lower than class C compounds.
Class D
LCL85
LCL120
LCL420
LCL82
ꢀ5.30
ꢀ5.67
ꢀ5.42
ꢀ5.03
1.13
2.33
2.58
0.85
1.73
3.29
4.00
1.25
3. Summary and conclusions
Based on the chemical structure differences and cell
activity presented here, compounds were grouped into
4 classes (A, B, C, and D). Class A: N-acyl analogs (neu-
tral); class B: urea analogs (neutral); class C: N-alkyl
analogs (lysosomotropic analogs); class D: x-cationic
analogs (mitochondriotropic analogs).
The most active were analogs from classes C and D,
with GI50 values from ꢀ5.03 to ꢀ5.77 (1.71–9.2 lM).
Class C analogs had GI50 values ꢀ5.58 to ꢀ5. 77 (2.61–
1.71 lM) with a broad spectrum of d values (0.15–2.26),
ranging from 0.94 to 3.27. From this group, LCL284,
LCL346, LCL286, and LCL347 had d > 1 indicating
cancer cell specificity. Remaining analogs showed a
broad spectrum of antitumor activity against the nine
tumor subpanels tested with LCL385 being the most
potent.
All new analogs showed an increased inhibitory effect on
MCF7 cell growth, compared to their parent com-
pounds. Class A and class B were active with
IC50 > 10 lM, whereas analogs from classes C and D
were active with IC50 < 10 lM. New analogs of inactive
L-e-MAPP were also potent.
Class D, x-cationic ceramidoids: LCL82, LCL85,
LCL120, and LCL420 had GI50 values from ꢀ5.03 to
ꢀ5.67, d ꢁ 1 (0.85, 1.13, 2.33, and 2.58, respectively)
and range values >1 (1.25, 1.73, 3.29, and 4.0, respec-
tively) indicating cancer cell specificity. Again, the least
potent was LCL82 (ꢁ9.2 lM).
An SAR study from the NCI’s 60 cell-based assay
showed that all tested compounds were active, with
GI50 values from ꢀ4.02 to ꢀ5.77; d values from 0.11
to 3.03, and a range values of 0.3–3.30. These results
indicate promising activity against all cancer cell lines
and specificity of particular analogs to certain cancers.
Again, the most active analogs were from classes C
and D, with GI50values from ꢀ5.03 to ꢀ5.77 (ꢁ1–
10 lM). Cationic analogs were more specific for certain
cell lines. Analogs from the B13 family had a non-selec-
tive spectrum of activity, whereas analogs of the D-e-
MAPP family were selective for particular cancer cell
lines. Compounds LCL85, LCL420, LCL120, LCL385,
LCL204, LCL286, LCL289, and LCL284 proved to be
the most promising derivatives identified from this ser-
ies. LCL85, LCL120, LCL420, and LCL385 had potent
anticancer activity and lower toxicity, and LCL284,
LCL289, and LCL286 had potent anticancer activity
and cancer specificity. These attributes, combined with
A full screen on the in vitro tumor 50% growth inhibi-
tion (GI50, M) for the selected analogs from classes C
and D, LCL284, LCL289, LCL246, LCL204, LCL385,
and LCL120, LCL420, LCL85 is shown in Table 2.
We can conclude that all presented analogs were potent
at the level of ꢁ1 lM with broad-spectrum antitumor
activity. LCL284 was specifically active in HL-60 (TB)
leukemia cells (ꢀ7.06, 86 nM) but showed low activity
in SK-MEL-5 cells (ꢀ4.97, 11 lM). Low activity in
SK-MEL-5 cells (ꢀ4.88, 12 lM) was also detected for
LCL289, the enantiomer of LCL284. LCL286, an
isomer of LCL284 containing the primary OH group

1022
Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
Table 2. Full screen, in vitro tumor 50% growth inhibition (log10 GI50
)
Cell line
Panel
LCL284
LCL289
LCL286
LCL204
LCL385
LCL120
LCL420
LCL85
Leukemia
CCRF-CEM
HL-60(TB)
K-562
ꢀ5.84
ꢀ7.06
ꢀ5.85
ꢀ6.06
ꢀ5.74
ꢀ5.62
ꢀ5.70
ꢀ5.78
ꢀ5.64
ꢀ5.70
ꢀ5.76
ꢀ5.75
ꢀ5.74
ꢀ5.69
ꢀ5.81
ꢀ5.70
ꢀ5.69
ꢀ5.73
ꢀ5.79
ꢀ5.66
ꢀ5.80
ꢀ5.48
ꢀ5.65
ꢀ5.68
ꢀ5.78
<ꢀ8.00
ꢀ5.95
ꢀ6.08
ꢀ5.60
ꢀ6.54
ND
5.00
ꢀ4.84
ꢀ5.43
ꢀ4.97
ꢀ4.70
ꢀ5.83
ꢀ6.04
ꢀ5.31
ND
MOLT-4
RPMI-8226
Non-small cell lung cancer
A549/ATCC
EKVS
HOP-62
ꢀ5.76
ꢀ5.72
ꢀ5.70
ꢀ5.71
ꢀ5.82
ꢀ5.71
ꢀ5.74
ꢀ5.69
ꢀ5.72
ꢀ5.69
ꢀ5.69
ꢀ5.33
ꢀ5.91
ꢀ5.91
ꢀ5.72
ꢀ5.68
ꢀ5.70
ꢀ5.74.
ꢀ5.68
ꢀ5.45
ꢀ5.70
ꢀ5.72
ꢀ5.90
ꢀ4.98
ꢀ4.84
ꢀ5.32
ꢀ5.72
ꢀ5.70
ꢀ5.77
ꢀ5.50
ꢀ5.83
ꢀ5.85
ꢀ5.49
ꢀ5.45
ꢀ5.67
ꢀ5.73
ꢀ5.70
ꢀ5.36
ꢀ5.59
ꢀ5.57
ꢀ7.39
ꢀ5.38
ꢀ5.53
ꢀ4.96
ꢀ5.53
ꢀ5.83
ꢀ5.29
ꢀ5.44
ꢀ5.15
ND
ꢀ5.40
ꢀ4.89
ꢀ5.86
ꢀ5.68
ꢀ4.95
ꢀ4.74
ꢀ5.41
ꢀ6.43
ꢀ5.53
ꢀ5.79
ꢀ5.73
ꢀ5.81
ꢀ5.73
ꢀ5.67
ꢀ5.72
ꢀ5.75
ꢀ5.72
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
ꢀ5.19
ꢀ5.53
ꢀ4.84
ꢀ5.55
ꢀ5.69
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
ꢀ5.72
ꢀ5.73
ꢀ5.73
ꢀ5.74
ꢀ5.75
ꢀ5.74
ꢀ5.67
ꢀ5.76
ꢀ5.74
ꢀ5.73
ꢀ5.72
ꢀ5.73
ꢀ5.73
ꢀ5.69
ꢀ5.78
ꢀ5.65
ꢀ5.69
ꢀ5.77
ꢀ5.69
ꢀ5.73
ꢀ5.70
ꢀ5.75
ꢀ5.62
ꢀ5.71
ꢀ5.73
ꢀ5.73
ꢀ5.73
ꢀ5.70
ꢀ5.73
ꢀ5.78
ꢀ5.76
ꢀ5.72
ꢀ5.77
ꢀ5.73
ꢀ5.62
ꢀ5.86
ꢀ5.82
ꢀ5.47
ꢀ4.75
ꢀ5.80
ꢀ5.76
ꢀ5.37
ꢀ5.98
ꢀ5.81
ꢀ5.46
ꢀ4.24
ꢀ5.88
ꢀ5.56
ꢀ5.16
ꢀ5.64
ꢀ5.87
ꢀ5.78
ꢀ5.14
ꢀ5.84
ꢀ5.93
ꢀ4.94
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
ꢀ5.75
ꢀ5.74
ꢀ5.87
ꢀ5.76
ꢀ5.68
ꢀ5.73
ꢀ5.75
ꢀ5.75
ꢀ5.75
ꢀ5.77
ꢀ5.77
ꢀ5.74
ꢀ5.75
ꢀ5.75
ꢀ5.77
ꢀ5.77
ꢀ5.66
ꢀ5.76
ꢀ5.68
ꢀ5.65
ꢀ5.78
ꢀ5.76
ꢀ5.40
ꢀ5.73
ꢀ5.78
ꢀ5.73
ꢀ5.81
ꢀ5.75
ꢀ5.64
ꢀ5.75
ꢀ5.78
ꢀ5.08
ꢀ5.69
ꢀ5.80
ꢀ5.53
ꢀ5.85
ꢀ5.49
ꢀ4.84
ꢀ5.47
ꢀ5.51
ꢀ5.77
ꢀ5.53
ꢀ4.89
ꢀ5.57
ꢀ5.07
ꢀ4.90
ꢀ4.80
ꢀ5.25
Melanoma
LOX IMVI
MALME-3M
M14
ꢀ5.74
ꢀ5.71
ꢀ5.73
ꢀ5.70
ꢀ5.73
ꢀ4.97
ꢀ5.78
ꢀ5.74
ꢀ5.75
ꢀ5.69
ꢀ5.71
ꢀ5.75
ꢀ5.72
ꢀ4.88
ꢀ5.73
ꢀ5.74
ꢀ5.80
ꢀ5.70
ꢀ5.65
ꢀ5.57
ꢀ5.72
ꢀ5.75
ꢀ5.68
ꢀ5.70
ꢀ5.76
ꢀ5.61
ꢀ5.69
ꢀ5.48
ꢀ5.72
ꢀ5.61
ꢀ5.37
ꢀ5.19
ꢀ5.75
ꢀ5.68
ꢀ5.73
ꢀ5.67
ꢀ5.74
ꢀ5.67
ꢀ5.77
ꢀ5.74
ꢀ5.45
ꢀ5.78
ꢀ5.74
ꢀ5.66
ꢀ5.77
ꢀ5.80
ꢀ5.80
ꢀ5.83
ꢀ5.39
ꢀ5.61
ꢀ5.20
ꢀ5.54
ꢀ5.63
ꢀ5.79
ꢀ5.65
ꢀ5.73
ꢀ5.66
ꢀ4.82
ꢀ5.32
ꢀ5.40
ꢀ5.47
ꢀ4.92
ꢀ4.79
ꢀ4.87
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
ꢀ5.79
ꢀ5.76
ꢀ5.67
ꢀ5.70
ꢀ5.74
ꢀ5.63
ꢀ5.74
ꢀ5.78
