Design, synthesis, and evaluation of novel mutual prodrugs (hybrid drugs) of all-trans-retinoic acid and histone deacetylase inhibitors with enhanced anticancer activities in breast and prostate cancer cells in vitro

Article abstract of DOI:10.1021/jm8001839

Novel mutual prodrugs (MPs) of ATRA (all-trans-retinoic acid) and HDIs (histone deacetylase inhibitors) (10, 13, 17-19) connected via glycine acyloxyalkyl carbamate linker (AC linker) or through a benzyl ester linker (1,6-elimination linker) were rationally designed and synthesized. Most of our novel MPs were potent inhibitors of growth of several hormone-insensitive/drug resistant breast cancer cell lines and the hormone-insensitive PC-3 prostate cancer cell line. The novel MPs exhibited differential antiproliferative potencies in both MDA-MB-231 and PC-3 cell lines. Whereas 19 (VNLG/124) [4-(butanoyloxymethyl)phenyl(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6, 6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI₅₀ of 10 nM was the most potent MP versus the MDA-MB-231 cells, 13 (VNLG/66) [{N-[N-{2-[4-{[3-pyridylmethoxy)carbonyamino]-methyl}phenyl)carbonylamino] phenyl} carbamoylcarbamoyloxy}methyl(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6- trimethyl cyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI₅₀ = 40 nM was the most potent versus the PC-3 cells. MP 19 exhibited the most benefit because its GI₅₀ of 10 nM versus MDA-MB-231 cells was remarkably 1085-fold lower than that of parent ATRA and over 100000-fold lower than butyric acid (BA).

Full text of DOI:10.1021/jm8001839

J. Med. Chem. 2008, 51, 3895–3904
3895
Design, Synthesis, and Evaluation of Novel Mutual Prodrugs (Hybrid Drugs) of
All-trans-Retinoic Acid and Histone Deacetylase Inhibitors with Enhanced Anticancer Activities
in Breast and Prostate Cancer Cells in Vitro
Lalji K. Gediya,†,§ Aakanksha Khandelwal,†,§ Jyoti Patel,†,| Aashvini Belosay,†, Gauri Sabnis,† Jhalak Mehta,†
Puranik Purushottamachar,† and Vincent C. O. Njar*,†
Department of Pharmacology & Experimental Therapeutics, UniVersity of Maryland School of Medicine, 685 West Baltimore Street, Baltimore,
Maryland 21201-1559, The UniVersity of Maryland Marlene and Stewart Greenebaum Cancer Center, School of Medicine, Baltimore,
Maryland 21201-1559
ReceiVed February 20, 2008
Novel mutual prodrugs (MPs) of ATRA (all-trans-retinoic acid) and HDIs (histone deacetylase inhibitors)
(10, 13, 17-19) connected via glycine acyloxyalkyl carbamate linker (AC linker) or through a benzyl ester
linker (1,6-elimination linker) were rationally designed and synthesized. Most of our novel MPs were potent
inhibitors of growth of several hormone-insensitive/drug resistant breast cancer cell lines and the hormone-
insensitive PC-3 prostate cancer cell line. The novel MPs exhibited differential antiproliferative potencies
inbothMDA-MB-231andPC-3celllines.Whereas19(VNLG/124)[4-(butanoyloxymethyl)phenyl(2E,4E,6E,8E)-
3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI50 of 10 nM was the most
potent MP versus the MDA-MB-231 cells, 13 (VNLG/66) [{N-[N-{2-[4-{[3-pyridylmethoxy)carbonyamino]-
methyl}phenyl) carbonylamino]phenyl} carbamoylcarbamoyloxy}methyl(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-
trimethyl cyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI50 ) 40 nM was the most potent versus the
PC-3 cells. MP 19 exhibited the most benefit because its GI50 of 10 nM versus MDA-MB-231 cells was
remarkably 1085-fold lower than that of parent ATRA and over 100000-fold lower than butyric acid (BA).
Introduction
The discovery of recruitment of histone deacetylase (HDAC)
enzyme by nuclear receptors in cancer has provided a rationale
for using inhibition of HDAC activity to release transcriptional
repression as a viable option toward achieving eventual thera-
peutic benefit.9 Histone deacetylase inhibitors (HDIs) block
deacetylation function, causing cell cycle arrest, differentiation,
and/or apoptosis of many tumors.10 Silencing of genes that affect
growth and differentiation has been shown to occur by aberrant
DNA methylation in promoter region and by changes in
chromatin structure that involve histone deacetylation.11,12
Recent studies have established a link between oncogene-
mediated suppression of transcription and recruitment of HDAC
into the nuclear complex.13–15 Several laboratories have reported
that the translocation-generated fusion oncogenes (PML-RAR
and PLZF-RAR) in APL suppress transcription as a result of
sequestering HDAC enzyme.16,17 Resistance to ATRA of human
APL cell lines could be overcome by addition of HDAC
inhibitors (HDIs).16,17 Of particular importance is the observa-
tion that an APL patient, who failed multiple therapies and was
highly resistant to ATRA, responded to the combination
treatment of ATRA and phenylbutyric acid.18
Cancer cells show various degrees of differentiation, and there
is normally an inverse relation between the degree of cell
differentiation and the clinical aggressiveness of cancer.1
Differentiation induction of malignant cells is defined by the
ability of an agent to induce a more normal or benign phenotype
in these cells. Certain retinoids (e.g., all-trans-retinoic acid
(ATRA)a and its isomers) are among the better known dif-
ferentiation-inducing agents. These retinoids binds with RAR
and RXR receptors, and the ligand-receptor complex interaction
with retinoid responsive DNA sequence leads to activation of
target genes: transcription.2 Retinoids are currently in clinical
use for the treatment of cancers such as acute promyelocytic
leukemia (APL) and neuroblastoma.3,4 The clinical development
of retinoids in the treatment of epithelial tumors has been
hampered by the development of resistance.5 Loss of retinoids
sensitivity has been associated with lack of RARꢀ2 expression.6
Recently, it is reported that lack of RARꢀ2 expressions in
retinoid resistant tumors is associated with RARꢀ2 promoter
hypermethylation and histone deacetylation.7,8
Furthermore, in the presence of retinoids, HDIs induce
acetylation in RARꢀ2 hypermethylated promoters leading to
the re-expression of RARꢀ2 in RARꢀ2-negative retinoid-
resistant tumor cells resulting in an additive inhibitory effect
on tumor cell growth in vitro and in vivo.7,19,20 Our group
recently reported that the combination of several HDIs with
either retinoids or our atypical retinoic acid metabolism blocking
agents (RAMBAs) resulted in additive/synergistic growth
inhibitionofhumanprostatecancercellsandtumorxenografts.21,22
Combination treatment with 2 (MS-275) [(N-(2-aminophenyl)4-
[N-(pyridine-3-ylmethoxycarbonyl)aminomethyl]benzamide, a
HDI in several phase 2 clinical trials23 and 13-cis-retinoic acid
restored retinoid sensitivity in human prostate carcinoma cell
* To whom correspondence should be addressed. Phone: (410) 706 5885.
Fax: (410) 706 0032. E-mail: vnjar001@umaryland.edu.
†
University of Maryland School of Medicine, Baltimore.
These authors contributed equally to this work.
