Discovery of a Fluorinated Enigmol Analog with Enhanced in Vivo Pharmacokinetic and Anti-Tumor Properties

Article abstract of DOI:10.1021/acsmedchemlett.6b00113

The orally bioavailable 1-deoxy-sphingosine analog, Enigmol, has demonstrated anticancer activity in numerous in vivo settings. However, as no Enigmol analog with enhanced potency in vitro has been identified, a new strategy to improve efficacy in vivo by increasing tumor uptake was adopted. Herein, synthesis and biological evaluation of two novel fluorinated Enigmol analogs, CF₃-Enigmol and CF₂-Enigmol, are reported. Each analog was equipotent to Enigmol in vitro, but achieved higher plasma and tissue levels than Enigmol in vivo. Although plasma and tissue exposures were anticipated to trend with fluorine content, CF₂-Enigmol absorbed into tissue at strikingly higher concentrations than CF₃-Enigmol. Using mouse xenograft models of prostate cancer, we also show that CF₃-Enigmol underperformed Enigmol-mediated inhibition of tumor growth and elicited systemic toxicity. By contrast, CF₂-Enigmol was not systemically toxic and demonstrated significantly enhanced antitumor activity as compared to Enigmol.

Full text of DOI:10.1021/acsmedchemlett.6b00113

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Fluorinated Enigmol Analog with Enhanced in Vivo
Pharmacokinetic and Anti-Tumor Properties
†
‡
†
∥
∥
Eric J. Miller, Suzanne G. Mays, Mark T. Baillie, Randy B. Howard, Deborah G. Culver,
∥
⊥
†
†
∥
Manohar Saindane, Sarah T. Pruett, Jason J. Holt, David S. Menaldino, Taylor J. Evers,
∥
∥
∥
‡,§,#,∇
G. Prabhakar Reddy, Richard F. Arrendale, Michael G. Natchus, John A. Petros,
and Dennis C. Liotta*
,†
†
Department of Chemistry, Emory University, 1521 Dickey Drive NE, Atlanta, Georgia 30322, United States
‡
Emory University School of Medicine, Department of Urology, 1365 Clifton Road NE, Atlanta, Georgia 30322, United States
Emory University School of Medicine, Department of Pathology and Laboratory Medicine, 1364 Clifton Road NE, Atlanta, Georgia
§
3
0322, United States
∥
Emory Institute for Drug Development (EIDD), 954 Gatewood Road NE, Atlanta, Georgia 30329, United States
⊥
Yerkes National Primate Research Center, Biomarkers Core Laboratory, Emory University, 954 Gatewood Road NE, Atlanta,
Georgia 30329, United States
#
Winship Cancer Institute, Emory University, 1365 Clifton Road NE, Atlanta, Georgia 30322, United States
∇Atlanta Veterans Affairs Medical Center, Atlanta, Georgia 30033, United States
*
S Supporting Information
ABSTRACT: The orally bioavailable 1-deoxy-sphingosine analog, Enigmol, has
demonstrated anticancer activity in numerous in vivo settings. However, as no
Enigmol analog with enhanced potency in vitro has been identified, a new strategy to
improve efficacy in vivo by increasing tumor uptake was adopted. Herein, synthesis and
biological evaluation of two novel fluorinated Enigmol analogs, CF -Enigmol and CF -
3
2
Enigmol, are reported. Each analog was equipotent to Enigmol in vitro, but achieved
higher plasma and tissue levels than Enigmol in vivo. Although plasma and tissue
exposures were anticipated to trend with fluorine content, CF -Enigmol absorbed into
2
tissue at strikingly higher concentrations than CF -Enigmol. Using mouse xenograft
3
models of prostate cancer, we also show that CF -Enigmol underperformed Enigmol-
3
mediated inhibition of tumor growth and elicited systemic toxicity. By contrast, CF2-
Enigmol was not systemically toxic and demonstrated significantly enhanced antitumor
activity as compared to Enigmol.
KEYWORDS: Sphingolipids, Enigmol, fluorination, prostate cancer
1
7
phingolipids are a structurally diverse class of lipids
endogenous sphingoid bases is limited by phosphorylation,
8
S
containing a common scaffold that is exemplified by
inhibition of SK represents a promising anticancer strategy.
sphingosine (Figure 1a), a ubiquitous intermediate in the
biosynthesis of a wide array of sphingolipids, many of which are
By contrast, we envisioned that a sphingosine mimic
incapable of phosphorylation could reestablish the proper
sphingosine/S1P balance, and thereby, suppress cancer growth.