ꢀ5.70
ꢀ5.77
ꢀ5.72
ꢀ5.63
ꢀ5.72
ꢀ5.78
ꢀ5.72
ꢀ5.62
ꢀ5.72
ꢀ5.66
ꢀ5.72
ꢀ5.09
ꢀ5.72
ꢀ5.43
ꢀ5.64
ꢀ5.05
ꢀ5.81
ꢀ5.72
ꢀ5.72
ꢀ5.68
ꢀ5.73
ꢀ5.17
ꢀ5.79
ꢀ5.96
ꢀ5.77
ꢀ5.28
ꢀ5.56
ꢀ5.51
ꢀ5.50
ꢀ5.88
ꢀ5.78
ꢀ4.96
ꢀ5.41
ꢀ5.48
ꢀ5.60
ꢀ5.38
ꢀ4.95
ꢀ5.50
ꢀ5.42
ꢀ4.70
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
A498
ꢀ5.76
ꢀ5.50
ꢀ5.73
ꢀ5.71
ꢀ5.86
ꢀ5.75
ꢀ5.73
ꢀ5.76
ꢀ5.80
ꢀ5.55
ꢀ5.75
ꢀ5.71
ꢀ5.78
ꢀ5.74
ꢀ5.70
ꢀ5.76
ꢀ5.74
ꢀ4.84
ꢀ5.77
ꢀ5.78
ꢀ5.77
ꢀ5.73
ꢀ5.69
ꢀ5.36
ꢀ5.79
ꢀ5.17
ꢀ5.73
ꢀ5.12
ꢀ5.74
ꢀ5.66
ꢀ4.98
ꢀ5.37
ꢀ5.79
ꢀ4.74
ꢀ5.76
ꢀ5.78
ꢀ5.94
ꢀ5.76
ꢀ5.70
ꢀ5.42
ꢀ5.58
ꢀ4.89
ꢀ4.77
ꢀ5.86
ꢀ5.34
ꢀ5.76
ꢀ5.31
ꢀ4.72
ꢀ4.99
ꢀ4.75
ꢀ4.66
ꢀ4.85
ꢀ4.92
ꢀ5.46
ꢀ4.84
ꢀ4.54
ꢀ5.77
ꢀ5.05
ꢀ5.41
ꢀ5.85
ꢀ5.30
ꢀ5.49
ꢀ5.60
ꢀ4.79
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC3
DU-145
ꢀ5.73
ꢀ5.83
ꢀ5.69
ꢀ5.73
ꢀ5.79
ꢀ5.84
ꢀ5.76
ꢀ5.81
ꢀ5.80
ꢀ5.78
ꢀ5.86
ꢀ5.46
ND
ꢀ4.90
ꢀ5.69
ꢀ5.63

Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
1023
Table 2 (continued)
Cell line
Panel
LCL284
LCL289
LCL286
LCL204
LCL385
LCL120
LCL420
LCL85
Breast cancer
MCF7
ꢀ5.74
ꢀ5.74
ꢀ5.76
ꢀ5.60
ꢀ5.75
ꢀ5.95
ꢀ5.64
ꢀ5.73
ꢀ5.77
ꢀ5.72
ꢀ5.66
ꢀ5.75
ꢀ5.82
ꢀ5.68
ꢀ5.76
ꢀ5.73
ꢀ5.70
ꢀ5.62
ꢀ5.76
ꢀ8.00
ꢀ5.78
ꢀ5.72
ꢀ5.69
ꢀ5.70
ꢀ5.68
ꢀ5.72
ꢀ5.82
ꢀ5.42
ꢀ5.74
ꢀ5.68
ꢀ5.76
ꢀ5.55
ꢀ5.75
ꢀ5.89
ꢀ5.72
ꢀ5.80
ꢀ4.71
ꢀ5.76
ꢀ5.75
ꢀ5.80
ꢀ7.16
ꢀ5.81
ꢀ5.83
>ꢀ4.00
ꢀ5.49
ꢀ5.62
ꢀ5.58
ꢀ8.00
ꢀ6.03
ꢀ5.39
ꢀ4.79
ꢀ5.62
ꢀ4.93
ꢀ5.63
ꢀ5.48
ꢀ4.79
NCI-ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
T-47D
MG-MID
Delta
Range
ꢀ5.75
ꢀ5.71
ꢀ5.74
ꢀ5.58
ꢀ5.70
ꢀ5.67
ꢀ5.42
ꢀ5.30
1.31
2.09
0.11
0.94
2.26
3.16
0.32
1.06
0.24
1.20
2.33
3.29
2.58
4.00
1.13
1.73
the synthetic ease of their preparation, suggest that these
analogs are promising lead structures for cancer chemo-
therapy development.
4.1.1. General procedure for the preparation of LCL11–
14 (class A). To a well-stirred solution of an appropriate
2-N-methylamino-1-phenyl-1-propanol (1–4, 0.83 g,
5.0 mmol) or their hydrochloride salts (1.0 g, 5.0 mmol)
in a mixture of 50% aqueous solution of sodium acetate
(24 mL) and THF (50 mL), myristoyl chloride (1.23 g,
5.0 mmol) was added dropwise at room temperature
(rt). The reaction mixture was stirred for 4 h. The organ-
ic phase was separated and the aqueous layer was ex-
tracted twice with ethyl acetate (2 · 10 mL). The
combined organic phases were dried over anhydrous
magnesium sulfate and evaporated under reduced pres-
sure to dryness to give a crude product. This material
was purified by flash column chromatography (elution
with CHCl3/MeOH/concd NH4OH, 10:2:0.01, v/v/v) fol-
lowing crystallization from suitable solvents.
4. Experimental
4.1. Chemistry
All solvents, general reagents, and starting amino alco-
hols 1–13 (Scheme 2) were purchased from Aldrich-Sigma
& Fluka. D-e-MAPP, L-e-MAPP, (1R,2R)-B13, and
(1S,2S)-B13 were prepared from the corresponding ami-
no alcohols 5, 6, 10 and 11 as described previously.20,21
1
Purity of these compounds was confirmed by H NMR
23
D
and the optical rotation values: D-e-MAPP: ½aꢂ þ 14:1
23
365
(c 1, MeOH); ½aꢂ
þ 43:9 (c 1, MeOH); L-e-MAPP:
23
D
23
365
4.1.1.1. (1S,2R)-2-(N-Methyl,N-tetradecanoylamino)-
1-phenyl-1-propanol (LCL11). Prepared from 1 in 78%
yield after crystallization from hexane–ethyl acetate
(1:1, v/v) as a white microcrystalline powder, mp 34–
36 ꢁC; TLC (CHCl3/MeOH, 5:1, v/v), Rf 0.74;
½aꢂ ꢀ 14:9 (c 1, MeOH); ½aꢂ
24
ꢀ 45:6 (c 1, MeOH).
24
D
(1R,2R)-B13: ½aꢂ ꢀ 29:3 (c 1, CHCl3); (1S,2S)-B13:
½aꢂ þ 29:6 (c 1, CHCl3). Reaction progress was moni-
D
tored with 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) or
0.150 mm C18-silica gel (Sorbent Technologies). Detec-
tion was performed with PMA reagent (ammonium hep-
tamolybdate tetrahydrate cerium sulfate, 5:2, g/g) in
125 mL of 10% H2SO4 and the Dragendorff reagent (Flu-
ka) following heating of the TLC plates at 170 ꢁC or with
UV (254 nm). Flash chromatography was performed
using EM Silica Gel 60 (230–400 mesh) with the indicated
eluent systems. Melting points were determined in open
capillaries on an Electrothermal IA 9200 melting point
apparatus and are reported uncorrected. Optical rotation
data were acquired using a Jasco P-1010 polarimeter. 1H
NMR spectra were recorded 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 chloro-
form (7.26 ppm). Mass spectral data were recorded in a
positive ion electrospray ionization (ESI) mode on Ther-
mo Finnigan TSQ 7000 triple quadrupole mass spectrom-
eter. Samples were infused in a methanol solution with an
ESI voltage of 4.5 kV and capillary temperature of
200 ꢁC.55 Specific structural features were established by
fragmentation pattern upon electrospray (ESI/MS/MS)
conditions, specific for each of the compound studied.
25
D
25
365
½aꢂ þ 10:2 (c 1, MeOH); ½aꢂ
þ 29:8 (c 1, MeOH);
1H NMR (500 MHz, CDCl3)d 7.34 (m, 4H, 2,3,5,6-
ArH), 7.27 (m, 1H, 4-ArH), 4.84 (m, 0.8H, 1-H), 4.67
(m, 0.2H, 1-H), 4.47 (m, 0.8H, 2-H), 4.0 (m, 0.2H, 2-H),
2.83 (s, 0.6H, NCH3), 2.67 (s, 2.4H, NCH3), 2.25 (m,
2H, COCH2), 1.58 (m, 2H, COCH2CH2), 1.23 (m, 20H,
CH2), 1.19 (d, 3H, J = 6.8, CHCH3) 0.87 (t, 3H, J = 7.0,
CH3); ESI-MS (CH3OH, relative intensity, %) m/z 376.6
(MH+, 100). Calcd for [C24H42NO2]+ m/z 376.31.
4.1.1.2. (1R,2S)-2-(N-Methyl,N-tetradecanoylamino)-
1-phenyl-1-propanol (LCL12). Prepared from 2 in 76%
yield after crystallization from n-hexane–ethyl acetate
25
(1:1, v/v) as a white powder; ½aꢂ ꢀ 10:9 (c 1, MeOH);
D
25
365
½aꢂ
ꢀ 31:2 (c 1, MeOH). Remaining data are identical
as reported for LCL11.