Present Address: Laboratory of Cancer Biology and Genetics, National
§
|
Institutes of Health, Bethesda, Maryland.
Present Address: Department of Food Science and Human Nutrition,
University of Illinois, 905 South Goodwin Avenue, Urbana-Champaign,
Illinois 61801.
a Abbreviations: APL, acute promyelocytic leukemia; ATRA, all-trans-
retinoic acid; BA, butyric acid; FDA, Food and Drug Administration; GI50,
concentration of compound that cause 50% growth inhibition; HDAC,
histone deacetylase; HDI, histone deacetylase inhibitor; MP, mutual prodrug;
PD, prodrug; RAMBAs, retinoic acid metabolism blocking agents; SAHA,
suberoyl hydroxamic acid.
10.1021/jm8001839 CCC: $40.75
2008 American Chemical Society
Published on Web 06/11/2008

3896 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Gediya et al.
Chart 1. Chemical Structures of 1-3, Butyric Acid (BA), and All-trans-Retinoic Acid (ATRA)
Scheme 1. Synthesis of p-Nitrophenyl Retinoyloxymethyl Carbonatea
a Reagents and conditions: (i) ClCO2CH2Cl, py, CHCl3, rt, 16 h; (ii) NaI, acetone, rt, 24 h; (iii) ATRA, AgCO3, acetone, reflux, 6 h.
lines and had a greater inhibitory effect on tumor cell growth
than single agents in vitro and in vivo.24 Indeed, on the basis
of this preclinical study, a phase 1 clinical trial has now been
initiated by Pili and colleagues.25 It should be pointed out that
several HDIs are at various stages of clinical development,10,26
and one of the early HDIs, N-hydroxy-N1-phenylacetanediamide,
also called suberoylanilide hydroxamic acid (SAHA),27,28 was
recently (2006) approved by the U.S. Food and Drug Admin-
istration (FDA) for the treatment of advanced cutaneous T-cell-
lymphoma.10
connected via an acyloxyalkyl carbamate (10 and 12), and the
second class is based on the 1,6-elimination concept (17-19).
The new compounds were evaluated against several breast
(MCF-7, MCF-7TAMR, MCF-7HOXB7 LTLC, LTLT-Ca, and
MDA-MB-231) and prostate (PC-3) cancer cell lines, most of
which are generally resistant to most therapeutic agents and were
found to be potent antineoplastic agents. Importantly, the MPs
possess enhanced anticancer activities compared to the parent
compounds or their combinations. A preliminary account of part
of this work has been presented.31
The putative synergistic interaction between retinoids and
HDIs has provided the impetus for synthesis and evaluation of
mutual prodrugs (MPs) of these agents with hopes of attainment
of superior therapeutic efficacy. It is relevant to state here that
a mutual prodrug (hybrid drug) is a type of carrier linked
prodrug where the carrier used is another pharmacologically
active compound instead of some inert molecule. Typically,
when two synergistic agents are administered individually but
simultaneously, they will be transported to the site of action
with different efficiencies. However, when it is desirable to have
the two agents reach a site simultaneously, the MP strategy may
be used to an advantage. Indeed, the MP of ATRA and butyric
acid (BA) called retinoyloxymethyl butyrate, 3 (RN1) has been
shown to function at lower concentrations than ATRA or BA
alone in the ATRA-sensitive leukemia cell line HL-60.29 In a
recent study, RN1 was found to exhibit significant growth
inhibitory activity in both ATRA-sensitive and -resistant APL
cells.30 The chemical structures of 1 (CI-994) [N-(2aminophe-
nyl)-4-acetylaminobenzamide], 2, BA, ATRA, and 3 are pre-
sented in Chart 1.
Chemistry. (Acyloxy)alkyl Carbamate Mutual Pro-
drugs. The (acyloxy) alkyl ester linker has been successfully
used for carboxylic acid containing agents to prepare prodrugs
(PDs) and MPs, as this linker is very labile and is cleaved by
the esterase enzyme. MP of ATRA and butyric acid (3) has
been prepared using this concept by Nudelman and collea-
gues.29,32,33 In addition, amine containing drugs have also been
converted into their corresponding acyloxyalkyl carbamates and
found to be excellent bioreversible prodrugs.34,35
To prepare mutual prodrugs of ATRA and 1 or 2 using this
strategy, we first synthesized p-nitrophenyl retinoyloxymethyl
(7) carbonate in three steps (Scheme 1). Thus, treatment of
p-nitrophenol (4) with chloromethyl chloroformate in the
presence of pyridine as base gave chloromethyl-p-nitrophenyl
carbonate (5) in good yield (52%). Compound 5 was converted
to the corresponding iodomethyl p-nitrophenyl carbonate (6)
following treatment with NaI. Treatment of 6 with ATRA in
the presence of silver carbonate as base in acetone afforded the
desired p-nitrophenyl retinoyloxymethyl carbonate (7) in 26%
yield. Attempts to condense aromatic amino group of 1 or 2
with compound 7 were unsuccessful. The reason for this is
unknown at this time but may be due to low nucleophilicity/
steric hindrance of the aromatic amine moiety in 1 and 2. On
In continuation of our research in this area, we have designed
and synthesized two classes of novel MPs of ATRA and HDIs
1, 2, or BA. One class is glycine linked 1 or 2 with ATRA

NoVel Mutual Prodrugs of ATRA and HDIs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3897
Scheme 2. Synthesis of Mutual Prodrug of ATRA and 1 with (Acyloxy)alkyl Carbamate Linkera
a Reagents and conditions: (i) Boc-Gly-OH, DCC, HOBt, DMF, rt, 18 h; (ii) TFA, CH Cl2, 2 h; (iii) 7, HMPA, TEA, rt, 24 h.
2
Scheme 3. Synthesis of Mutual Prodrug of ATRA and 2 with (Acyloxy)alkyl Carbamate Linkera
a Reagents and conditions: (i) Boc-Gly-OH, DCC, HOBt, DMF, rt, 18 h; (ii) TFA, CH Cl2, 2 h; (iii) 7, HMPA, TEA, rt, 24 h.
2
Scheme 4. Drug Release from Prodrug via 1,6-Elimination
the basis of previous studies,36 we decided to prepare amino
Mechanism
acid derivative (prodrugs) of CI-994 and MS-275 for coupling
with compound 7. First, we prepared a glycine derivative of
compound 1 (9) in two steps. N-Boc-glycine was coupled to 1
to give 8 (70% yield) by the active ester method using
dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole
(HOBt) followed by deprotection with trifluroacetic acid (TFA)
to give the glycine derivative of compound 1 (9). Treatment of
9 with p-nitrophenyl retinoyloxymethyl carbonate (7) afforded
the desired MP 10 in good yield (60%) (Scheme 2). A similar
synthetic procedure was used to synthesize the MP of ATRA
and 2, compound 13, as outlined in Scheme 3.