Specifically, sphingoid base analogs lacking the C-1 hydroxyl
group were hypothesized to adopt the cytotoxic signaling
2
second messengers. For example, amino group acylation by
ceramide synthase (CS) produces ceramides, which facilitate
signaling events from lipid rafts. Alternatively, phosphorylation
of the primary hydroxyl group by sphingosine kinase (SK)
generates sphingosine-1-phosphate (S1P), the endogenous
3
profile of sphingosine without the counterweight of S1P
4
9,10
metabolites. Evaluation of 1-deoxy-sphingoid bases
led to
agonist for seven-transmembrane S1P receptors. The sphingo-
lipid biosynthetic and metabolic pathways include numerous
pleiotropic signaling molecules involved in complex equilibria.
Notable among these are the opposing roles of sphingosine and
S1P: S1P promotes cell survival, whereas sphingosine promotes
the identification of Enigmol (Figure 1b), which exhibited
broad-spectrum cytotoxicity (0.4 μM ≤ IC ≤ 14 μM) against
5
0
11
57 human cancer cell lines, in addition to LNCaP (IC = 9.7
5
0
± 0.3 μM) and PC-3 (IC50 = 10.3 ± 0.2 μM) human prostate
5
cell death. While healthy cells benefit from a proper balance of
Received: March 14, 2016
Accepted: March 21, 2016
6
S1P and sphingosine, many cancer cell types overexpress SK,
which tips the balance in favor of S1P. Since the cytotoxicity of
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00113
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Scheme 1. Synthesis of (a) CF -Enigmol and (b) CF -
3
2
Enigmol
Figure 1. Sphingosine-S1P axis and fluorinated Enigmol analogs.
cLogP was calculated using QikProp in Maestro.
1
2
cancer cell lines, in vitro. This activity profile was attributed to
1
1
10
9
(
“
1) inhibition of SK, CS, and protein kinase C (PKC), (2)
11
immunity” to SK and other kinases, and (3) slow CS-
10
mediated N-acylation. Sluggish metabolism endows Enigmol
with a longer lifetime than its endogenous counterparts,
thereby extending the duration of its therapeutic action.
While most compounds with micromolar anticancer activity
in vitro cannot achieve effective tumor concentrations in vivo,
Enigmol robustly partitions into tissue, building up drug
12
reservoirs after multiple oral (p.o.) doses. This combination
of long lifetime and extensive tissue uptake enabled moderately
cytotoxic Enigmol to significantly inhibit tumor growth in
multiple cancer models in vivo without causing systemic
1
1
toxicity. In a mouse xenograft model of prostate cancer, for
example, Enigmol exhibited comparable efficacy to surgical
hormone deprivation and to docetaxel, which are the current
clinical standards of care for androgen-dependent and
12
androgen-independent prostate cancer, respectively.
As evidenced by striking in vivo efficacy and low systemic
toxicity, Enigmol’s modest in vitro potency is leveraged by
favorable pharmacokinetic (PK) properties in vivo. These
results encouraged synthesis and evaluation of various Enigmol
1
2,13
analogs,
a process facilitated by two diastereoselective
Addition of the aldehyde to a preformed, (−)-DiPCl-stabilized
14,12
14
approaches to Enigmol developed in our laboratory.
enolate of methyl ketone 5 produced the aldol adduct, which
However, because the maximum potential of these compounds
could only be determined with time- and resource-intensive in
vivo studies, we decided to increase lipophilicity with the
expectation of enhanced anticancer activity due to improved
oral absorption and elevated tumor exposure. Specifically,
replacing selected hydrogens in Enigmol (cLogP = 3.55) with
fluorines was anticipated to increase hydrophobicity without
was immediately subjected to reduction with sodium
borohydride, yielding syn-diol 6 in 50% yield over three steps
(d.r. = 99:1). Finally, a one-pot alkyne hydrogenation/N,N-
dibenzylamino hydrogenolysis furnished CF -Enigmol. CF -
3
2
Enigmol was prepared using a modified aldol approach
involving α,α-difluoro methyl ketone 9 and α-aminoaldehyde
1
9
20
10 (Scheme 1b). Tandem hydrozirconation/iodination of
alkyne 7 gave the corresponding terminal vinyl iodide, which
15,16
substantially perturbing other physicochemical properties.