4.1.1.3. (1S,2S)-2-(N-Methyl,N-tetradecanoylamino)-
1-phenyl-1-propanol (LCL13). Prepared from 3 in 78%
yield after crystallization from hexane–ethyl acetate
(1:1, v/v) as a white microcrystalline powder, mp 30–
32 ꢁC; TLC (CHCl3/MeOH, 5:1, v/v), Rf 0.66;
25
D
25
365
½aꢂ þ 69:2 (c 1, MeOH); ½aꢂ
þ 264:0 (c 1, MeOH);
1H NMR (500 MHz, CDCl3) d 7.33 (m, 4H, 2,3,5,6-

1024
Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
Table 3. Full screen, in vitro total growth inhibition (Log10 TGI)
Cell line
Panel
LCL284
LCL289
LCL286
LCL204
LCL385
LCL120
LCL420
LCL85
Leukemia
CCRF-CEM
HL-60(TB)
K-562
ꢀ5.48
ꢀ5.60
ꢀ5.55
ꢀ5.66
ꢀ5.44
ꢀ5.29
ꢀ5.40
ꢀ5.45
ꢀ5.33
ꢀ5.36
ꢀ5.43
ꢀ5.39
ꢀ5.39
ꢀ5.36
ꢀ5.47
ꢀ5.40
ꢀ5.32
ꢀ5.42
ꢀ5.45
ꢀ5.33
ꢀ5.45
ꢀ5.43
ꢀ5.31
ND
ND
ND
ꢀ4.62
ꢀ4.52
ꢀ4.90
ꢀ4.62
ꢀ4.41
ꢀ5.50
ꢀ5.40
ꢀ5.19
ꢀ5.72
ꢀ5.36
ꢀ4.80
ꢀ4.73
ND
MOLT-4
RPMI-8226
ꢀ5.44
Non-small cell lung cancer
A549/ATCC
EKVS
HOP-62
ꢀ5.48
ꢀ5.44
ꢀ5.42
ꢀ5.46
ꢀ5.51
ꢀ5.42
ꢀ5.45
ꢀ5.43
ꢀ5.46
ꢀ5.41
ꢀ5.33
ꢀ4.77
ꢀ5.59
ꢀ5.55
ꢀ5.42
ꢀ5.35
ꢀ5.41
ꢀ5.47
ꢀ5.40
ꢀ4.71
ꢀ5.37
ꢀ5.46
ꢀ5.56
ꢀ4.46
ꢀ4.46
ꢀ4.83
ꢀ5.45
ꢀ5.40
ꢀ5.49
ꢀ4.76
ꢀ5.55
ꢀ5.54
ꢀ4.93
ꢀ5.01
ꢀ5.41
ꢀ5.46
ꢀ5.42
ꢀ4.36
ꢀ5.06
ꢀ5.05
ꢀ6.04
ꢀ4.64
ꢀ4.93
ꢀ4.64
ꢀ4.94
ꢀ5.44
ꢀ4.66
ꢀ4.83
ꢀ4.69
ND
ꢀ4.77
ꢀ4.55
ꢀ5.55
ꢀ5.25
ꢀ4.56
ꢀ4.46
ꢀ4.88
ꢀ5.78
ꢀ5.13
ꢀ5.51
ꢀ5.48
ꢀ5.52
ꢀ5.44
ꢀ5.39
ꢀ5.48
ꢀ5.48
ꢀ5.45
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
ꢀ4.57
ꢀ4.81
ꢀ4.50
ꢀ4.88
ꢀ4.88
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
ꢀ5.47
ꢀ5.46
ꢀ5.49
ꢀ5.49
ꢀ5.49
ꢀ5.50
ꢀ5.38
ꢀ5.48
ꢀ5.49
ꢀ5.46
ꢀ5.46
ꢀ5.45
ꢀ5.47
ꢀ5.50
ꢀ5.43
ꢀ5.43
ꢀ5.50
ꢀ5.40
ꢀ5.47
ꢀ5.39
ꢀ5.45
ꢀ5.25
ꢀ5.46
ꢀ5.46
ꢀ5.46
ꢀ5.47
ꢀ5.33
ꢀ5.47
ꢀ5.51
ꢀ5.50
ꢀ5.47
ꢀ5.51
ꢀ5.48
ꢀ5.52
ꢀ5.52
ꢀ4.88
ꢀ4.49
ꢀ5.46
ꢀ5.50
ꢀ4.76
ꢀ5.31
ꢀ5.13
ꢀ4.83
>ꢀ4.00
ꢀ4.93
ꢀ4.94
ꢀ4.70
ꢀ5.30
ꢀ5.54
ꢀ5.52
ꢀ4.70
ꢀ5.48
ꢀ5.61
ꢀ4.62
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
ꢀ5.46
ꢀ5.47
ꢀ5.57
ꢀ5.51
ꢀ5.41
ꢀ5.48
ꢀ5.47
ꢀ5.47
ꢀ5.47
ꢀ5.50
ꢀ5.47
ꢀ5.49
ꢀ5.45
ꢀ5.48
ꢀ5.47
ꢀ5.50
ꢀ5.32
ꢀ5.49
ꢀ5.31
ꢀ5.33
ꢀ5.50
ꢀ5.45
ꢀ4.75
ꢀ5.46
ꢀ5.49
ꢀ5.45
ꢀ5.51
ꢀ5.49
ꢀ5.25
ꢀ5.50
ꢀ5.50
ꢀ4.62
ꢀ5.26
ꢀ5.51
ꢀ5.27
ꢀ5.56
ꢀ4.88
ꢀ4.56
ꢀ4.85
ꢀ4.88
ꢀ4.95
ꢀ4.86
ꢀ4.55
ꢀ4.99
ꢀ4.68
ꢀ4.59
ꢀ4.40
ꢀ4.70
Melanoma
LOX IMVI
MALME-3M
MI4
ꢀ5.48
ꢀ5.47
ꢀ5.47
ꢀ5.40
ꢀ5.49
ꢀ5.46
ꢀ5.48
ꢀ5.46
ꢀ5.49
ꢀ5.44
ꢀ5.45
ꢀ5.43
ꢀ5.46
ꢀ4.32
ꢀ5.45
ꢀ5.47
ꢀ5.52
ꢀ5.45
ꢀ5.41
ꢀ5.26
ꢀ5.46
ꢀ5.49
ꢀ5.32
ꢀ5.40
ꢀ5.49
ꢀ5.31
ꢀ5.43
ꢀ5.09
ꢀ5.47
ꢀ5.15
ꢀ4.68
ꢀ4.62
ꢀ5.50
ꢀ5.42
ꢀ5.48
ꢀ5.32
ꢀ5.49
ꢀ5.39
ꢀ5.46
ꢀ5.43
ꢀ4.87
ꢀ5.51
ꢀ5.41
ꢀ5.36
ꢀ5.50
ꢀ5.53
ꢀ5.47
ꢀ5.54
ꢀ4.83
ꢀ5.23
ꢀ4.71
ꢀ5.13
ꢀ5.22
ꢀ5.41
ꢀ5.08
ꢀ5.24
ꢀ5.31
ꢀ4.54
ꢀ4.75
ꢀ4.86
ꢀ4.88
ꢀ4.61
ꢀ4.42
ꢀ4.57
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
ꢀ5.51
ꢀ5.49
ꢀ5.42
ꢀ5.46
ꢀ5.42
ꢀ5.32
ꢀ5.45
ꢀ5.50
ꢀ5.43
ꢀ5.50
ꢀ5.39
ꢀ5.32
ꢀ5.43
ꢀ5.52
ꢀ5.46
ꢀ5.26
ꢀ5.42
ꢀ5.34
ꢀ5.44
ꢀ4.51
ꢀ5.45
ꢀ4.85
ꢀ5.25
ꢀ4.56
ꢀ5.49
ꢀ5.39
ꢀ5.46
ꢀ5.42
ꢀ5.42
ꢀ4.61
ꢀ5.48
ꢀ5.62
ꢀ5.27
ꢀ4.74
ꢀ4.87
ꢀ4.87
ꢀ4.80
ꢀ5.19
ꢀ4.91
ꢀ4.62
ꢀ4.64
ꢀ4.92
ꢀ5.19
ꢀ4.78
ꢀ4.56
ꢀ5.13
ꢀ4.77
ꢀ4.33
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
A498
ꢀ5.50
ꢀ5.19
ꢀ5.48
ꢀ5.47
ꢀ5.56
ꢀ5.46
ꢀ5.48
ꢀ5.49
ꢀ5.51
ꢀ5.00
ꢀ5.48
ꢀ5.47
ꢀ5.50
ꢀ5.46
ꢀ5.46
ꢀ5.47
ꢀ5.49
ꢀ4.56
ꢀ5.50
ꢀ5.51
ꢀ5.46
ꢀ5.43
ꢀ5.43
ꢀ4.67
ꢀ5.51
ꢀ4.57
ꢀ5.47
ꢀ4.62
ꢀ5.41
ꢀ5.32
ꢀ4.58
ꢀ4.73
ꢀ5.52
ꢀ4.37
ꢀ5.50
ꢀ5.45
ꢀ5.61
ꢀ5.48
ꢀ5.33
ꢀ4.75
ꢀ5.07
ꢀ4.55
ꢀ4.48
ꢀ4.52
ꢀ4.62
ꢀ5.44
ꢀ4.80
ꢀ4.38
ꢀ4.65
ꢀ4.49
ꢀ4.35
ꢀ4.56
ꢀ4.53
ꢀ4.83
ꢀ4.55
ꢀ4.02
ꢀ5.50
ꢀ4.68
ꢀ4.87
ꢀ5.25
ꢀ4.67
ꢀ4.87
ꢀ5.19
ꢀ4.51
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate caner
PC-3
DU-145
ꢀ5.48
ꢀ5.55
ꢀ5.43
ꢀ5.46
ꢀ5.55
ꢀ5.51
ꢀ5.50
ꢀ5.54
ꢀ5.53
ꢀ5.50
ꢀ5.40
ꢀ4.82
ND
ꢀ4.60
ꢀ5.30
ꢀ4.96

Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
1025
Table 3 (continued)
Cell line
Panel
LCL284
LCL289
LCL286
LCL204
LCL385
LCL120
LCL420
LCL85
Breast cancer
MCF7
ꢀ5.47
ꢀ5.45
ꢀ5.47
ꢀ5.26
ꢀ5.50
ꢀ5.62
ꢀ5.38
ꢀ5.47
ꢀ5.47
ꢀ5.45
ꢀ5.26
ꢀ5.49
ꢀ5.53
ꢀ5.40
ꢀ5.49
ꢀ5.46
ꢀ5.44
ꢀ5.30
ꢀ5.50
ꢀ5.75
ꢀ5.48
ꢀ5.46
ꢀ5.37
ꢀ5.41
ꢀ5.28
ꢀ5.48
ꢀ5.54
ꢀ4.85
ꢀ5.48
ꢀ5.42
ꢀ5.47
ꢀ5.19
ꢀ5.50
ꢀ5.58
ꢀ5.44
ꢀ5.43
ꢀ4.39
ꢀ5.44
ꢀ5.39
ꢀ5.53
ꢀ5.97
ꢀ5.19
ꢀ5.32
>ꢀ4.00
ꢀ4.84
ꢀ4.87
ꢀ5.14
ꢀ6.07
ꢀ4.97
ND
NCI-ADR-RES
MDA-MB-231/ATCC
HS 578T
ꢀ4.49
ꢀ5.21
ꢀ4.51
ꢀ5.17
ꢀ4.80
ꢀ4.47
MDA-MB-435
BT-549
T-47D
MG-MID
Delta
Range
ꢀ5.45
ꢀ5.42
ꢀ5.40
ꢀ5.21
ꢀ5.38
ꢀ4.96
ꢀ4.84
ꢀ4.87
0.21
1.20
0.11
1.21
0.35
1.19
0.35
1.10
0.23
1.24
1.79
2.64
1.23
2.07
0.91
1.45
ArH), 7.24 (m, 1H, 4-ArH), 4.58 (m, 0.8H, 1-H), 4.40
(m, 1.2H, 1-H and 2-H), 4.0 (m, 0.2H, 2-H), 2.90 (s,
0.6H, NCH3), 2.79 (s, 2.4H, NCH3), 2.30 (m, 2H,
COCH2), 1.60 (m, 2H, COCH2CH2), 1.25 (m, 20H,
CH2), 1.11 (d, 2.4H, J = 7.0, CHCH3), 0.98 (d, 0.6H,
J = 6.7, CHCH3), 0.87 (t, 3H, J = 7.0, CH3); ESI-MS
(CH3OH, relative intensity, %) m/z 376.5 (MH+, 100).