1,6-Elimination Based Prodrugs. Generally, 1,4- or 1,6-
elimination of HX (where X is a good leaving group like halide,
functionalized oxygen derivatives such as carboxylates, or a
carbamic acid anion) from benzyl compounds bearing strong
targeted drug delivery,38 there are Very few examples of MPs
electron-releasing o- or p-hydroxy or -amino substituents is a
fast reaction that occurs under mildly basic conditions. Con-
comitantly, quinone methides and quinonimine methides are
produced (Scheme 4).37 Although many prodrugs have been
prepared using the 1,6-elimination concept, especially for tumor
based on this concept.39,40
First, we prepared all-trans-retinoic acid benzyl alcohol ester
(16), a key intermediate for the synthesis of MPs of various
HDIs based on the 1,6-elimination concept (Scheme 5). p-

3898 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Scheme 5. Synthesis of Retinoic Acid Benzyl Alcohol Estera
Gediya et al.
a Reagents and conditions: (i) ATRA, DCC, DMAP, DMF, rt, 24 h: (ii) NaBH , CHCl3: IPA [1:5], 0 °C, 1 h.
4
Scheme 6. Synthesis of Mutual Prodrugs of ATRA and HDIs based on 1,6-Elimination Concepta
a Reagents and conditions: (i) triphosgene, Na 2CO3, PhCH3, 0 °C, 6 h; (ii) 2, TEA, THF, 0 °C, rt, 16h; (iii) 1, TEA, THF, 0 °C, rt, 16 h; (iv) butyric acid,
DCC, DMAP, rt, 24 h.
Table 1. GI50 (Growth Inhibitory Activity) of ATRA and HDIs and
Hydroxybenzaldehyde was coupled with ATRA using DCC/
Mutual Prodrugs from Dose-Response Curves in PC-3 and
DMAP in DMF to yield benzaldehyde ester of ATRA (15).
Compound 12 was readily reduced with NaBH4 to the corre-
sponding alcohol (16), which was then converted to the
corresponding chloroformate intermediate by treatment with
triphosgene and Na2CO3 in toluene. This chloroformate was used
in the subsequent step without purification. Reaction of the
chloroformate with 2 using triethylamine (TEA) as base in THF
produced the desired MP 17. A similar reaction of the
chloroformate derivative with 1 gave MP 18. Finally, reaction
of alcohol (16) with butyric acid in the presence of DCC/DMAP
yielded MP 19.
MDA-MB-231 Cell Lines
GI50 values (µM)a
MDA-MB-231
PC-3 prostate breast cancer
compounds
cancer cells
cells
ATRA
1
2
7.6
0.29
0.19
72.44
4.27
0.04
0.18
0.87
1.02
10.85
0.17
0.009
>1000
sodium butyrate (BA)
10, (VNLG/60), ATRA-1 (AC linker)b
13, (VNLG/66), ATRA-2 (AC linker)b
17, (VNLG/114) ATRA-2 (1,6-E linker)b
18, (VNLG/122), ATRA-1 (1,6-E linker)b
19, (VNLG/124), ATRA-BA (1,6-E linker)b
0.63
0.94
0.17
0.02
0.01
To be effective MPs, these modified novel compounds must
revert rapidly and quantitatively to ATRA and the corresponding
HDIs in animal tissues and cell cultures. Thus, we developed
HPLC methods (see Experimental Section) to briefly study the
cleavage of compounds 10, 12, and 17-19 in fresh mouse
plasma. We observed that all MPs were completely cleaved to
their parent compounds within 1 h of incubations at 37 °C.
Furthermore, the stability of mutual prodrugs was studied in
0.02 M phosphate buffer (pH ) 7.2) and also in 80% human
serum (obtained from Sigma) containing 20% 0.02 M phosphate
buffer as previously described.41 The MPs were each incubated
at 37 °C for 24 h, extracted and analyzed by HPLC. We found
that all the MPs were stable under these conditions. The
difference in stabilities of the MPs in fresh mouse plasma and
commercial human serum may be due to deactivation of certain
enzymes such as esterases and peptidases required for cleavage
in human serum.
a The GI50 values were determined from dose-response curves (by
nonlinear regression analysis using GraphPad Prism) compiled from at least
three independent experiments, SEM < 10%, and represents the compound
concentration (µM) required to inhibit cell growth by 50%. b AC linker )
acyloxymethylcarbamate linker and 1,6-E linker ) 1,6-elimination linker.
with some HDIs cause additive/synergistic inhibition of growth
of these cancer cell lines. In an effort to improve delivery
(efficacy) of each agent, we have designed and synthesized novel
MPs of ATRA and three HDIs, including BA, 1, and 2 as
described above. We hypothesize that the MPs might exert
stronger antiproliferative activity in cancer cells than cotreat-
ments of the two agents that comprise the MPs, or than each of
the two agents alone.
MPs Inhibit Proliferation of MDA-MB-231 Breast and
PC-3 Prostate Cancer Cells. First, the growth inhibitory effect
of ATRA and different HDIs were evaluated and GI50 values
(concentrations that cause 50% growth inhibition) were obtained
from the dose-response curve and are presented in Table 1.
The cell growth inhibitory potencies elicited by ATRA were
similar in both MDA-MB-231 (GI50 ) 10.8 µM) and PC-3 (GI50
Biological Studies. Most retinoids, including ATRA, are
generally weak inhibitors of proliferation of hormone-insensitive
breast and prostate cancer cells.1,3 We22,31,42 and others19,23,24,29,30
have demonstrated that cotreatment of combinations of retinoids

NoVel Mutual Prodrugs of ATRA and HDIs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3899
Figure 2. Growth inhibitory effect of 3 and MP 19 in MDA-MB-231
breast cancer cell line. Data are means (( SEM) of at least three
independent experiments.
Figure 1. Concentration-dependent curve showing the antiproliferative
effect of MP 18 on human prostate cancer PC-3 cells. Data are means
(( SEM) of at least three independent experiments. The experiments
with the other compounds gave plots that were similar to that shown
above.
either ATRA (543-fold lower) or 1 (8.5-fold lower). Indeed,
this gain in function of 19 in this cell line is by far superior
to that previously reported by Nudelman and Rephaeli29 for
retinoyloxymethyl butyrate (3, an MP derived from ATRA
and BA with an acyloxyalkyl linker) in myeloid leukemia
cell line HL-60. It might be unexpected that the coupling of
ATRA to BA would cause such a large increase in activity,
considering the low potency of BA. As previously reported,29
the results may be explained by a combination of two factors:
(i) the ATRA fragment of 19 imparts lipophilicity and
facilitates the penetration of BA to the cellular target site,
and (ii) the intracellularly released ATRA and BA affect the
cells synergistically.
) 7.6 µM) cells. With respect to the HDIs, both cell lines were
sensitive to 2, and as expected, BA was a very weak inhibitor
of cell growth. In both cell lines, the same order of potency, 2
> 1 > ATRA . BA, was observed. In addition, significant
differences between the two cell lines were (i) the gigantic
difference between the potencies of BA in PC-3 (GI50 ) 72.44
µM) versus MDA-MB-231 (GI50 > 1 mM) and (ii) sensitivity
of MDA-MB-231 to 2 (GI50 ) 9.0 nM) compared to PC-3 (GI50
) 190 nM), a 21-fold difference.
To assess the effect of MPs on cell growth, PC-3 and MDA-
MB-231 cells were treated with MPs for 4 or 6 days,
respectively. A typical dose response curve for the antiprolif-
erative effect of MP 18 is presented in Figure 1. Relative to
other MPs, 13 (ATRA-2 with AC linker) was the most potent
at inhibiting PC-3 cell growth (GI50 ) 40.0 nM) while 10
(ATRA-1 with AC linker) was the least potent (GI50 ) 4.27
µM). In this cell line, the order of potency was 13 > 17 > 18
> 19 > 10. The efficacies of MPs were compared to the
efficacies of ATRA or HDIs alone in PC-3 prostate cancer cells.