13
21
Accordingly, we prepared two analogs (Figure 1b), CF3-
Enigmol (cLogP = 4.25, trifluorination at C-18) and CF2-
Enigmol (cLogP = 3.88, difluorination at C-6), and evaluated:
was subjected to a copper-mediated coupling with ethyl
bromodifluoroacetate to give α,α-difluoro ester 8. The resulting
22
olefin was reduced via palladium-catalyzed hydrogenation and
subsequently converted to ketone 9 using a two-step Weinreb
(
(
1) cytotoxicity against cultured human prostate cancer cells,
2) plasma and tissue PK properties, and (3) antitumor activity
23
ketone synthesis. Achiral borane-mediated aldol reaction with
19
in xenograft models of prostate cancer.
aldehyde 10 resulted in a chromatographically separable
mixture of diastereomers (d.r. = 2:1, 36% and 20% isolated
The synthesis of CF -Enigmol was accomplished using our
3
14
previously reported diastereoselective aldol route (Scheme
a). Homopropargyllic alcohol 1 was converted to THP-ether 2
via an alkyne zipper reaction, followed by protection of the
primary alcohol. The resulting terminal alkyne was then
yields). Although TiCl led to improved syn-diastereoselectivity
4
1
(d.r. = 4:1, Supporting Information Table 1), isolated yield
suffered substantially, and the use of either (+)-DiPCl or
(−)-DiPCl did not significantly improve syn-stereoinduction.
Attempted syn-reduction of the resulting β-hydroxy ketone
17
1
8
alkylated with bromide 3, followed by THP group removal
24
and Dess-Martin oxidation to produce the desired aldehyde.
using Et BOMe and NaBH4 gave disappointing selectivity as
2
B
DOI: 10.1021/acsmedchemlett.6b00113
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Table 1. Cytotoxicity against Cultured Human Prostate Cancer Cells
LNCaP Cells
PC-3 Cells
Drug
IC50 (μM)
IC90 (μM)
IC50 (μM)
IC90 (μM)
Enigmol
11.7 ± 1.10
11.3 ± 1.10
13.4 ± 1.07
19.6 ± 1.10
19.2 ± 1.30
16.7 ± 1.20
12.1 ± 1.02
21.4 ± 1.17
11.7 ± 1.05
14.0 ± 1.00
24.4 ± 1.20
15.3 ± 1.10
CF -Enigmol
3
CF -Enigmol
2
a
LNCaP or PC-3 cells were incubated with compound for 24 h (n = 3−4), and viability was assessed using WST-1. Data represent the mean ± SEM.
well (Supporting Information Table 2). Interestingly, while
Subsequent PK experiments were designed to compare
plasma absorption and tissue distribution properties after oral
administration. Accordingly, SD rats were dosed once via oral
reduction with DiBAL-H (d.r. = 1:5) favored the anti-diol,
25
DiBAL-H reduction with additive ZnBr2 (d.r. = 5:2) furnished
the desired syn-diol 11 in 56% isolated yield. Finally,
deprotection of the N,N-dibenzylamino group via hydro-
gavage with 10 mg/kg of either Enigmol (n = 4), CF -Enigmol
3
(n = 4) or CF -Enigmol (n = 4), and analog concentrations in
2
genolysis produced CF -Engimol.
isolated plasma and various tissues were quantified using LC-
MS/MS. Counter to results from i.v. experiments, systemic
2
26
CF -Enigmol and CF -Enigmol were assayed for cytotoxicity
3
2
toward cultured androgen-dependent (LNCaP) and androgen-
independent (PC-3) human prostate cancer cells by measuring
exposure after p.o. dose, as indicated by Cmax and AUC, trended
with cLogP (Figure 3a). Furthermore, CF -Enigmol required
3
12
mitochondrial activity (Supporting Information Figure 2). As
expected, the IC50 values (Table 1) of CF -Enigmol and CF -
3
2
Enigmol were comparable to Enigmol, indicating that fluorine
incorporation did not substantially alter potency, and
suggesting that any differences in in vivo activity would stem
from altered PK properties. IC90 values were also calculated to
indicate therapeutically effective concentrations desired in
tumors.
With therapeutic concentrations established, PK studies were
designed to evaluate the effect of fluorination on plasma
elimination, oral absorption, and tissue distribution. As rapid
intravenous (i.v.) injection effectively normalizes plasma
absorption profiles, Sprague−Dawley (SD) rats were injected
i.v. with a single 2 mg/kg dose of either Enigmol (n = 3), CF -
3
Enigmol (n = 4), or CF -Enigmol (n = 4), facilitating reliable
2
assessment of terminal half-life (t1/2) and rate of clearance (Cl).