Calcd for [C24H42NO2]+ m/z 376.31.
4.1.2.2. (1S,2R)-3-Dodecyl-1-(10-hydroxy-20-methyl-
10-phenylethyl)-urea (LCL16). Prepared from 5 in 73%
yield after recrystallization from n-hexane–diethyl ether
(1:2, v/v) as white microcrystalline needles, mp 68.5–
70 ꢁC; TLC (CHCl3–MeOH, 5:1, v/v) Rf 0.36; TLC
(n-hexane–ethyl acetate, 2:1, v/v) Rf 0.48 (lit. Rf
20
20
0.46)49; ½aꢂ þ 12:0 (c 1, MeOH), (lit. ½aꢂ þ 15:81, c
D
D
20
365
0.2, MeOH)49; ½aꢂ
þ 25:4 (c 1, MeOH); 1H NMR
(500 MHz, CDCl3) d 7.30 (m, 4H, 2,3,5,6-ArH), 7.25
(m, 1H, 4-ArH), 4.77 (d, 1H, J = 1.3, 1-H), 4.55 (br s,
2H, NH or OH), 4.35 (1H, NH or OH), 4.17 (m, 1H,
2-H), 3.12 (m, 2H, NHCH2), 1.47 (m, 2H,
NHCH2CH2), 1.23(m, 18H, CH2), 0.98 (d, 3H, J = 6.8,
CHCH3) 0.87 (t, 3H, J = 7.0, CH3); ESI-MS (CH3OH,
relative intensity, %) m/z 363.2 (MH+, 20), 345.2
([MHꢀH2O]+, 100). Calcd for [C22H39N2O2]+ m/z
363.29.
4.1.1.4. (1R,2R)-2-(N-Methyl,N-tetradecanoylamino)-
1-phenyl-1-propanol (LCL14). Prepared from 4 in 72%
yield after crystallization from n-hexane–ethyl acetate
25
(1:1, v/v) as a white powder; ½aꢂ ꢀ 68:7 (c 1, MeOH);
D
25
365
as reported for LCL13.
½aꢂ
ꢀ 263:0 (c 1, MeOH). Remaining data are identical
4.1.2. General procedure for the preparation of LCL15-17
and LCL81 (class B). To a well-stirred solution of the
appropriate 2-amino-1-phenyl-1-propanol or 2-amino-
1-phenyl-1,3-propandiol (5, 6, 10, or 11, 3.0 mmol) in
anhydrous tetrahydrofuran (12 mL) and anhydrous eth-
anol (1.0 mL), dodecyl isocyanate (98%, 0.90 mL,
3.75 mmol) was added dropwise over 1 min. Reaction
mixture was stirred under dry nitrogen at rt for 4 h
and evaporated under reduced pressure to dryness,
and the residue was dried in a high vacuum at rt for
2 h. The crude product was purified by gradient flash
column chromatography (ethyl acetate–n-hexane, 6:1,
v/v, following pure ethyl acetate) and recrystallized from
a suitable solvent.
4.1.2.3. (1R,2S)-3-Dodecyl-1-(10-hydroxy-20-methyl-
10-phenylethyl)urea (LCL17). Prepared from 6 in 75%
21
D
21
365
yield, ½aꢂ ꢀ 11:5 (c 1, MeOH); ½aꢂ
ꢀ 27:5 (c 1,
20
D
ing analytical data are identical as reported for LCL16.
MeOH), (lit. ½aꢂ ꢀ 27:27, c 0.1, MeOH).49 The remain-
4.1.2.4. (1S,2S)-3-Dodecyl-1-[10-hydroxy-20-hydroxy-
methyl-10-(400-nitrophenyl)ethyl]-urea (LCL81). Prepared
21
D
from 11 in 70% yield. ½aꢂ þ 46:4 (c 1, MeOH), (lit.
21
½aꢂ þ 52:15, c 0.3, MeOH).49 The remaining analytical
D
data are identical as reported for LCL15.
4.1.3. General procedure for the preparation of class C
analogs: LCL18, 204, 284, 286, 289, 343, 346, 347, 378,
381, 385, and 397. To a well-stirred mixture of the
appropriate 2-amino-1-phenyl-propanols (4.4 mmol)
and aldehydes (3.5 mmol) in methanol (15 mL) and gla-
cial acetic acid (1.0 mL), sodium cyanoborohydride
(300 mg, 4.77 mmol) was added portion-wise over
1 min at room temperature and reaction mixture was
stirred for an additional 30 min. The reaction mixture
was evaporated under a reduced pressure to dryness
and the obtained residue was dried under high vacuum
(ꢁ1 Torr at room temperature over 6 h). The crude
product was purified by flash column chromatography
using chloroform–methanol–concentrated ammonium
hydroxide (5:1:0.1; v/v/v) to give a pure product as a
4.1.2.1. (1R,2R)-3-Dodecyl-1-[10-hydroxy-20-hydroxy-
methyl-10-(400-nitrophenyl)ethyl]-urea (LCL15). Prepared
from 10 in 69% yield after recrystallization from n-hex-
ane–diethyl ether (1:3, v/v) as a pale yellow microcrystal-
line powder, mp 82–84 ꢁC; TLC (ethyl acetate) Rf 0.17 (lit.
20
20
Rf 0.14)49; ½aꢂ ꢀ 53:6 (c 1, MeOH) (lit. ½aꢂ ꢀ 50:98, c
D
D
1
0.4, MeOH); H NMR (500 MHz, CD3OD) d 8.17 (d,
2H, J = 8.9, 3,5-ArH), 7.63 (d, 2H, J = 8.3, 2,6-ArH),
5.11 (d, 1H, J = 2.4, 1-H), 3.90 (m, 1H, 2-H), 3.69 (dd,
1H, J = 10.6 and 7.9, 3-Ha), 3.56 (dd, 1H, J = 10.6 and
5.5, 3-Hb), 2.90 (m, 2H, NHCH2), 1.28 (m, 20H, CH2),
0.89 (t, 3H, J = 7.0, CH3); ESI-MS (CH3OH, relative
intensity, %) m/z 424.2 (MH+, 15), 267.1 (100). Calcd
for [C22H38N3O5]+ m/z 424.3.