In general, the GI50 values of all MPs were 1.8- to 190-fold
lower than that of ATRA and 17- to 1811-fold lower that of
BA. Comparing the efficacies of MPs with either of the HDIs,
MP 16 (ATRA-BA, with 1,6-elimination linker) exhibited the
most benefit because its GI50 of 1.02 µM was 74-fold lower
than BA. Given the potent cell growth inhibition (GI50 ) 190
nM) caused by compound 2, it is remarkable that MP 13
(ATRA-2 with AC linker) was still very potent with a GI50 of
40 nM, 4.75-fold lower than 2. In contrast, MPs with HDI 1,
10, and 18, with GI50 values of 4.27 and 0.87 µM, respectively,
were each less potent than 1 (GI50 ) 0.29 µM). The reason(s)
for these differential potencies of the different MPs in PC-3
cells are unknown at this time, but may be idiosyncratic, possibly
due to extents and efficiencies of cell membrane penetration
and/or intracellular cleavage of MPs.
Compared to their efficacies in PC-3 cells, the MPs exhibited
different potencies in the MDA-MB-231 cells. Relative to other
MPs, 19 (ATRA-BA with 1,6-elimination linker) was the most
potent at inhibiting MDA-MB-231 cell growth (GI50 ) 10.0
nM), while 13 (ATRA-2 with AC linker) was the least potent
(GI50 ) 940 nM). In this cell line, the order of potency was 19
> 18 > 17 > 10 > 13. Other notable observations on the
antiproliferative effects of ATRA, HDIs, and MPs in this cell
line were: (i) that MP 19 exhibited the most benefit because its
GI50 of 10 nM was remarkably 1085-fold lower that that of
ATRA and over 100000-fold lower than BA, (ii) MP 18
(ATRA-1 with 1,6-E linker) with GI50 ) 20 nM is superior to
related MP 10 (ATRA-1 with AC linker), GI50 ) 630 nM, a
robust 31.5-fold difference, and (iii) 18 is also more potent than
Furthermore, in our desire to compare the efficacy of 3 with
that of our closely related MP 19, we synthesized compound 3
as previously described29 and assessed their antiproliferative
activities head-to-head in MDA-MB-231 cells. As shown in
Figure 2, the GI50 of 19 for inhibition of growth of MDA-MB-
231 cells was 48 nM, 25-fold lower than that of 3 (GI50 ) 1.18
µM). Together, these data suggest that the acyloxymethycar-
bamate linker is superior to the acyloxyalkyl linker. On the basis
of the mean GI50 values of all MPs obtained for the two cell
lines, it tempting to suggest that that the 1,6-elimination linker
with mean GI50 ) 0.035 µM (n ) 6) is superior to AC linker
with mean GI50 ) 1.47 µM (n ) 4) (see Table 1). Validation
of this assertion would probably require analysis of larger data
set. It should be stated that some byproduct resulting from
intracellular cleavage of MPs such as formaldehyde (generated
from MPs with acyloxylalkyl linker)29,43 or quinine methide
(generated from MPs with 1,6-elimination type linker)39 have
also been implicated in the anticancer activities of the two
components of the MPs. Experiments to assess the possible
involvement of byproduct of our MPs are envisioned in future
mechanistic studies.
Based on these encouraging results with the novel MPs, we
wished to assess further possible advantage of the MPs over
simultaneous treatment of components of the MPs. Using
representative MPs (17-19), we observed that the antiprolif-
erative activities in both cell lines elicited by the MPs were
each greater than those of the combined parent ATRA and HDIs
(Figure 3a-e). Treatment of PC-3 cells with 20 µM 19 resulted
in significantly potent growth inhibition (∼80%) compared to
a mixture of 10 µM ATRA and 10 µM BA (Figure 3a).
Furthermore, using dose-response curves, the antiproliferative
activity elicited by 19 (GI50 ) 1.7 µM) was 15-fold lower than
the combination of increasing concentrations of ATRA and BA
(10.0 µM) (GI50 ) 25.7 µM) (Figure 3b). Similar results were
also obtained for 18 versus parent ATRA (increasing concentra-
tions) and 1 (0.2 µM) (Figure 3c) and 17 versus parent ATRA

3900 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Gediya et al.
Figure 3. (a) Effect of ATRA and butyric acid administered alone or in combination and of mutual prodrug 19 on PC-3 cell growth. ** indicates
a significant increase from control and BA, ATRA, or BA + ATRA treatments (P < 0.001). (b) Growth inhibitory effect of ATRA and sodium
butyrate in combination and of corresponding mutual prodrug 19 in PC-3 cell line. Data are means (( SEM) of at least three independent experiments.
(c) Effect of ATRA and 1 administered alone or in combination and of corresponding mutual prodrug 18 on PC3 cell growth. Data are means (SEM
< 10%) of at least three independent experiments. (d) Effect of ATRA and 2 administered alone or in combination and of corresponding mutual
prodrug 17 on MDA-MB-231 cell growth. * indicates a significant increase from control and 2, ATRA, or 2 + ATRA treatments (P < 0.01). (e)
Effect of ATRA and butyric acid administered alone or in combination and of mutual prodrug 19 on MDA-MB-231 cell growth. *** indicates a
significant increase from ATRA (5 nM) + BA (5 nM) treatment (P < 0.0001). ### indicates a significant increase from ATRA (10 nM) + BA (10
nM) treatment (P < 0.0001).
and 2 (Figure 3d) in PC-3 cells, and also for 19 versus parent
ATRA and BA in MDA-MB-231 (Figures 3e).
of these resistant cell lines, with GI50 values in the low
nanomolar range.
MPs Also Inhibit Proliferation of Drug-Resistant
Breast Cancer Cell Lines. Given the exquisite potency of most
of our novel MPs in MDA-MB-231 and PC-3 cell lines, it
seemed logical to investigate their effects on the growth of some
known drug-resistant breast cancer cell lines, including MCF-
Conclusion
We have developed rationale strategies that allowed us to
synthesize novel mutual prodrugs (MPs) of ATRA and three
promising HDIs, BA, 1, and 2. Most of these novel MPs were
shown to possess potent antiproliferative activity versus hormone/
drug-resistant breast and prostate cancer cell lines. The unique-
ness of these novel MPs stem from the combination of two
moieties, ATRA and HDI (BA or 1 or 2), each affecting
7
TAMR, MCF-7HOX-B7, LTLC, and LTLT-Ca (see Experimental
Section for description of cell lines phenotypes) compared to
parental MCF-7 cells. As presented in Table 2, most of the MPs
tested, including 10, 13, 17, and 18 resulted in potent inhibition

NoVel Mutual Prodrugs of ATRA and HDIs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3901
Table 2. GI50 Values Obtained from Dose-Response Curves in Other
18-CH3), 2.02 (s, 3H, 19-CH3), 2.04 (m, 2H, CH2), 2.40 (s, 3H,
20-CH3), 5.82 (s, 1H, 14-H), 5.94 (s, 2H, CH2), 6.23 (m, 4H, 7, 8,
10 and 12-Hs), 7.09 (dd, 1H, J ) 13.5 Hz, 11H), 7.42 (d, 2H, J )