Analog concentrations in isolated plasma were quantified using
liquid chromatography/tandem-mass spectrometry (LC-MS/
MS) analysis (Figure 2). Although plasma elimination was
Figure 3. Plasma pharmacokinetics and tissue distribution (p.o.). (a)
SD rats (n = 4 per group) were orally administered 10 mg/kg of either
Enigmol (vehicle =90:10 olive oil/EtOH), CF -Enigmol (vehicle
3
=
90:10 olive oil/EtOH), or CF -Enigmol (vehicle =95:5 PEG400/
2
Tween80), and plasma concentration was measured periodically using
LC-MS/MS. (b) After 24 h, tissues were harvested, and drug levels
were measured by LC-MS/MS. Data represent the mean ± SEM. CF2-
Enigmol t1/2 is reported with the exclusion of one rat due to
inappropriate semilog plots of the terminal 2, 3, or 4 nonzero data
2
points (positive linear slopes or R = 0.038).
the longest amount of time (Tmax) to reach Cmax, and also
demonstrated substantially improved oral bioavailability (68%)
Figure 2. Plasma pharmacokinetics (i.v.). SD rats (n = 3−4 per group)
were treated with compound (2 mg/kg) via i.v. injection (vehicle
as compared to Enigmol (11%) and CF -Enigmol (10%).
2
=
40:10:3:47 PEG400/EtOH/Tween80/H O), and plasma concen-
Exponential decay of CF -Enigmol plasma concentration after
Cmax was likely not observed due to a relatively high t1/2 and a
long absorption phase that overlaps with the elimination phase.
2
2
tration was measured periodically using LC-MS/MS. Data represent
the mean ± SEM.
Notably, Enigmol and CF -Enigmol were not observed in
3
anticipated to trend inversely with cLogP due to plasma protein
binding, t1/2 and Cl values, as well as the maximum plasma
concentration (Cmax) and area under the curve (AUC),
plasma for >1 h, nor was CF -Enigmol for >2 h. With plasma
2
elimination virtually complete after 24 h, the accumulation of
each compound in red blood cells, brain, lung, liver, kidney, and
prostate was measured (Figure 3b). Although tissue distribu-
indicated that CF -Enigmol was removed from plasma
3
substantially faster than both Enigmol and CF -Enigmol. By
tion was anticipated to correlate with cLogP, CF -Enigmol (n =
4) achieved substantially higher concentrations than both
2
2
contrast, CF -Enigmol followed the expected trend, exhibiting
2
lower Cl and higher t1/2, AUC, and Cmax than Enigmol.
Enigmol (n = 4) and CF -Enigmol (n = 4) in all tissues
3
C
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ACS Medicinal Chemistry Letters
Letter
Figure 4. CF -Enigmol and CF -Enigmol in mouse xenograft models of prostate cancer. Nude mice (n = 10−11 per group) with palpable tumors
3
2
derived from PC-3 human prostate cancer cells were treated once daily via oral gavage with either vehicle (95:5 olive oil/EtOH), Enigmol (3 or 10
mg/kg), or CF -Enigmol (3 or 10 mg/kg) in the first study (a−c), or with either vehicle (95:5 PEG400/Tween80), Enigmol (10 mg/kg), or CF -
3
2
Enigmol (10 or 30 mg/kg) in the second study (d−f). Tumor volume and change in body weight were measured periodically. At the end of each
study, tumors were harvested, and accumulation was measured using LC-MS/MS. Data represent the mean ± SEM. Statistical significance (*p <
0
.05, **p < 0.01, or ***p < 0.001) of tumor growth inhibition was assessed via a linear mixed model for repeated measurement with the
autoregressive covariance structure using Statistical Analysis System (SAS) software. Significance of change in body mass was determined using One-
Way ANOVA followed by Sidak’s multiple comparison test.
analyzed. While Enigmol never reached its IC level (≥14 μM)
mg/kg Enigmol did not achieve IC90PC‑3 concentrations
9
0
PC‑3
in any tissue type, CF -Enigmol did so in lung (≥19 μM).
(=14 μM) in tumor, 10 mg/kg CF -Enigmol (IC
= 24
3
3
90
Remarkably, CF -Enigmol achieved IC concentrations (≥15
μM) did. A related boost in antitumor activity, however, was
unexpectedly not observed.