1026
Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
Table 4. Full screen, in vitro tumor 50% lethal concentration (log10 LC50
)
Cell line
Panel
LCL284
LCL289
LCL286
LCL204
LCL385
LCL120
LCL420
LCL85
Leukemia
CCRF-CEM
HL-60(TB)
K-562
ꢀ5.12
ꢀ5.18
ꢀ5.25
ꢀ5.31
ꢀ5.14
>ꢀ4.00
ꢀ5.09
ꢀ5.13
ND
ꢀ5.11
ꢀ5.03
ꢀ5.03
ꢀ5.03
ꢀ5.13
ND
ꢀ5.10
ꢀ5.07
>ꢀ4.00
ND
ND
ND
ND
ꢀ4.24
ꢀ4.20
ꢀ4.41
ꢀ4.27
ꢀ4.12
ꢀ4.82
ꢀ5.11
ꢀ5.12
>ꢀ4.00
ꢀ4.50
ꢀ4.11
ꢀ4.20
ND
>ꢀ4.00
>4.00
ꢀ5.29
MOLT-4
RPM1-8226
ND
ꢀ5.11
Non-small cell lung cancer
A549/ATCC
EKVS
HOP-62
ꢀ5.20
ꢀ5.22
ꢀ5.23
ꢀ5.23
ꢀ5.l5
ꢀ5.10
ꢀ5.23
ꢀ5.21
ꢀ5.18
ꢀ5.15
ND
ꢀ4.85
ꢀ4.34
ꢀ5.27
ꢀ5.19
ND
>ꢀ4.00
ꢀ5.05
ꢀ5.20
ꢀ5.21
ꢀ4.00
ꢀ4.08
ꢀ4.37
ND
ꢀ5.22
ꢀ4.04
ꢀ5.27
ꢀ5.23
>ꢀ4.00
ꢀ4.19
ꢀ5.14
ND
>ꢀ4.00
>ꢀ4.00
ꢀ4.42
ꢀ5.24
>ꢀ4.00
ꢀ4.25
ꢀ4.31
>ꢀ4.00
ND
ꢀ4.17
ꢀ4.37
ꢀ4.35
ꢀ4.17
ꢀ4.22
ꢀ5.24
ꢀ4.68
ꢀ4.17
ꢀ4.19
ꢀ4.44
ꢀ4.96
ꢀ4.51
ꢀ5.21
ND
ND
HOP-92
NCI-H226
NCI-H23
MCI-H322M
MCI-H460
MCI-H522
ꢀ4.05
ꢀ4.31
ꢀ4.28
ꢀ4.38
ꢀ4.30
ꢀ5.17
ꢀ5.18
ꢀ5.20
ꢀ5.13
ꢀ5.01
ꢀ5.13
ꢀ5.21
ND
ND
ND
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
ꢀ5.21
ꢀ5.19
ꢀ5.24
ꢀ5.23
ꢀ5.24
ꢀ5.25
ꢀ5.10
ꢀ5.21
ꢀ5.24
ꢀ5.19
ꢀ5.20
ꢀ5.17
ꢀ5.20
>ꢀ4.00
ꢀ5.22
ꢀ5.21
ꢀ5.17
ꢀ5.23
ꢀ5.11
ꢀ5.21
ꢀ5.09
ND
ꢀ5.21
ꢀ5.25
ꢀ5.25
ꢀ5.22
ꢀ5.25
ꢀ5.23
>ꢀ4.00
ND
ꢀ4.50
ꢀ4.51
ꢀ4.41
>ꢀ4.00
ꢀ4.46
ꢀ4.44
ꢀ4.34
ꢀ4.87
ꢀ5.21
ꢀ5.26
ꢀ4.33
ꢀ5.12
ꢀ5.30
ꢀ4.31
ꢀ4.53
ꢀ5.21
ND
ꢀ5.22
ꢀ4.23
ꢀ4.23
ꢀ5.12
ꢀ5.24
ꢀ4.18
ND
KM12
SW-620
ꢀ5.21
ꢀ4.00
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
ꢀ5.17
ꢀ5.21
ꢀ5.26
ND
ꢀ5.18
ꢀ5.19
ND
ꢀ5.15
ꢀ5.21
ꢀ5.17
ꢀ5.23
ꢀ4.85
ND
>ꢀ4.00
ꢀ5.01
ꢀ5.22
ND
ND
ꢀ5.22
ꢀ4.20
>ꢀ4.00
ND
ꢀ4.36
ꢀ4.27
ꢀ4.40
ꢀ4.42
ꢀ4.42
ꢀ4.41
ꢀ4.21
ꢀ4.42
ꢀ4.34
ꢀ4.28
>ꢀ4.00
ꢀ4.23
ꢀ5.17
ꢀ5.22
ND
ꢀ5.23
ꢀ5.18
ꢀ5.23
ꢀ5.15
ꢀ5.23
ꢀ4.22
ND
ꢀ4.56
ꢀ5.25
ꢀ5.01
ꢀ5.28
Melanoma
LOX IMVI
MALME-3M
M14
ꢀ5.23
ꢀ5.22
ꢀ5.21
ꢀ5.09
ꢀ5.24
ꢀ4.00
ꢀ5.19
ꢀ5.18
ꢀ5.23
ꢀ5.19
ꢀ5.20
ND
ꢀ5.24
ꢀ5.20
ꢀ5.17
>ꢀ4.00
ꢀ5.20
ꢀ5.22
ꢀ4.75
ꢀ5.10
ꢀ5.22
ꢀ5.02
ꢀ5.16
>ꢀ4.00
ꢀ5.22
>ꢀ4.00
ꢀ4.03
ꢀ4.12
ꢀ5.24
ꢀ5.17
ꢀ5.23
>ꢀ4.00
ꢀ5.25
ND
ꢀ4.29
ꢀ5.24
ꢀ5.08
ND
ꢀ4.35
ꢀ4.70
ꢀ4.33
ꢀ4.22
ꢀ4.69
ꢀ5.02
ꢀ4.41
ꢀ4.60
ꢀ4.91
ꢀ4.27
ꢀ4.31
ꢀ4.03
ꢀ4.44
ꢀ4.31
ꢀ4.05
ꢀ4.26
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
ꢀ5.20
>ꢀ4.00
ꢀ5.17
ꢀ5.20
ꢀ5.24
ꢀ5.26
ND
ꢀ5.14
ꢀ5.12
ꢀ5.24
Ounrijifj cancer
1GROV1
ꢀ5.24
ꢀ5.22
ꢀ5.17
ꢀ5.21
ND
ND
ND
ND
ND
ND
ND
ND
ND
ꢀ4.24
ꢀ4.58
ꢀ4.37
ꢀ4.29
>ꢀ4.00
ꢀ4.45
ꢀ4.65
ꢀ4.26
ꢀ4.18
ꢀ4.61
ꢀ4.09
>ꢀ4.00
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
ꢀ5.26
ꢀ5.20
ꢀ4.80
ND
ꢀ4.00
ꢀ5.17
ꢀ4.14
>ꢀ4.00
ꢀ4.10
ꢀ5.16
ꢀ5.24
ND
ND
ꢀ5.15
ND
>ꢀ4.00
ꢀ4.30
>ꢀ4.00
ꢀ4.22
ꢀ5.01
ꢀ5.01
ꢀ5.03
ꢀ4.10
Renal cancer
786-0
A498
ꢀ5.24
ꢀ4.13
ꢀ5.23
ꢀ5.24
ꢀ5.26
ꢀ5.17
ꢀ5.24
ꢀ5.23
ꢀ5.23
>ꢀ4.00
ꢀ5.22
ꢀ5.23
ꢀ5.22
ꢀ5.17
ꢀ5.21
ꢀ5.18
ꢀ5.23
ꢀ4.28
ꢀ5.23
ꢀ5.25
ꢀ5.16
ꢀ5.13
ꢀ5.17
>ꢀ4.00
ꢀ5.24
ꢀ4.03
ꢀ5.21
ꢀ4.17
ꢀ5.08
>ꢀ4.00
ꢀ4.18
ꢀ4.06
ꢀ5.25
>ꢀ4.00
ꢀ5.25
ꢀ5.11
ꢀ5.29
ꢀ5.19
ꢀ4.86
ꢀ4.10
ꢀ4.46
ꢀ4.21
ꢀ4.19
ꢀ4.18
ꢀ4.01
ꢀ5.11
ꢀ4.39
ꢀ4.03
ꢀ4.31
ꢀ4.24
ꢀ4.04
ꢀ4.27
ꢀ4.13
ꢀ4.32
ꢀ4.25
>ꢀ4.00
ꢀ5.24
ꢀ4.33
ꢀ4.44
ꢀ4.59
ꢀ4.20
ꢀ4.38
ꢀ4.65
ꢀ4.22
ACHN
CAK1-1
RXF393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
ꢀ5.23
ꢀ5.28
ND
ꢀ5.19
ꢀ5.25
ꢀ5.22
ND
ꢀ5.27
ꢀ5.26
ꢀ5.21
ꢀ4.82
ꢀ4.31
ND
ꢀ4.30
ꢀ4.80
ꢀ4.48

Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
1027
Table 4 (continued)
Cell line
Panel
LCL284
LCL289
LCL286
LCL204
LCL385
LCL120
LCL420
LCL85
Breast cancer
MCF7
ꢀ5.20
ND
ꢀ5.21
ND
ꢀ5.22
ꢀ5.20
ꢀ5.18
ꢀ4.96
ꢀ5.25
ꢀ5.34
ꢀ5.16
ND
ND
ꢀ5.07
ꢀ4.07
ꢀ5.12
ꢀ5.04
ꢀ5.26
ꢀ5.47
>ꢀ4.00
ꢀ4.68
>ꢀ4.00
ꢀ4.37
>ꢀ4.00
ꢀ4.59
ꢀ5.06
ꢀ4.23
ꢀ4.37
ꢀ4.20
ꢀ4.56
ꢀ4.09
ꢀ4.61
ꢀ4.39
ꢀ4.16
NCI-ADR-RES
MDA-MB-231/ATCC
HS 578T
ꢀ5.05
ꢀ5.11
ꢀ4.00
ꢀ5.24
ꢀ5.25
ꢀ4.06
ꢀ5.16
ꢀ5.18
>ꢀ4.00
ꢀ5.25
ꢀ5.26
ND
ꢀ5.19
ꢀ4.33
ꢀ5.25
ꢀ5.30
ꢀ5.11
ꢀ5.19
>ꢀ4.00
ꢀ5.24
ꢀ5.24
ꢀ5.12
MDA-MB-435
BT-549
T-47D
MG-MID
Delta
Range
ꢀ5.15
ꢀ5.06
ꢀ5.07
ꢀ4.61
ꢀ4.93
0.36
1.29
ꢀ4.23
ꢀ4.35
ꢀ4.44
0.16
1.31
0.18
1.24
0.27
1.34
0.66
1.27
1.78
2.41
0.71
1.06
0.86
1.30
white microcrystalline powder or a waxy solid. These
products were dissolved in anhydrous ethyl acetate
(15 mL) and 1 M HCl solution in a dry diethyl ether
(7.0 mL) was added dropwise at +4 ꢁC. The reaction
mixture was stirred at rt for an additional 10 min. The
mixture was evaporated under reduced pressure to dry-
ness and the afforded residue was dried in a high vacuum
for 2 h at room temperature. The crude hydrochlorides
were recrystallized from a suitable solvent as listed
below.
intensity, %) m/z 364.4 (M+, 100). Calcd for
[C23H42NO2]+m/z 364.32.