9.5 Hz, Ar-Hs), 8.29 (d, 2H, J ) 9 Hz, Ar-Hs).
Breast Cancer Cell Lines
GI50 values (µM)a
compounds MCF-7 LTLC LTLT-Ca MCF-7TAMR MCF-7HOX-B7
N-(2-{[4-(Acetylamino)phenyl]carbonylamino}phenyl)-2-[(tert-
butoxy)carbonylamino] acetamide (8). Boc-glycine (0.210 g, 1.2
mmol) and 1-hydroxybenzotriazole (HOBt) (0.162 g, 1.2 mmol)
were dissolved in DMF (5 mL) and stirred at 0-5 °C. To this added
solution was added 1 (0.269 g, 1 mmol), followed by dicyclohexy-
lcarbodiimide (DCC) (0.248 g, 1.2 mmol). The cooling bath was
removed after 30 min, and the reaction mixture was stirred at rt
for 18 h. The reaction mixture was poured into ice-cold water and
extracted with EtOAc. The organic layer was washed with brine,
dried over anhydrous Na2SO4, and evaporated to dryness. The crude
product was purified by FCC [CH2Cl2/EtOH, (9:1)] to afford 300
10
13
17
18
0.25
0.15
0.06
0.52
n/d
0.02
n/d
n/d
0.65
n/d
0.17
0.02
0.21
0.08
1.20
0.006
0.72
8.13
n/d
n/d
a The GI50 values were determined from dose-response curves (by
nonlinear regression analysis using GraphPad Prism) compiled from at least
three independent experiments, SEM < 10%, and represent the compound
concentration (µM) required to inhibit cell growth by 50%. n/d ) not
determined.
distinctive cellular targets and when released simultaneously
inside the cancer cells probably act synergistically. Evaluation
of their mechanisms of action and in vivo antitumor efficacies
of some of these novel agents in breast and prostate tumor
xenograft models are currently underway in our laboratory.
1
mg pure compound 8 (70%); mp: 125-126 °C. H NMR: δ 1.34
(s, 9H, CH3), 2.08 (s, 3H, CH3), 3.73 (s, 2H, CH2) 7.21 (s, 2H,
Ar-Hs), 7.59 (s, 1H, Ar-H), 7.63 (s, 1H, Ar-H), 7.72 (d, 2H, J )
7.5 Hz, Ar-Hs), 7.94 (d, 2H, J ) 8.5 Hz, Ar-Hs), 9.51 (s, 1H, NH),
9.82 (s, 1H, NH), 10.22 (s, 1H, NH).
N-(2-{[4-(Acetylamino)phenyl]carbonylamino}phenyl)-2-ami-
noacetamide (9). To an ice-cold solution of compound 8 (250 mg,
0.586 mmol) in CH2Cl2 (4 mL) was added TFA (4 mL), followed
by stirring at 0-5 0 °C for 2 h. The reaction mixture was evaporated
to dryness; acetone was added and stirred for 30 min. The white
precipitate that formed was filtered and dried under vacuum to give
Experimental Section
Chemistry. General procedures and techniques were identical
with those previously reported.22 1H NMR spectra were recorded
in CDCl3 or DMSO-d6 at 500 MHz with Me4Si as an internal
standard using a Varian Inova 500 MHz spectrometer. High-
resolution mass spectra (HRMS) were determined on a Bruker
12Tesla APEX-Qe FTICR-MS by positive ion ESI mode by Susan
A. Hatcher, Facility Director, College of Sciences Major Instru-
mentation Cluster, Old Dominion University, Norfolk, VA. Ret-
inoids (all-trans-retinoic acid from LKT Laboratories, Inc., St. Paul,
MN). Compounds 1 and 2 were synthesized in our laboratory as
previously reported.21 All other reagents were purchased from
Sigma-Aldrich. Although the retinoidal intermediates and final
products appeared to be relatively stable to light, precautions were
taken to minimize exposure to any light source and to the
atmosphere. Thus, all operations were performed in dim light, with
reaction vessels wrapped with aluminum foil. All compounds were
stored in an atmosphere of argon and in the cold (-20 or -80 °C).
2-Chloromethyl-p-nitrophenyl Carbonate (5). To an ice-cold
mixture of p-nitrophenol (4, 1.39 g, 10 mmol) and pyridine (0.8 g,
10 mmol) in CHCl3 (50 mL) was added chloromethyl chloroformate
(1.41 g, 11 mmol). After approximately 30 min at 0-4 °C, the
reaction mixture was stirred further for 16 h at rt. Following
successive washing with 0.5% aq NaOH and water, the CHCl3 layer
was dried over anhydrous Na2SO4 and evaporated to give thick
yellow oil. This crude product was purified using flash column
chromatography [FCC, pet. ether/EtOAc, (9:1)] to obtain 5 (1.2 g,
52%); mp 44-45 °C. 1H NMR (CDCl3): δ 5.85 (s, 2H, CH2), 7.43
(d, 2H, J ) 7.5 Hz, Ar-Hs), 8.31 (d, 2H, J ) 9 Hz, Ar-Hs).
2-Iodomethyl-p-nitrophenyl Carbonate (6). Compound 5 (2.0
g, 8.63 mmol) dissolved in acetone was treated with NaI (2.16 g,
14.42 mmol) and then stirred at rt for 24 h. The reaction mixture
was evaporated and the residue was dissolved in CH2Cl2, followed
by washing with saturated solution of sodium bisulfite and water.
The organic layer was dried over anhydrous Na2SO4 and evaporated
to obtain thick brown oil 6 (2.3 g). The crude product was used as
1
pure compound 9 (148 mg, 77%); mp: 215-218 °C. H NMR: δ
2.13 (s, 3H, CH3), 3.42 (s, 1H, NH2), 3.86 (s, 2H, CH2), 7.28 (s,
1H, Ar-H), 7.68 (s, 1H, Ar-H), 7.77 (d, 2H, J ) 9 Hz, Ar-Hs),
7.99 (d, 2H, J ) 9 Hz, Ar-Hs), 8.17 (s, 2H, Ar-Hs), 9.71 (s, 1H,
NH), 9.80 (s, 1H, NH), 10.30 (s, 1H, NH).
(N-{[N-(2-{[4-(Acetylamino)phenyl]carbonylamino}phenyl)-
carbamoyl]methyl}carbamoyloxy) methyl (2E,4E,6E,8E)-3,7-
dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetr-
aenoate (10). To the solution of compound 7 (50 mg, 0.101 mmol)
in hexamethylphosphoramide (HMPA) (1 mL) was added com-
pound 9 (49 mg, 0.15 mmol) and Et3N (210 µL, 0.15 mmol), and
the reaction mixture was stirred at rt for 24 h. The reaction mixture
was poured into ice-cold water and extracted with CH2Cl2. The
organic layer was dried with anhydrous Na2SO4 and evaporated to
dryness. The crude product was purified using FCC [CH2Cl2/EtOH,
(20:1)] to give pure 10 (42 mg, 60%); mp: 32-33 °C. IR (CHCl3):
3429, 1734, 1676, 1599, 1508, 1457, 1335, 1297, 1214, 1066, 986,
754, 668 cm-1. 1H NMR (DMSO-d6): δ 1.03 (s, 6H, 16,17-CH3),
1.71 (s, 3H, 18-CH3), 2.02 (s, 3H, 19-CH3), 2.13 (s, 3H, CH3),
2.40 (s, 3H, 20-CH3), 3.90 (s, 2H, CH2), 5.59 (s, 2H, CH2), 5.80
(s, 1H, 14-H), 6.28 (m, 4H, 7-, 8-, 10- and 12-Hs), 7.20 (dd, 1H,
J ) 14.7 Hz, 11-H), 7.23 (s, 2H, Ar-H), 7.61 (s, 1H, Ar-H), 7.63
(s, 1H, 9Ar-H), 7.72 (d, 2H, J ) 7.5 Hz, Ar-Hs), 7.94 (d, 2H, J )
8.5 Hz, Ar-Hs), 8.1 (s, 1H, NH), 9.817 (s, 1H, NH), 10.3 (s, 1H,
NH). HRMS calcd 683.3439 (C39H46N4O7H+), found 683.3448.