2
90
μM) in lung, brain, and kidney. Also notable are the relative
drug levels in prostate, where CF -Enigmol achieved 2-fold and
Thus, focus was shifted to CF -Enigmol, which was
2
2
9
-fold higher concentrations than CF -Enigmol and Enigmol,
compared head-to-head with Enigmol in a subsequent
xenograft study. After tumors were established, once daily
oral gavage of vehicle (95:5 PEG400/Tween80, n = 9 or 10,
one mouse died on day 32), 10 mg/kg Enigmol (n = 9 or 10,
3
respectively.
Although tissue accumulation did not trend with cLogP,
fluorinated analogs reached higher tissue levels than Enigmol.
Therefore, CF -Enigmol and CF -Enigmol were expected to
one mouse died on day 36), 10 mg/kg CF -Enigmol (n = 10),
3
2
2
achieve elevated tumor concentrations and enhanced antitumor
efficacy. To test this hypothesis, nude mice were injected
subcutaneously with PC-3 cells, and tumors were established
until palpable before beginning once daily oral gavage of either
or 30 mg/kg CF -Enigmol (n = 10) was initiated (Figure 4d).
2
Gratifyingly, 10 mg/kg CF -Enigmol significantly inhibited
2
tumor growth relative to both control (p = 0.0003) and 10 mg/
kg Enigmol (p = 0.04), and neither 10 mg/kg nor 30 mg/kg
Enigmol (3 mg/kg or 10 mg/kg), CF -Enigmol (3 mg/kg or 10
CF -Enigmol caused significant weight loss (Figure 4e).
3
2
mg/kg), or vehicle (95:5 olive oil/EtOH). Tumor volume was
measured to quantify drug-induced growth inhibition (Figure
Consistent with tissue distribution, 10 mg/kg CF -Enigmol
2
PC‑3
(IC90
= 15 μM) achieved strikingly higher tumor
4a). While 3 mg/kg dosing regiments were ineffective for both
concentrations (24 μM) than 10 mg/kg Enigmol (Figure 4f).
Enigmol (n = 11) and CF -Enigmol (n = 10), 10 mg/kg
Although 30 mg/kg CF -Enigmol reached extraordinary tumor
3
2
Enigmol (n = 11) significantly inhibited tumor growth (p =
levels (62 μM), it underperformed relative to 10 mg/kg CF2-
Enigmol, suggesting that a therapeutically saturating concen-
tration in tumor cells can be achieved after 2 weeks of once
daily oral dosing at <10 mg/kg.
0
.02) versus control (n = 11); 10 mg/kg CF -Enigmol (n = 11)
3
surprisingly did not (p = 0.17), despite substantially elevated
tissue concentrations. While no weight loss (Figure 4b) was
observed among control, 3 mg/kg Enigmol, 10 mg/kg Enigmol,
or 3 mg/kg CF -Enigmol treatment groups, 10 mg/kg CF -
CF -Enigmol exhibited slower plasma elimination kinetics
2
(i.v.), more robust plasma absorption (p.o.), and more
pronounced tissue accumulation than Enigmol and CF3-
Enigmol. Although oral absorption trended with cLogP, the
effects of fluorination on plasma elimination and tissue
accumulation followed entirely different trends. Since cLogP
can be an insufficient representation of fluorinated small
3
3
Enigmol caused significant weight loss (11.6 ± 1.32%) over the
treatment course (p = 0.0037). To evaluate the relationship
between efficacy and local drug concentrations, tumor
accumulation of Enigmol and CF -Enigmol was quantified
3
using LC-MS/MS at the end of the study (Figure 4c). While 10
D
DOI: 10.1021/acsmedchemlett.6b00113
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ACS Medicinal Chemistry Letters
Letter
molecule lipophilicity, an astute reviewer suggested that
empirical measurements of LogD would help to clarify the
role of lipophilicity. Therefore, LogD (pH = 7.4, n = 2, mean ±
SEM) of each analog was measured empirically (Absorption
Systems, Exton, PA), indicating an unexpected trend in
AUTHOR INFORMATION
■
*
Corresponding Author
E-mail: dliotta@emory.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. Questions regarding synthesis, pharmacokinetic
analysis, and characterization of anticancer activity should be
addressed to Eric J. Miller (ejmill2@emory.edu), G. Prabhakar
Reddy (pgrudda@emory.edu), or Suzanne G. Mays (smays@
emory.edu), respectively.