4.1.3.4. (1S,2S)2-(N-Tetradecylamino)-1-phenyl-1,3-
propandiol hydrochloride (LCL18). Prepared from 13
21
D
and tetradecyl aldehyde in 60% yield, ½aꢂ þ 38:5 (c 1,
MeOH). The remaining analytical data are identical as
reported for LCL385.
4.1.3.5. (1S,2R)-2-N-(Tetradecylamino)-1-phenyl-1-
propanol hydrochloride (LCL284). Prepared from 5 and
tetradecyl aldehyde in 64% yield. Analytical sample
was obtained by crystallization from anhydrous ethyl
acetate as a white powder, mp > 177 ꢁC (with decomp.);
4.1.3.1. (1R,2R)-2-(N-Tetradecylamino)-1-(40-nitro-
phenyl)-1,3-propandiol hydrochloride (LCL204). Pre-
pared from 10 and tetradecyl aldehyde in 57% yield.
Analytical sample of LCL204 was obtained by crystalliza-
tion from ethyl acetate–n-hexane (1:1, v/v) as a white pow-
TLC (CHCl3/MeOH/concd NH4OH, 8:1:0.1, v/v/v)
20
D
Rf = 0.65; ½aꢂ þ 14:5 (c 1, MeOH) and ꢀ16.7 (c 1,
20
365
der, mp 101–103 ꢁC. TLC (CHCl3/MeOH/concd
CHCl3); ½aꢂ
þ 41:2 (c 1, MeOH) and ꢀ58.0 (c 1,
20
1
NH4OH, 5:1:0.1, v/v/v) Rf = 0.35; ½aꢂ ꢀ 30:5 (c 1,
CHCl3); H NMR (500 MHz, CD3OD) d 7.40 (m, 4H,
2,3,5,6-ArH), 7.38 (m, 1H, 4-ArH), 5.13 (d, 1H,
J = 3.0, 1-H), 3.46 (dd, 1H, J = 3.0, 6.7, 2-H), 3.09 (t,
2H, J = 4.3, NHCH2), 1.74 (m, 2H, NHCH2CH2), 1.30
(m, 22H, CH2), 1.05 (d, 3H, J = 6.8, CHCH3) 0.89 (t,
3H, J = 7.1, CH3); ESI-MS (CH3OH, relative intensity,
%) m/z 348.3 (M+, 100). Calcd for [C23H42NO]+ m/z
348.33. Anal. Calcd for C23H42ClNO (384.04): C,
71.93; H, 11.02; N, 3.65; Cl, 9.23. Found: C, 71.77; H,
11.08; N, 3.69; Cl, 9.53.
D
1
MeOH); H NMR (500 MHz, CD3OD) d 8.28 (d, 2H,
J = 8.8, ArH), 7.72 (d, 2H, J = 8.8, ArH), 5.02 (d, 1H,
J = 9.2, 1-H), 3.67 (dd, 1H, J = 12.5, 3.4, 1-Ha), 3.38
(dd, 1H, J = 12.5, 4.1, 1-Hb), 3.34 (m, 1H, 2-H), 3.15 (m,
2H, NHCH2), 1.73 (m, 2H, NHCH2CH2), 1.30 (m, 22H,
CH2), 0.89 (t, 3H, J = 7.1, CH3); ESI-MS (CH3OH, rela-
tive intensity, %) m/z 409.5 (M+, 100), 391 ([MꢀH2O]+,
10). Calcd for [C23H41N2O4]+ m/z 409.31. Anal. Calcd
for C23H41ClN2O4(445.0): C, 62.07; H, 9.29; N, 6.29; Cl,
7.97. Found: C, 61.29; H, 9.22; N, 6.21; Cl, 7.86.
4.1.3.6. (1R,2S)-2-(N-Tetradecylamino)-1-phenyl-1-
propanol hydrochloride (LCL289). Prepared from 6 and
4.1.3.2.
(1S,2S)-2-(N-Tetradecylamino)-1-(40-nitro-
phenyl)-1,3-propandiol hydrochloride (LCL343). Pre-
pared from 11 and tetradecyl aldehyde in 61% yield,
20
D
tetradecyl aldehyde in 62% yield, ½aꢂ ꢀ 13:3 (c 1,
20
365
MeOH) and +16.0 (c 1, CHCl3); ½aꢂ
ꢀ 40:5 (c 1,
20
½aꢂ þ 31:5 (c 1, MeOH). The remaining analytical data
MeOH) and +59.0 (c 1, CHCl3). Remaining analytical
data are identical as reported for LCL284.
D
are identical as reported for LCL204.
4.1.3.3. (1R,2R)-2-N-(Tetradecylamino)-1-phenyl-1,3-
propandiol hydrochloride (LCL385). Prepared from 12
and tetradecyl aldehyde in 59% yield. Analytical sample
was obtained by crystallization from n-hexane–ethyl
acetate (4:2, v/v) as a white powder, mp > 170 (with de-
4.1.3.7. (1R,2R)-2-(N-Tetradecylamino)-1-phenyl-1-
propanol hydrochloride (LCL346). Prepared from 7 and
tetradecyl aldehyde in 60% yield. Analytical sample
was obtained by crystallization from anhydrous ethyl
23
D
acetate as a white powder, mp > 155ꢁC; ½aꢂ ꢀ 33:2 (c
21
23
365
1
1
comp.); ½aꢂ ꢀ 40:0 (c 1, MeOH). H NMR (500 MHz,
1, MeOH) and ½aꢂ
ꢀ 107:3 (c 1, MeOH); H NMR
D
CDCl3) d 7.38 (d, 1H, J = 6.8, NH), 7.30 (m, 5H, ArH),
5.13 (d, 1H, J = 9.4, 1-H), 3.73 (dd, 1H, J = 13.4, 2.3, 1-
Ha), 3.53 (dd, 1H, J = 13.4, 4.7, 1-Hb), 3.16 (m, 1H, 2-
H), 3.08 (m, 2H, NHCH2), 1.85 (m, 2H, NHCH2CH2),
1.30 (m, 2H, NHCH2CH2CH2), 1.20 (m, 20H, CH2),
0.82 (t, 3H, J = 7.1, CH3); ESI-MS (CH3OH, relative
(500 MHz, MeOD) d 9.84 (br s, 1H, NH), 8.33 (br s,
1H, NH), 7.35 (m, 5H, 2,6-ArH), 4.83 (d, 1H, J = 7.6,
1-H), 3.40 (m, 1H, NHCH2), 3.16 (m, 1H, NHCH2),
2.90 (m, 1H, 2-H), 1.94 (m, 2H, NHCH2CH2), 1.61
(m, 2H, CH2), 1.25 (m, 24H, CH3 and CH2), 0.87 (t,
3H, J = 7.1, CH3); ESI-MS (CH3OH, relative intensity,

1028
Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
1
%) m/z 348.3 (M+, 100). Calcd for [C23H42NO]+ m/z
348.33.
(c 0.5, MeOH); H NMR (500 MHz, CD3OD) d 7.41
(m, 4H, 2,3,5,6-ArH), 7.37 (m, 1H, 4-ArH), 5.12(d,
1H, J = 3.0, 1-H), 3.44 (dd, 1H, J = 3.1, 6.7, 2-H), 3.08
(t, 2H, J = 4.2, NHCH2), 1.73 (m, 2H, NHCH2CH2),
1.30 (m, 24H, CH2), 1.04 (d, 3H, J = 6.8, CHCH3)
0.89 (t, 3H, J = 7.1, CH3); ESI-MS (CH3OH, relative
intensity, %) m/z 376.5 (M+, 100). Calcd for
[C25H46NO]+ m/z 376.36.
4.1.3.8. (2S)-2-N-(Tetradecylamino)-1-phenyl-3-pro-
panol hydrochloride (LCL286). Prepared from 8 and
tetradecyl aldehyde in 65% yield. Analytical sample
was obtained by crystallization from anhydrous ethyl
acetate as a white powder, mp >165 ꢁC (with decomp.);
24
D
24
365
½aꢂ ꢀ 7:70 (c 1, MeOH) and ½aꢂ
ꢀ 25:6 (c 1,
MeOH); 1H NMR (500 MHz, CDCl3) d 9.25 (br s,
1H, NH), 8.89 (br s, 1H, NH), 7.27 (m, 5H, 2-6-ArH),
3.81 (dd, 1H, J = 2.4 and 13.0, 1-Ha), 3.72 (dd, 1H,
J = 6.6 and 13.0, 1-Hb), 3.26 (m, 1H, 2-H), 3.19 (dd,
1H, J = 5.4 and 13.6, 3-Ha), 3.01 (dd, 1H, J = 9.4 and
13.6, 3-Hb), 2.93 (m, 1H, NHCH2), 2.83 (m, 1H,
NHCH2) 1.82 (m, 2H, NHCH2CH2), 1.18 (m, 23H,
CH2), 0.81 (t, 3H, J = 7.1, CH3); ESI-MS (CH3OH, rel-
ative intensity, %) m/z 348.4 (M+, 100). Calcd for
[C23H42NO]+ m/z 348.33.
4.1.4. General procedure for the preparation of LCL82,
LCL85, LCL120 and LCL420 (class D)
• Preparation of x-Br analogs 14–17.
4.1.4.1. (1S,2R)-2-N-(160-Bromohexadecanoylamino)-
1-phenyl-1-propanol (14). Prepared from 1 and 16-
bromohexadecanoyl chloride17 according to the general
procedure for class A. The crude product was purified
by flash column chromatography (CHCl3/MeOH/concd
NH4OH, 10:1:0.05, v/v/v) to give a pure bromo-analog
14 in 79% yield. Analytical sample of 14 was obtained
by recrystallization from n-hexane–ethyl acetate (3:1,
v/v) to give a white microcrystalline powder, mp 77–
78.5ꢁC; TLC Rf (CHCl3/MeOH, 8:1, v/v) Rf 0.68.