2-[(tert-Butoxy)carbonylamino]-N-{2-[(4-{[(3-pyridylmethoxy)-
carbonylamino]methyl} phenyl)carbonylamino]phenyl}acetamide
(11). Boc-glycine (0.210 g, 1.2 mmol) and HOBt (0.162 g, 1.2
mmol) was dissolved in DMF (5 mL) and stirred at 0-5 °C. To
this was added 2 (0.376 g, 1 mmol), followed by DCC (0.247 g,
1.2 mmol). The cooling bath was removed after 30 min and reaction
mixture was stirred at rt for 18 h. The reaction mixture was poured
into ice-cold water and extracted with EtOAC. The organic layer
was washed with brine and dried over anhydrous Na2SO4 and then
evaporated to dryness. The crude product was purified by FCC
[CH2Cl2/EtOH (9:1)] to afford 380 mg (71%) of pure compound
11 as a low melting solid. 1H NMR (CDCl3): δ 1.23 (s, 9H, CH3),
3.87 (s, 2H, CH2), 4.39 (d, 2H, J ) 5.5, CH2), 5.14 (s, 2H, CH2),
7.15 (m, 2H, Ar-Hs), 7.29 (d, 2H, J ) 7.5 Hz, Ar-Hs), 7.61 (d, H,
J ) 8 Hz, Ar-Hs), 7.72 (d, 1H, J ) 7 Hz, Ar-Hs), 7.79 (m, 1H,
Ar-H), 7.83 (d, 2H, J ) 8.5 Hz, Ar-Hs), 8.52 (s, 1H, NH), 8.58 (s,
1H, NH2), 8.92 (s, 1H, NH), 9.24 (s, 1H, NH).
1
such without further purification. H NMR: δ 6.07 (s, 2H, CH2),
7.43 (d, 2H, J ) 8.0 Hz, Ar-Hs), 8.31 (d, 2H, J ) 8.5 Hz, Ar-Hs).
(4-Nitrophenoxycarbonyloxy)methyl(2E,4E,6E,8E)-3,7-dimethyl-
9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate (7). ATRA
(0.3 g, 1 mmol) was dissolved in acetone (15 mL) and to this was
added Ag2CO3 (303 mg, 1.1 mmol) and refluxed for 1 h. The
reaction mixture was cooled to rt (mixture A). Crude compound 6
(0.388 g) was dissolved in acetone (10 mL) separately and stirred
at rt. Mixture A was added slowly to the solution of 6 followed by
refluxing for 6 h. The reaction mixture was cooled to rt, filtered,
and the filtrate was evaporated to dryness. The crude product was
purified by FCC [pet. ether/EtOAc, (9.5:0.5)] to obtain pure 7 (129
2-Amino-N-{2-[(4{[(3-pyridylmethoxy)carbonylamino]meth-
yl}phenyl)carbonylamino]phenyl} acetamide (12). To an ice-cold
solution of compound 11 (300 mg, 0.562 mmol) dissolved in
1
mg, 26%); mp: 35-36 °C. H NMR (CDCl3): δ 1.03 (s, 6H, 16
and 17-CH3), 1.46 (m, 2H, CH2), 1.60 (m, 2H, CH2), 1.71 (s, 3H,

3902 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Gediya et al.
CH2Cl2 (4 mL) was added TFA (4 mL) and stirred at 0-5 °C for
2 h. The reaction mixture was evaporated to dryness and to this
was added acetone followed by stirring for 30 min. The white
precipitate was filtered and dried under vacuum to give pure 12 as
a low melting solid (169 mg, 69%). 1H NMR (DMSO-d6): δ 3.82
(s, 2H, CH2), 4.28 (d, 2H, J ) 6 Hz, CH2), 5.11 (s, 2H, CH2), 7.24
(d, 1H, J ) 7 Hz, Ar-Hs), 7.39 (d, 1H, J ) 8 Hz, Ar-H), 7.45 (m,
1H, Ar-H), 7.65 (s, H, Ar-H), 7. 83 (s, 1H, Ar), 7.95 (d, 2H, J )
8 Hz, Ar-Hs), 8.08 (s, 1H, Ar-H), 8.25 (s, 1H, Ar-H), 8.623 (s,1H,
Ar-H), 8.563 (s,1H, Ar-H), 9.55 (s, 1H, NH), 9.77 (s, 1H, NH),
10.02 (s, 1H, NH).
oil that was reconstituted in THF (5 mL). This THF solution was
added to the solution of 2 (124 mg, 0.33 mmol) and TEA (55 µL,
0.39 mmol) in THF (5 mL) at 0 °C and then stirred further at rt for
16 h. The reaction mixture was evaporated and purified by FCC
[CH2Cl2/EtOH, 9:1] to give compound 17 (110 mg, 40%); mp:
128-130 °C. IR (CHCl3): 3306, 1725, 1694, 1555, 1458, 1324,
1259, 1213, 1129, 1073, 749 cm-1 1H NMR (300 MHz, DMSO-
.
d6): δ 1.02 (s, 6H, 16, 17-CH3), 1.48 (m, 2H, CH2), 1.62 (m, 2H,
CH2), 1.70 (s, 3H, 18-CH3), 2.01 (s, 3H, 19-CH3), 2.34 (s, 3H,
20-CH3), 4.28 (d, 2H, J ) 6 Hz, CH2), 5.09 (s, 2H, CH2), 5.14 (s,
2H, CH2), 6.04 (s, 1H, 14-H), 6.25 (m, 4H, 7,8,10,12-Hs), 6.537
(s,1H, Ar-H), 6.507 (s, 1H, Ar-H), 7.16 (m, 4H, 11-H and Ar-Hs),
7.39 (d, 2H, J ) 7 Hz, Ar-Hs), 7.44 (d, 2H, J ) 8.5 Hz, Ar-Hs),
7.53 (d, 1H, J ) 8 Hz, Ar-Hs), 7.61 (d, 1H, J ) 8 Hz, Ar-Hs),
7.76 (d, 1H, J ) 7 Hz, Ar-Hs), 7.90 (d, 2H, J ) 7.5 Hz, Ar-Hs),
7.95 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 8.53 (s, 1H, Ar-H), 8.59 (s,
1H, NH), 9.05 (s, 1H, NH), 9.78 (s, 1H, NH). HRMS calcd
809.3908 (C49H52N4O7H+), found 809.3898.