lipophilicity: Enigmol (3.50 ± 0.09) > CF -Enigmol (3.24 ±
3
0
.06) > CF -Enigmol (1.73 ± 0.06). Counter to the Hansch
2
15
hydrophobicity parameters and the average increase in LogD
upon fluorine incorporation, fluorination sometimes actually
16
decreases lipophilicity due to the interplay between change in
27
15
volume and dipole moment. For example, trifluoroethanol
LogP = 0.36) is more lipophilic than ethanol (LogP = −0.32)
(
Funding
because the volume change upon fluorination overrides the
change in dipole moment. Conversely, larger trifluoromethyl-
hexanol (LogP = 1.14) is less lipophilic than hexanol (LogP =
Work presented herein was funded internally.
Notes
The authors declare no competing financial interest.
2.03) because the volume change is insignificant compared to
the change in dipole moment. In the case of fluorinated
Enigmol analogs, an apparent increase in dipole moment upon
fluorination appears to override the relatively small increase in
volume, leading to decreased lipophilicity. Furthermore,
ACKNOWLEDGMENTS
■
The authors would like to thank Dr. Jinjing Gao (Winship
Cancer Institute, Emory University) for conducting statistical
analysis for the xenograft studies, as well as Dr. George Painter
(Emory Institute for Drug Development), Dr. Jim Snyder, Dr.
Bryan Cox, Dr. Terry Moore, Dr. Valerie Truax, Dr. Tim Acker,
Dr. John DiRaddo, Cynthia Gaillard (Department of
Chemistry, Emory University), and Kristin Jones (Biomarkers
Core Laboratory, Yerkes National Primate Research Center)
for thoughtful insights and extensive discussion concerning data
presented in this manuscript.
increased acidity of the CF -Enigmol C-5 alcohol likely leads
2
16
to improved hydrogen bonding with water, combining with
28
the inherent polarity of the difluoromethylene unit to
decrease LogD. Notably, the conventional wisdom that
lipophilicity is a strong determinant of systemic exposure
after oral administration did not hold up here: tissue
accumulation actually trended inversely with LogD.
Ultimately, the flat in vitro SAR observed with Enigmol
analogs was overcome by modulating PK properties via fluorine
installation. Since CF -Enigmol and CF -Enigmol exhibited
ABBREVIATIONS
■
3
2
CS, ceramide synthase; SK, sphingosine kinase; S1P,
sphingosine-1-phosphate; PK, pharmacokinetic; i.p., intra-
peritoneal; i.v., intravenous; p.o., per os (oral); NOE, Nuclear
Overhauser Effect; WST-1, 2-(4-iodophenyl)-3-(4-nitrophen-
yl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt;
SD, Sprague−Dawley; LC-MS/MS, liquid chromatography/
tandem-mass spectrometry
similar cytotoxicity to Enigmol against human prostate cancer
cells in vitro, we conclude that differences in antitumor efficacy
resulted from changes in PK. As no trend between LogD and
any PK parameter (other than an inverse trend with tissue
accumulation) was observed, the PK results presented herein
indicate that physicochemical properties other than lipophilicity
contribute to the plasma elimination, oral absorption, and tissue
distribution of these compounds. Despite achieving IC90 levels
REFERENCES
■
in tumor, CF -Enigmol did not significantly inhibit tumor
3
(1) Pruett, S. T.; Bushnev, A.; Hagedorn, K.; Adiga, M.; Haynes, C.
A.; Sullards, M. C.; Liotta, D. C.; Merrill, A. H. Biodiversity of
Sphingoid Bases (“Sphingosines”) and Related Amino Alcohols. J.
Lipid Res. 2008, 49, 1621−1639.
(2) Hannun, Y. A.; Obeid, L. M. Principles of Bioactive Lipid
Signalling: Lessons from Sphingolipids. Nat. Rev. Mol. Cell Biol. 2008,
growth and caused significant weight loss. Alternatively, CF2-
Enigmol statistically suppressed tumor growth better than
Enigmol, likely because of elevated tumor concentrations. Since
12
Enigmol was equally efficacious to docetaxel, CF -Enigmol
2
may offer an advantage over taxane-based chemotherapeutics
for hormone-refractory prostate cancer, especially given the lack
of systemic toxicity. Furthermore, due to enormous CF2-
Enigmol concentrations in lung, brain, and kidney, antitumor
efficacy could prove to be even more profound for the
treatment of solid tumors residing in these tissues.
9
(
, 139−150.