4.1.3.9. (2R)-2-(N-Tetradecylamino)-1-phenyl-3-pro-
panol hydrochloride (LCL347). Prepared from 9 and
20
D
tetradecyl aldehyde in 61% yield, ½aꢂ þ 5:22 (c 1,
20
365
lytical data are identical as reported for LCL286.
MeOH) and ½aꢂ
þ 19:4 (c 1, MeOH). Remaining ana-
22
22
½aꢂ þ 13:4 (c 1, MeOH); ½aꢂ
þ 37:8 (c 1, MeOH);
D
365
1H NMR (500 MHz, CDCl3) d 7.32 (m, 4H, 2,3,5,6-
ArH), 7.27 (m, 1H, 4-ArH), 5.46 (d, 1H, J = 7.4, NH),
4.83 (d, 1H, J = 2.8, 1-H), 4.34 (m, 1H, 2-H), 3.39 (t,
2H, J = 6.9, CH2Br), 2.17 (t, 2H, J = 7.1, COCH2),
1.84 (p, 2H, J = 7.2, CH2CH2Br), 1.61 (m, 2H,
COCH2CH2), 1.40 (m, 2-H, CH2CH2CH2Br), 1.25(m,
20H, CH2), 1.01 (d, 3H, J = 6.9, CHCH3); ESI-MS
(CH3OH, relative intensity, %) m/z 468.3 (MH+, 100).
Calcd for [C25H43BrNO2]+ m/z 468.24.
4.1.3.10. (1S,2R)-2-(N-Methyl,N-tetradecylamino)-1-
phenyl-1-propanol hydrochloride (LCL381). Prepared
from 1 and tetradecyl aldehyde in 59% yield. An analyt-
ical sample was obtained by crystallization from anhy-
drous ethyl acetate as a white powder, mp 107–109 ꢁC;
24
D
20
365
½aꢂ þ 5:54 (c 1, MeOH) and ½aꢂ
þ 18:2 (c 1,
1
MeOH). H NMR (500 MHz, CD3OD) d.43 (m, 4H,
2,3,5 and 6-ArH), 7.35 (m, 1H, 4-ArH), 5.21 (d, 1H,
J = 2.1, 1-H), 3.53 (m, 1H, 2-H), 2.96 (s, 3H, NHCH3),
1.78 (m, 2H, NHCH2), 1.42 (m, 4H, CH2 and
NHCH2CH2), 1.28 (m, 23H, CH2), 1.14 (d, 3H,
J = 6.8, CHCH3) 0.89 (t, 3H, J = 7.1, CH3); ESI-MS
(CH3OH, relative intensity, %) m/z 362.3 (M+, 100).
Calcd for [C24H44NO]+ m/z 362.34.
4.1.4.2. (1R,2S)-2-N-(160-Bromohexadecanoylamino)-
1-phenyl-1-propanol (15). Prepared from 2 and 16-
bromohexadecanoyl chloride17 in 75% yield according
20
D
to the general procedure for class A. ½aꢂ ꢀ 13:2 (c 1,
20
365
MeOH); ½aꢂ
ꢀ 38:5 (c 1, MeOH). Remaining analyti-
cal data are identical as reported for 14.
4.1.3.11. (1S,2R)-2-(N-Dodecylamino)-1-phenyl-1-pro-
panol hydrochloride (LCL378). Prepared from 5 and
dodecyl aldehyde in 58% yield. Analytical sample was ob-
tained by crystallization from anhydrous ethyl acetate as
a white powder, mp >198 ꢁC (with decomp.); TLC
(CHCl3/MeOH/concd NH4OH, 8:1:0.1, v/v/v) Rf = 0.60;
4.1.4.3. (1R,2R)-2-N-(160-Bromohexadecanoylamino)-
1-(40-nitrophenyl)-1,3-propandiol (16). Prepared from 10
and 16-bromohexadecanoyl chloride17 in 72% yield
according to the general procedure for class A. The
crude product was purified by flash column chromatog-
raphy (CHCl3/MeOH/concd NH4OH, 10:1:0.05, v/v/v)
23
23
½aꢂ þ 8:3 (c 0.5, MeOH) and ½aꢂ
þ 32:0 (c 0.5,
D
365
1
MeOH); H NMR (500 MHz, CD3OD) d 7.39 (m, 4H,
2,3,5,6-ArH), 7.36 (m, 1H, 4-ArH), 5.11(d, 1H, J = 3.1,
1-H), 3.43 (dd, 1H, J = 3.1, 6.8, 2-H), 3.10 (t, 2H,
J = 4.5, NHCH2), 1.72 (m, 2H, NHCH2CH2), 1.30 (m,
20H, CH2), 1.03 (d, 3H, J = 6.8, CHCH3) 0.88 (t, 3H,
to give a pure corresponding bromo-analog 16
(457 mg, 75% yield) as a pale yellow powder. Analytical
sample of 16 was obtained by recrystallization from
n-hexane–ethyl acetate (2:1, v/v) to give a white micro-
crystalline powder, mp 79-81ꢁC; TLC Rf (CHCl3/
20
1
J = 7.1, CH3); ESI-MS (CH3OH, relative intensity, %)
MeOH, 8:1, v/v) Rf 0.38. ½aꢂ ꢀ 1:90 (c 1, MeOH); H
NMR (500 MHz, CDCl3) dD8.19 (d, 2H, J = 8.8, 3,5-
ArH), 7.55 (d, 2H, J = 8.4, 2,6-ArH), 6.08 (d, 1H,
J = 7.9, NH), 5.20 (d, 1H, J = 4.7, 1-H), 4.13 (m, 1H,
2-H), 3.88 (d, 2H, J = 4.3, 3-Hab), 3.40 (t, 2H, J = 6.8,
CH2Br), 2.11 (m, 2H, COCH2), 1.83 (p, 2H, J = 7.2,
CH2CH2Br), 1.47(m, 2H, COCH2CH2), 1.43 (m, 2-H,
CH2CH2CH2Br), 1.23 (m, 20H, CH2); ESI-MS
(CH3OH, relative intensity, %) m/z 529.3 (MH+, 100).
Calcd for [C25H42BrN2O5]+ m/z 529.22.
m/z 320.4 (M+, 100). Calcd for [C21H38NO]+ m/z 320.29.
4.1.3.12. (1S,2R)-2-(N-Hexadecylamino)-1-phenyl-1-
propanol hydrochloride (LCL397). Prepared from 5 and
hexadecyl aldehyde in 62% yield. Analytical sample
was obtained by crystallization from anhydrous ethyl
acetate as a white powder, mp >195 ꢁC (with decomp.);
TLC (CHCl3/MeOH/concd NH4OH, 8:1:0.1, v/v/v)
23
D
20
365
Rf = 0.67; ½aꢂ þ 14:8 (c 0.5, MeOH) and ½aꢂ
þ 38:6

Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
1029
4.1.4.4. (1R,2R)-2-N-(Tetradecanoylamino)-1-(40-ami-
nophenyl)-1,3-propandiol (17). To a well-stirred and
cooled mixture of Zn dust (1.1 g) and 80% acetic acid
(20 mL), B13 (1.05 g, 2.5 mmol) was added portion-wise
over 5 min. The mixture was stirred for an additional
10 min and then gently heated to 50 ꢁC to dissolve the
formed white solid. The hot reaction mixture was fil-
tered off and the filtrate was cooled down to rt and
refrigerated for 30 min. The formed precipitate was sep-
arated by filtration and the filtrate was kept in the free-
zer (ꢀ10 ꢁC) overnight. The formed crude product was
separated by filtration, washed with 0.5 N NH4OH solu-
tion, water and dried at rt for 24 h. This material was
recrystallized twice from ethanol–water (3:4, v/v) to give
pure 17 (0.340 g, 29%) as a pale yellow microcrystalline
combined and evaporated under reduced pressure at
room temperature. The obtained residue was lyophilized
in a high vacuum to give a pure LCL120 in 81% as a col-
orless oil. Analytical sample of LCL120 was prepared by
recrystallization from anhydrous acetone–ethanol (8:1,
v/v) to give a colorless semi-solid. TLC (CHCl3/
(CH3)2CO/MeOH/CH3COOH/H2O, 20:8:8:2:1, v/v) Rf
0.49; RP TLC (C18 Silica, CH3CN/MeOH/1 M NH4Cl
21
D
(aq), 4:1:1.5, v/v) Rf 0.46; ½aꢂ þ 9:1 (c 1, MeOH);
21
365
½aꢂ
þ 30:4 (c 1, MeOH); 1H NMR (500 MHz, CD3OD)
d 9.00 (d, 2H, J = 5.6, 2,5-HPy) 8.59 (t, 1H, J = 7.9, 4-
HPy), 8.11 (t, 2H, J = 7.0, 3,5-HPy), 7.36 (m, 2H, ArH),
7.28 (m, 2H, ArH), 7.21 (m, 1H, ArH), 4.62 (m, 3H, 1-
H and C(16)H2-pyridinium ring), 4.12 (m, 1H, 2-H),
2.07 (m, 2H, COCH2), 2.01 (m, 2H, C(15)H2C(16)H2-
pyridinium ring), 1.45 (m, 2H, COCH2CH2), 1.38 (m,
2H, C(14)H2C(15)H2C(16)H2-pyridinium ring), 1.25 (m,
20H, CH2), 1.08 (d, 3H, J = 6.8, CH3); ESI-MS (CH3OH,
relative intensity, %) m/z 467.3 (M+, 100). Calcd for
[C30H47N2O2]+ m/z 467.36.
powder, mp 124–126ꢁC. TLC (CHCl3/MeOH/concd
24
NH4OH, 4:1:0.1, v/v/v) Rf = 0.41; ½aꢂ ꢀ 16:5 (c 0.5,
D
1
MeOH); H NMR (500 MHz, CD3OD) d 7.55 (d, 2H,
J = 8.1, ArH), 7. 31 (d, 2H, J = 8.5, ArH), 5.02 (d, 1H,
J = 3.2, 1-H), 4.00 (m, 1H, 2-H), 3.71 (dd, 1H, J = 7.1
and 10.8, 3-Ha), 3.51(dd, 1H, J = 5.7 and 10.8, 3-Hb),
2.09 (m, 2H, COCH2), 1.43 (m, 2H, COCH2CH2), 1.28
(m, 23H, CH2), 0.89 (t, 3H, J = 7.1, CH3); ESI-MS
(CH3OH, relative intensity, %) m/z 393.4. (MH+, 100).