{N-[N-{2-[4-{[3-Pyridylmethoxy)carbonyamino]methyl}phen-
yl)carbonylamino]phenyl}carbamoyloxy}methyl(2E,4E,6E,8E)-
3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tet-
raenoate (13). To the solution of 7 (50 mg, 0.101 mmol) in HMPA
was added 12 (64.98 mg, 0.15 mmol) and Et3N (210 µL, 0.15
mmol) and the reaction mixture was stirred at rt for 24 h. The
reaction mixture was poured into ice-cold water and extracted with
CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and
evaporated to dryness. The crude product was purified using FCC
[CH2Cl2/EtOH, (20:1)] to yield compound 13 (60 mg, 65%); mp:
56-58 °C. IR (CHCl3): 3684, 1715, 1651, 1592, 1519, 1477, 1336,
4-{[N-2-{[4-(Acetylamino)phenyl]carbonylamino}phenylcar-
bamoyloxy]methyl}phenyl (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-
trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate (18). To a
solution of triphosgene (118 mg, 0.39 mmol) in dry toluene (10
mL) at 0 °C was added NaHCO3 (42 mg, 0.39 mmol), and the
reaction mixture was stirred for 1 h. Compound 16 (135 mg, 0.32
mmol) dissolved in dry toluene (5 mL) was added dropwise over
30 min, and the resulting reaction mixture was further stirred at 0
°C for 16 h. The reaction mixture was filtered, and filtrate was
evaporated to obtain dark-brown oil, which was reconstituted in
THF (5 mL). This THF solution was added to the solution of 1 (89
mg, 0.33 mmol) and TEA (55 µL, 0.39 mmol) in THF (5 mL) at
0 °C and then stirred further at rt for 16 h. The reaction mixture
was evaporated and purified by FCC [CH2Cl2/EtOH, 9:1] to give
compound 18 (98 mg, 42%); mp: 123-124 °C. IR (CHCl3): 3310,
1296, 1214, 1123, 988, 754, 668 cm-1 1H NMR (300 MHz,
.
CDCl3): δ 1.02 (s, 6H, 16,17-CH3), 1.47 (m, 2H, CH2), 1.61 (m,
2H, CH2), 1.70 (s, 3H, 18-CH3), 1.99 (s, 3H, 19-CH3), 2.29 (s, 3H,
20-CH3), 4.0 (s, 2H, CH2), 4.43 (s, 2H, CH2), 5.16 (s, 2H, CH2),
5.71 (s, 1H, 4-H), 5.58 (s, 2H, CH2), 5.80 (s, 1H, 14-H), 5.94 (s,
2H, CH2), 6.23 (m, 4H, 7-, 8-, 10- and 12-Hs), 7.09 (dd, 1H, J )
14.7 Hz, 11-H), 7.33 (m, 4H, Ar-Hs), 7.63 (d, 2H, J ) 7 Hz, Ar-
Hs), 7.72 (s, 1H, Ar--H), 7.87 (s, 2H, Ar-Hs), 8.11 (s, 1H, NH),
8.09 (s, 1H, NH), 8.63 (s, 1H, NH), 9.51 (s, 1H, NH), 9.81 (s, 1H,
NH), 10.21 (s, 1H, NH). HRMS calcd 790.3810 (C45H51N5O8Na+),
found 790.3810.
1
4-Formylphenyl(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimeth-
ylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate (15). ATRA (0.6 g,
2 mmol), 4-hydroxybenzaldehyde (14) (0.293 g, 2.4 mmol), and
DMAP (0.293 g, 2.4 mmol) were dissolved in dry DMF and to
this solution was added DCC (0.5 g, 2.4 mmol) at 0-10 °C. The
reaction mixture was stirred for 24 h at rt. The reaction mixture
was filtered, poured into ice-cold water and extracted with CH2Cl2.
The organic layer was dried over anhydrous Na2SO4 and evaporated
to give a crude product that was purified by FCC [CH2Cl2/EtOH,
9.5:0.5] to give the desired pure 15 (0.37 g, 91%); mp: 118-119
1718, 1654, 1600, 1508, 1312, 1215, 1125, 758 cm-1. H NMR
(300 MHz, DMSO-d6): δ 1.02 (s, 6H, 16,17-CH3), 1.45 (m, 2H,
CH), 1.57 (m, 2H, CH2), 1.70 (s, 3H, 18-CH3), 2.01 (s, 3H, 19-
CH3), 2.08 (s, 3H, CH3), 2.35 (s, 3H, 20-CH3), 5.14 (s, 4H, CH2),
6.09 (s, 1H, 14-H), 6.26 (m, 4H, 7-, 8-, 10- and 12-Hs), 6.54 (s,
1H, CH), 6.54 (s, 1H, CH), 7.11 (d, 2H, J ) 8 Hz, Ar-Hs), 7.17
(m, 2H, Ar-Hs), 7.43 (d, 2H, J ) 8 Hz, Ar-Hs), 7.51 (d, 1H, J )
7.5 Hz, Ar-Hs), 7.60 (d, 1H, J ) 7.5 Hz, Ar-Hs), 7.91 (d, 2H, J )
8.0 Hz, Ar-Hs), 8.31 (s, 2H, Ar), 9.03 (s, 1H, NH), 9.73 (s,1H,
NH), 10.22 (s, 1H, NH). HRMS calcd 702.3537 (C43H47N3O6H+),
found 702.3541.
1
°C. H NMR (CDCl3): δ 1.04 (s, 6H, 16, 17-CH3), 1.32 (m, 2H,
CH2), 1.72 (s, 3H, 18-CH3), 1.90 (m, 2H, CH2), 2.03 (s, 3H, 19-
CH3), 2.42 (s, 3H, 20-CH3), 5.99 (s, 1H, 14-H), 6.27 (m, 4H,
7,8,10,12-Hs), 7.10 (dd, 1H, 11-H), 7.31 (d, 2H, J ) 8.5 Hz, Ar-
Hs), 7.92 (d, 2H, J ) 8.5 Hz, Ar-Hs), 9.99 (s, 1H, CHO).
4-(Butanoyloxymethyl)phenyl(2E,4E,6E,8E)-3,7-dimethyl-9-
(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate (19). To
a solution of butyric acid (42 mg, 0.47 mmol) in DMF (5 mL) was
added compound 16 (200 mg, 0.47 mmol), DCC (108 mg, 0.52
mmol), and DMAP (63.75, 0.52 mmol), and the reaction mixture
was stirred at rt for 24 h. The reaction mixture was poured into
ice-cold water (50 mL) and extracted with CH2Cl2 (25 mL × 3),
and the organic layer was dried over anhydrous Na2SO4 and
evaporated to dryness. The crude product was purified by FCC [pet.
ether/EtOAc, (50:1)] pure 19 as a yellow oil (123 mg, 52%); mp:
38-40 °C. IR (CHCl3): 1723, 1577, 1353, 1234, 1214, 1123, 966,
753 cm-1. 1H NMR (300 MHz, DMSO-d6): δ 0.934 (t, 3H, J ) 7.