3) Zheng, W.; Kollmeyer, J.; Symolon, H.; Momin, A.; Munter, E.;
Wang, E.; Kelly, S.; Allegood, J. C.; Liu, Y.; Peng, Q.; Ramaraju, H.;
Sullards, M. C.; Cabot, M.; Merrill, A. H. Ceramides and Other
Bioactive Sphingolipid Backbones in Health and Disease: Lipidomic
Analysis, Metabolism, and Roles in Membrane Structure, Dynamics,
Signaling and Autophagy. Biochim. Biophys. Acta, Biomembr. 2006,
1
758, 1864−1884.
ASSOCIATED CONTENT
■
(4) Spiegel, S.; Milstien, S. Sphingosine-1-Phosphate: An Enigmatic
Signalling Lipid. Nat. Rev. Mol. Cell Biol. 2003, 4, 397−407.
*
S
Supporting Information
(
5) Merrill, A. H.; Nimkar, S.; Menaldino, D.; Hannun, Y. A.;
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00113.
Loomis, C.; Bell, R. M.; Tyagi, S. R.; Lambeth, J. D.; Stevens, V. L.;
Hunter, R.; Liotta, D. C. Stuctural Requirements for Long-Chain
(
Sphingoid) Base Inhibition of Protein Kinase C in Vitro and for the
Cellular Effects of These Compounds. Biochemistry 1989, 28, 3138−
145.
6) Pyne, N. J.; Pyne, S. Sphingosine 1-Phosphate and Cancer. Nat.
Rev. Cancer 2010, 10, 489−503.
7) Modrak, D. E.; Gold, D. V.; Goldenberg, D. M. Sphingolipid
Synthetic procedures utilized for the preparation of CF3-
3
(
Enigmol and CF -Enigmol, concentration−response
2
curves of Enigmol, CF -Enigmol, and CF -Enigmol,
2
3
and experimental methods and raw data from pharma-
cokinetic and xenograft experiments. (PDF)
(
Targets in Cancer Therapy. Mol. Cancer Ther. 2006, 5, 200−208.
E
DOI: 10.1021/acsmedchemlett.6b00113
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(
8) Plano, D.; Amin, S.; Sharma, A. K. Importance of Sphingosine
(27) Huchet, Q. A.; Kuhn, B.; Wagner, B.; Kratochwil, N. A.; Fischer,
H.; Kansy, M.; Zimmerli, D.; Carreira, E. M.; Muller, K. Fluorination
Patterning: A Study of Structural Motifs That Impact Physicochemical
Properties of Relevance to Drug Discovery. J. Med. Chem. 2015, 58,
9041−9060.
Kinase (SphK) as a Target in Developing Cancer Therapeutics and
Recent Developments in the Synthesis of Novel SphK Inhibitors. J.
Med. Chem. 2014, 57, 5509−5524.
(
9) Menaldino, D. S.; Bushnev, A.; Sun, A.; Liotta, D. C.; Symolon,
H.; Desai, K.; Dillehay, D. L.; Peng, Q.; Wang, E.; Allegood, J.;
Trotman-Pruett, S.; Sullards, M. C.; Merrill, A. H. Sphingoid Bases and
de Novo Ceramide Synthesis: Enzymes Involved, Pharmacology and
Mechanisms of Action. Pharmacol. Res. 2003, 47, 373−381.
́
(28) O’Hagan, D.; Wang, Y.; Skibinski, M.; Slawin, A. M. Z. Influence
of the Difluoromethylene Group (CF2) on the Conformation and
Properties of Selected Organic Compounds. Pure Appl. Chem. 2012,
8
4, 1587−1595.
(
10) Humpf, H.-U.; Schmelz, E.-M.; Meredith, F. I.; Vesper, H.;
Vales, T. R.; Wang, E.; Menaldino, D. S.; Liotta, D. C.; Merrill, A. H.
Acylation of Naturally Occurring and Synthetic 1-Deoxysphinganines
by Ceramide Synthase. J. Biol. Chem. 1998, 273, 19060−19064.
(
11) Symolon, H.; Bushnev, A.; Peng, Q.; Ramaraju, H.; Mays, S. G.;
Allegood, J. C.; Pruett, S. T.; Sullards, M. C.; Dillehay, D. L.; Liotta, D.
C.; Merrill, A. H. Enigmol: A Novel Sphingolipid Analogue with
Anticancer Activity against Cancer Cell Lines and In Vivo Models for
Intestinal and Prostate Cancer. Mol. Cancer Ther. 2011, 10, 648−657.