Calcd for [C23H41N2O3]+ m/z 393.3.
4.1.5.2.
(1R,2S)-2-N-[16-(10-Pyridinium)-hexadec-
anoylamino]-1-phenyl-1-propanol bromide (LCL420). Pre-
pared from 15 as described for LCL120 in 76% yield.
21
D
21
365
½aꢂ ꢀ 8:54 (c 1, MeOH); ½aꢂ
ꢀ 29:6 (c 1, MeOH).
Remaining analytical data are identical as reported for
LCL120.
4.1.4.5. (1R,2R)-2-N-(Tetradecanoylamino)-1-[40-N-
(600-bromohexanoyl)-aminophenyl]-1,3-propandiol (18).
Prepared from 17 and 6-bromohexanoyl chloride in
63% yield according to the general procedure for class
A. Analytical sample of 18 was obtained by recrystalli-
zation from ethyl acetate to give a white microcrystalline
powder, mp 131-133ꢁC. TLC (CHCl3/MeOH, 9:1, v/v)
Rf = 0.37; 1H NMR (500 MHz, CD3OD) d 7.55 (d,
2H, J = 8.1, ArH), 7. 31 (d, 2H, J = 8.5, ArH), 5.02 (d,
1H, J = 3.2, 1-H), 4.00 (m, 1H, 2-H), 3.71 (dd, 1H,
J = 7.1 and 10.8, 3-Ha), 3.51(dd, 1H, J = 5.7 and 10.8,
3-Hb), 2.09 (m, 2H, COCH2), 1.43 (m, 2H,
COCH2CH2), 1.28 (m, 23H, CH2), 0.89 (t, 3H, J = 7.1,
CH3); ESI-MS (CH3OH, relative intensity, %) m/z
569.4 (MH+, 100). Calcd for [C29H49BrN2O4]+m/z
569.29.
4.1.5.3.
anoylamino]-1-(400-nitrophenyl)-1,3-propandil
(1R,2R)-2-N-[16-(10-Pyridinium)-hexadec-
bromide
(LCL85). Obtained from 16. Crude product was dissolved
in distilled water (10 mL) and extracted with dichloro-
methane (2 · 4.0 mL). The organic fractions were ex-
tracted back with water (2 · 4 mL) and all the aqueous
fractions were combined and evaporated under reduced
pressure. The obtained residue was lyophilized in a high
vacuum to give a pure LCL85 as a pale yellow semi-solid
(220 mg, 75% yield). Analytical sample of LCL85 was
prepared by recrystallization from anhydrous acetone–
ethyl acetate (8:1, v/v) to give a pale yellow microcrystal-
line powder, mp 52–53.5 ꢁC. TLC (CHCl3/(CH3)2CO/
MeOH–CH3COOH/H2O, 20:8: 8:2:1, v/v) Rf 0.45;
RP TLC (C18 Silica, CH3CN/MeOH/1 M NH4Cl (aq),
25
D
4:1:1.5, v/v) Rf 0.51 ½aꢂ ꢀ 1:55 (c 1, MeOH); 1H NMR
• Preparation of aromatic ceramidoids (x-pyridinium
analogs).
(500 MHz, CD3OD) d 9.00 (dd, 2H, J = 6.6, 1.2, 2,5-
HPy) 8.59 (t, 1H, J = 7.8, 4-HPy), 8.16 (d, 2H, J = 8.9,
ArH), 8.11 (t, 2H, J = 7.2, 3,5-HPy), 7.63 (d, 2H, J = 8.9,
ArH), 5.13 (m, 1H, 1-H), 4.63 (t, 2H, J = 7.7, C(16)H2-
pyridinium ring), 4.18 (m, 1H, 2-H), 3.76 (dd, 1H,
J = 10.7, 7.6, 3-Ha), 3.56 (dd, 1H, J = 10.7, 6.0, 3-Hb),
2.07 (m, 2H, COCH2), 2.01 (m, 2H, C(15)H2C(16)H2-
pyridinium ring), 1.30 (m, 24H, COCH2CH2,
C(14)H2C(15)H2C(16)H2-pyridinium ring and CH2);
ESI-MS (CH3OH, relative intensity, %) m/z 528.5 (M+,
100). Calcd for [C30H46N3O5]+ m/z 528.34.
4.1.5. General procedure for cationization of 14–18. Mix-
tures of 14–16 and 18 (0.50 mmol), anhydrous pyridine
(2 mL), and anhydrous toluene (2 mL) were heated in
a sealed glass test-tube in an oil bath at 75–85 ꢁC over
6 h. After completion, the reaction mixture was cooled
and evaporated to dryness. The residues were dried un-
der the high vacuum (ꢁ1 Torr at rt over 6 h), washed
twice with ethyl acetate–n-hexane (4:1, v/v/,
2 · 10 mL), and the afforded crude products were puri-
fied as described below.
4.1.5.4.
(1R,2R)-2-N-(Tetradecanoylamino)-1-[40-N-
4.1.5.1.
(1S,2R)2-N-[16-(10-Pyridinium)-hexadec-
[(600-pyridinium)-hexanoyl]aminophenyl]-1,3-propandiol bro-
mide (LCL82). Obtained from 18. Crude product was
dissolved in a warm mixture of acetone–THF (1:1, v/v)
and kept in refrigerator overnight. The obtained solid
was separated by filtration, washed with ethyl acetate–ace-
tone (1:1, v/v) and dried in a high vacuum to give a pure
anoylamino]-1-phenyl-1-propanol bromide (LCL120).
Prepared from 14. Crude product was dissolved in dis-
tilled water (10 mL) and extracted with dichloromethane
(2 · 4.0 mL). The organic fractions were extracted back
with water (2 · 4 mL) and all the aqueous fractions were

1030
Z. M. Szulc et al. / Bioorg. Med. Chem. 16 (2008) 1015–1031
LCL82 in 84% yield as a white solid. Analytical sample of
LCL82 was prepared by recrystallization from anhydrous
acetone–THF (1:2, v/v) to give a white microcrystalline
powder, mp >122 ꢁC. TLC (CHCl3/(CH3)2CO/MeOH–
CH3COOH/H2O, 20:8:8:2:1, v/v) Rf 0.31; RP TLC (C18
Silica, CH3CN/MeOH/1 M NH4Cl (aq), 4:1:1.5, v/v) Rf
were solubilized in dimethyl sulfoxide at 400-fold the de-
sired final maximum concentration. Following drug
addition, the plates were incubated for an additional
48 h at 37 ꢁC, 5% CO2, 95% air, and 100% relative
humidity.50–54
25
25
0.38; ½aꢂ ꢀ 7:65 (c 0.5, MeOH) and ½aꢂ
ꢀ 11:7 (c
4.2.4. Cellular levels of exogenously added new analogs by
HPLC–MS analysis. Cellular levels of the new analogs
used for MCF7 cell treatment were established by
HPLC–MS performed by the Lipidomics Core at MUSC
on Thermo Finnigan TSQ 7000, triple-stage quadrupole
mass spectrometer operating in a Multiple Reaction
Monitoring (MRM) positive-ionization mode.55 Quanti-
tative analysis of the cellular level of D-e-MAPP, B13, and
their analogs was based on the calibration curves gener-
ated by spiking an artificial matrix with known amounts
of target standards and an equal amount of the internal
standard (IS) as described.56 The target analyte to IS peak
area ratios from the samples were similarly normalized to
their respective IS and compared to the calibration curves
using a linear regression model.
D
1
365
0.5, MeOH); H NMR (500 MHz, CD3OD) d 8.99 (dd,
2H, J = 6.5, 1.2, 2,5-HPy) 8.58 (t, 1H, J = 7.9, 4-HPy),
8.08 (t, 2H, J = 7.2, 3,5-HPy), 7.49 (d, 2H, J = 8.9,
ArH), 7.32 (d, 2H, J = 8.6, ArH), 4.92 (d, 1H, J = 3.8,
1-H), 4.65 (t, 2H, J = 7.5, C(6) H2-pyridinium ring), 4.08
(m, 1H, 2-H), 3.68 (dd, 1H, J = 10.9, 6.7, 3-Ha), 3.48
(dd, 1H, J = 10.7, 5.7, 3-Hb), 2.39 (t, 2H, J = 7.2, 4-
NHCOCH2), 2.13 (t, 2H, J = 7.6, 2-NHCOCH2), 2.07
(m, 2H, C(5) H2C(6)H2-pyridinium ring), 1.77 (m, 2H,
4-NHCOCH2CH2), 1.46 (m, 4H, 4-NHCOCH2CH2CH2
and C(4) H2C(5)H2C(6)H2-pyridinium ring), 1.27(m,
22H, CH2), 0.88 (t, 3H, J = 7.1, CH3); ESI-MS (CH3OH,
relative intensity, %) m/z 568.6 (M+, 100). Calcd for
[C34H54N3O4]+ m/z 568.41.
4.2. Biology
Acknowledgments
4.2.1. Cell culture. MCF7 cells (breast adenocarcinoma,
pleural effusion) were purchased 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
Biotechnology, CO, USA) and maintained under stan-
dard 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.
Financial support was provided by the NIH NCI—
PO1CA097132. Special acknowledgment is for
NCRR—CO6RR018823 providing laboratory space
for Lipidomics Core in CRI building. We thank Dr. Jen-
nifer Schnellmann for editorial assistance.
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