111-CH3), 1.03 (s, 6H, 16,17-CH3), 1.11 (m, 2H, CH2), 1. 53 (m,
2H, CH2), 1.68 (m, 2H, 101-CH2), 1.72 (s, 3H, 18-CH3), 2.02 (s,
3H, 19-CH3), 2.33 (s, 3H, 20-CH3), 2.46 (m, 2H, 91-CH2), 5.10 (s,
2H, CH2), 6.00 (s, 1H, 14-H), 6.27 (m, 5H, 7-, 8-, 10-, 11- and
12-Hs), 7.11 (d, 2H, J ) 8 Hz, Ar-Hs), 7.37 (d, 2H, J ) 8.5, Ar).
HRMS calcd 499.2818 (C31H40O4Na+), found 499.2822.
Kinetics of Hydrolysis in Plasma. Blood was collected from
male SCID mice (n ) 10) via cardiac puncture and centrifuged to
obtain plasma. Plasma (0.8 mL) was mixed with 0.2 mL of 0.02
M phosphate buffer (pH ) 7.2). Incubations were performed at 37
°C using a shaking water bath. The reaction was initiated by adding
25 µL of stock solution of mutual prodrugs (1 mg/mL in CH3CN)
to preincubated plasma, and aliquots were taken after 1 h for
processing and analyses. Plasma samples underwent solid-phase
extraction using 3 mL of C18 Bond Elut columns (Varian, Harbor
4-(Hydroxymethyl)phenyl (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-
trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate (16). Com-
pound 15 (0.35 g, 0.86 mmol) was dissolved in IPA:CHCl3 (1:5,
50 mL)) and cooled to 0 °C. NaBH4 (0.037 g) was then added to
this, and the reaction mixture was stirred for 1 h at 0 °C. The
reaction was quenched by addition of acetone (1 mL), evaporated
to dryness, and purified by FCC [CH2Cl2/EtOH, (9:1)] to give pure
1
16 (0.32 g, 78.8%); mp: 89-90 °C. H NMR (CDCl3): δ 1.03 (s,
6H, 16, 17-CH3), 1.48 (m, 2H, CH2), 1.62 (m, 2H, CH2), 1.72 (s,
3H, 18-CH3), 2.02 (s, 3H, 19-CH3), 2.40 (s, 3H, 20-CH3), 4.69 (s,
2H, CH2), 5.99 (s, 1H, 14-H), 6.26 (m, 4H, 7,8,10,12-Hs), 7.08
(dd, 1H, 11-H), 7.11 (d, 2H, J ) 8.5 Hz, Ar-Hs), 7.38 (d, 2H, J )
8.0 Hz, Ar-Hs).
4-{[N-(2-{[4-({[(3-Pyridylmethyl)oxycarbonyl]methyl}amino)-
phenyl]carbonylamino}phenyl)carbamoyloxy]methyl}phenyl(2E,
4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-
2,4,6,8-tetraenoate (17). To a solution of triphosgene (118 mg,
0.39 mmol) in toluene (10 mL) at 0 °C was added NaHCO3 (42
mg, 0.39 mmol), and the reaction mixture was stirred for an 1 h.
Compound 16 (135.0 mg, 0.32 mmol) dissolved in dry toluene (5
mL) was added dropwise over 30 min, and the resulting reaction
mixture was further stirred at 0 °C for 16 h. The reaction mixture
was filtered, and the filtrate was evaporated to obtain dark-brown

NoVel Mutual Prodrugs of ATRA and HDIs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3903
City, CA), which had previously been rinsed with methanol (3 mL)
and distilled water (3 mL). Then 500 µL of the sample were loaded,
the column was washed with 3 mL of distilled water, and the drug
was eluted with 2 mL of acetonitrile (in the case of MPs of CI-994
1:1 MeOH:CH3CN was used). Eluates were evaporated to dryness.
Samples were then reconstituted in 400 µL of acetonitrile and
filtered through a 0.45 µm filter (Ultrafree-MC; Millipore Corpora-
tion, Bedford, MA) before analyses by HPLC. Chromatographic
analysis was achieved by a reverse-phase HPLC method on a
Waters Novapak C18 column (3.9 mm × 150 mm) protected by
Waters guard cartridge packed with C18 as previously described.
The HPLC system used in this study consisted of a Waters solvent
delivery system, a Waters controller (Milford, MA) coupled to a
Waters 717plus autosampler, and a Waters 996 photodiode array
detector operating at 240.0 and 350 nm. A multilinear gradient
solvent system, (i) 20 mM aqueous ammonium acetate buffer/
methanol (50:50, v/v) (100-0%), and (ii) methanol (0-100%) at
a flow rate of 0.8 mL/min, was used. Retention times (mins) for
ATRA, HDIs, and prodrugs were as follows: ATRA (21.85), 2
(3.037), 1 (6.75), 10, 13, 17, 18, and 19 (23.97, 24.45, 25.63, 26.28,
and 26.93, respectively). ATRA and the mutual prodrugs were
detected at 350 nm, while HDIs were detected at 245 nm.
The stability of the mutual prodrugs were assessed in 0.02 M
phosphate buffer (pH ) 7.2) by incubations of each agent for 48 h
at 37 °C. Samples were processed and analyzed as described above.
Cell Culture. PC-3 (androgen receptor negative, AR -ve) cells
were obtained from American Type Culture Collection (ATCC,
Rockville, MD). Cells were maintained in RPMI 1640 medium
(Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal
bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 1%
penicillin/streptomycin. Cells were grown as a monolayer in T75
tissue culture flasks in a humidified incubator (5% CO2, 95% air)
at 37 °C.
Other cell lines used in this study including MDA-MB-231
(estrogen receptor negative, ER sve) and MCF-7 (ER +ve) were
also purchased from ATCC and were cultured as previously
described.44,45 LTLC and LTLT-Ca were kindly provided by Dr.
Angela Brodie, University of Maryland, Baltimore, and details of
their phenotypes and culturing conditions are as previously
reported.46,47 MCF-7TAM and MCF-7HOXB-7 were provided by Dr.
Saraswati Sukumar of Johns Hopkins University, Baltimore, and
details of their phenotypes and culturing conditions are as previously
reported.48 Except for MCF-7 breast cancer cell, all other breast
cancer cells used in this study are insensitive to endocrine
therapeutic agents and to most anticancer agents.
Cell Growth Inhibition (MTT Colorimetric Assay). PC-3 cells
were seeded in 24-well plates (Corning Costar) at a density of 2 ×
104 cells per well per 1 mL of medium. Cells were allowed to
adhere to the plate for 24 h and then treated with different
concentrations of ATRA, HDIs, or MPs dissolved in 10% DMSO,
90% ethanol. Cells were treated for five days with renewal of
prodrug and media on day 3. On the fifth day, medium was renewed
and 100 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide from Sigma) solution (0.5 mg MTT/mL of
media) was added to the medium such that the ratio of MTT:
medium was 1:10. The cells were incubated with MTT for 2 h.
The medium was then aspirated and 500 µL of DMSO was added
to solubilize the violet MTT-formazan product. The absorbance at
560 nm was measured by spectrophotometry (Victor 1420 multi-
label counted, Wallac). For each concentration of agent or MPs,
there were triplicate wells in each independent experiment. GI50
values were calculated by nonlinear regression analysis using
GraphPad Prism software.
Supporting Information Available: HPLC chromatograms and
high-resolution mass spectral data of MPs 10, 13, 17, 18, and 19.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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