(
12) Garnier-Amblard, E. C.; Mays, S. G.; Arrendale, R. F.; Baillie, M.
T.; Bushnev, A. S.; Culver, D. G.; Evers, T. J.; Holt, J. J.; Howard, R.
B.; Liebeskind, L. S.; Menaldino, D. S.; Natchus, M. G.; Petros, J. A.;
Ramaraju, H.; Reddy, G. P.; Liotta, D. C. Novel Synthesis and
Biological Evaluation of Enigmols as Therapeutic Agents for Treating
Prostate Cancer. ACS Med. Chem. Lett. 2011, 2, 438−443.
(
13) Liotta, D. C.; Holt, J. J.; Natchus, M. G.; Galinski, M. R.; Baillie,
M. T.; Miller, E. J. Sphingosine Analogs, Compositions, and Methods
Related Thereto. WO 2013/049280 A2, 2013.
(
14) Bushnev, A. S.; Baillie, M. T.; Holt, J. J.; Menaldino, D. S.;
Merrill, A. H.; Liotta, D. C. An Efficient Asymmetric Synthesis of
Enigmols (1-Deoxy-5-Hydroxysphingoid Bases), an Important Class
of Bioactive Lipid Modulators. ARKIVOC 2010, viii, 263−277.
(
15) Yamazaki, T.; Taguchi, T.; Ojima, I. Fluorine in Medicinal
Chemistry and Chemical Biology; Blackwell Publishing, Ltd: 2009.
16) Bohm, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.;
Muller, K.; Obst-Sander, U.; Stahl, M. Fluorine in Medicinal
Chemistry. ChemBioChem 2004, 5, 637−643.
17) Denmark, S. E.; Yang, S.-M. Intramolecular Silicon-Assisted
(
̈
̈
(
Cross-Coupling Reactions: General Synthesis of Medium Sized Rings
Containing a 1,3-Cis-Cis Diene Unit. J. Am. Chem. Soc. 2002, 124,
2
(
102−2103.
18) Takai, K.; Takagi, T.; Baba, T.; Kanamori, T. Synthesis and
Characterization of Partially Fluorinated Stearolic Acid Analogs: Effect
of Their Fluorine Content on the Monolayer at the Air-Water
Interface. J. Fluorine Chem. 2007, 128, 120−126.
(
19) Reetz, M. T. Synthesis and Diastereoselective Reactions of N,N-
Dibenzylamino Aldehydes and Related Compounds. Chem. Rev. 1999,
9, 1121−1162.
20) Moreno, M.; Murruzzu, C.; Riera, A. Enantioselective Synthesis
of Sphingadienines and Aromatic Ceramide Analogs. Org. Lett. 2011,
3, 5184−5187.
21) Sato, K.; Omote, M.; Ando, A.; Kumadaki, I. Reactions of Ethyl
9
(
1
(
Bromodifluoroacetate in the Presence of Copper Powder. J. Fluorine
Chem. 2004, 125, 509−515.
(
22) Morikawa, T.; Nishiwaki, T.; Nakamura, K.; Kobayashi, Y.
Studies on Organic Fluorine Compounds. LIV. Synthesis of 2,2-
Difluoroarachidonic Acid. Chem. Pharm. Bull. 1989, 37, 813−815.
(
23) Iseki, K.; Asada, D.; Kuroki, Y. Preparation of Optically Active
α,α-Difluoro-β-Hydroxyketones. J. Fluorine Chem. 1999, 97, 85−89.
(
24) Chen, K.-M.; Hardtman, G. E.; Prasad, K.; Repic, O.; Shapiro,
M. J. 1,3-Syn Diastereoselective Reduction of β-Hydroxyketones
Utilizing Alkoxydialkylboranes. Tetrahedron Lett. 1987, 28, 155−158.
(
25) Kuroboshi, M.; Ishihara, T. Diastereoselective Reduction of α,α-
Difluoro β-Hydroxy Ketones to Syn- and Anti-2,2-Difluoro-1,3-Diols.
Bull. Chem. Soc. Jpn. 1990, 63, 1185−1190.
(
26) Although the CF2-Enigmol Experiment Was Conducted Using
a PEG400/Tween80 Vehicle as Opposed to Olive oil/EtOH, These
Trends Held When Enigmol and CF3-Enigmol Were Formulated with
PEG400/Tween80 (Supporting Information Figure 3).
F
DOI: 10.1021/acsmedchemlett.6b00113
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX