Design, synthesis, and preliminary evaluation of doxazolidine carbamates as prodrugs activated by carboxylesterases

Article abstract of DOI:10.1021/jm060597e

The synthesis and tumor cell growth inhibition by doxazolidine carbamate prodrugs are reported. The carbamates were designed for selective hydrolysis by one or more human carboxylesterases to release doxazolidine (Doxaz), the formaldehyde-oxazolidine of d

Full text of DOI:10.1021/jm060597e

7002
J. Med. Chem. 2006, 49, 7002-7012
Design, Synthesis, and Preliminary Evaluation of Doxazolidine Carbamates as Prodrugs
Activated by Carboxylesterases
David J. Burkhart,†,‡ Benjamin L. Barthel,†,‡ Glen C. Post,† Brian T. Kalet,† Jordan W. Nafie,† Richard K. Shoemaker,† and
Tad H. Koch*,†,§
Department of Chemistry and Biochemistry, UniVersity of Colorado, Boulder, Colorado 80309-0215, and UniVersity of Colorado Cancer
Center, Aurora, Colorado 80010
ReceiVed May 18, 2006
The synthesis and tumor cell growth inhibition by doxazolidine carbamate prodrugs are reported. The
carbamates were designed for selective hydrolysis by one or more human carboxylesterases to release
doxazolidine (Doxaz), the formaldehyde-oxazolidine of doxorubicin that cross-links DNA to trigger cell
death. Simple butyl and pentyl, but not ethyl, carbamate prodrugs inhibited the growth of cancer cells that
overexpress carboxylesterase CES1 (hCE1) and CES2 (hiCE). Relative CES1 and CES2 expression levels
were determined by reverse transcription of the respective mRNAs, followed by polymerase chain reaction
amplification. More complex structures with a p-aminobenzyl alcohol (PABA) self-eliminating spacer showed
better growth inhibition (IC50 ) 50 nM for Hep G2 liver cancer cells) while exhibiting reduced toxicity
toward rat cardiomyocytes, relative to the parent drug doxorubicin. Pentyl 4-(N-doxazolidinylcarbony-
loxymethyl)phenylcarbamate, the lead compound for further investigation, appears to be activated in Hep
G2 cells that express both CES1 and CES2.
Introduction
Capecitabine is the pentyl carbamate of a 5-fluorocytidine
derivative that functions as a prodrug of the antitumor compound
5-fluorouracil (5-FU).5,6 In vivo activation requires three
enzymatic steps, the first of which is hydrolysis of the pentyl
carbamate functional group by a carboxylesterase (Scheme 1).
Capecitabine is a substrate for human carboxylesterases CES1
(hCE1) and CES2 (hiCE), with CES1 being the more active
enzyme.7 CES1 is a promiscuous serine hydrolase, overex-
pressed in numerous types of cancer cells and malignant tissue,
and it appears in both cytosolic and microsomal fractions.8,9
Irinotecan is a carbamate prodrug of a water soluble camptoth-
ecin and is hydrolyzed primarily in colon and primary colon
tumors by CES2, also a serine hydrolase.10,11 In spite of the
substantial structural differences between capecitabine and
irinotecan, they are both hydrolyzed by carboxylesterases CES1
and CES2, but in general CES1 prefers a smaller alcohol moiety
as in capecitabine and CES2, a larger alcohol moiety as in
irinotecan. Carboxylesterases also play a significant role in the
metabolism and elimination of numerous xenobiotics, including
cocaine and heroin.12 The substrate selectivity of carboxy-
lesterases stems from a pair of recognition pockets. One pocket
is a small, rigid cavity that commonly recognizes small alcohol
or ester functional groups and the other is a large flexible pocket
capable of accommodating numerous substrates for hydroly-
sis.13,14
Doxazolidine (Doxaza) is the oxazolidine derivative resulting
from the reaction of the antitumor drug doxorubicin (Dox) at
its vicinal aminol functional group with formaldehyde (Chart
1). Further reaction with formaldehyde couples two Doxaz
molecules at their oxazolidine nitrogens via a methylene group
to yield doxoform (Doxf, Chart 1).1,2 Circumstantial evidence
now implicates Doxaz as the active metabolite of Dox that
virtually cross-links DNA, leading to tumor cell death.2,3 Doxf
is a very labile prodrug of Doxaz. Doxf and, correspondingly,
Doxaz are 10- to 10 000-fold more active than Dox for growth
inhibition of sensitive and resistant cancer cells. Measurements
of the kinetics of spontaneous hydrolysis of Doxf to Doxaz to
Dox indicate that the half-lives of Doxf and Doxaz with respect
to hydrolysis to Dox under physiological conditions are ap-
proximately 1-3 min.2 Starting with Doxf, the slower of the
two steps is the hydrolysis of Doxaz to Dox. In spite of its short
half-life, Doxaz is taken up rapidly by cancer cells and exhibits
excellent cytotoxicity. For in vivo treatment of tumors, release
of Doxaz from a hydrolytically robust prodrug by an enzyme
overexpressed at the site of the tumor should increase tumor
response and minimize side effects, especially treatment-limiting
cardiotoxicity associated with doxorubicin therapy.4 Although
oxazolidine rings are known to hydrolyze rapidly in aqueous
media, their N-carbamate derivatives are quite stable (Chart 1).
The design of such a hydrolytically stable derivative is suggested
by the structures of two new clinical prodrugs, capecitabine and
irinotecan.
We began our search for a hydrolytically robust Doxaz
prodrug by synthesizing simple carbamates analogous to the
carbamate in capecitabine. Doxaz has a cytokinetic selectivity
for cancer cells, and this prodrug strategy has the potential for
additional selectivity because prodrug activation requires the
overexpression of a carboxylesterase. In addition, a small
lipophilic carbamate moiety could increase the rate of prodrug
uptake without completely sacrificing water solubility.
* To whom correspondence should be addressed. Phone: 303-492-6193.
Fax: 303-492-5894. E-mail: tad.koch@colorado.edu.
† University of Colorado.
‡ These authors contributed equally to this work.
§ Colorado Cancer Center.
a Abbreviations: Butyl PABC-Doxaz, butyl 4-(N-doxazolidinylcarbo-
nyloxymethyl)phenyl-carbamate; CES, carboxylesterase; Dox, doxorubicin;
Doxaz, doxazolidine, the formaldehyde oxazolidine derivative of doxoru-
bicin; Doxf, doxoform, N,N′-bis-doxazolidinylmethane; 5-FU, 5-fluorouracil;
PABA, p-aminobenzyl alcohol; PABC, p-aminobenzyloxycarbonyl; pentyl
PABC-Doxaz, pentyl 4-(N-doxazolidinylcarbonyloxymethyl)phenylcarbamate;
RT-PCR, reverse transcriptase-polymerase chain reaction.
Results and Discussion
Synthesis and Characterization of Simple Doxazolidine
Carbamates. The synthesis of simple Doxaz carbamates was
achieved by addition of the modestly nucleophilic Doxaz to the
10.1021/jm060597e CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/09/2006

Doxazolidine Carbamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7003
Chart 1Clinical Drug Doxorubicin, Potent Cytotoxins Doxoform and Doxazolidine (Doxaz), and Proposed Carbamate Prodrug of
Doxazolidine Rendering the Oxazolidine Ring Hydrolytically Stablea
a The numbering scheme adopted is that traditionally used for the anthracyclines.
Scheme 1. Proposed Enzymatic Activation of Capecitabine and Irinotecan by Human Carboxylesterases
Scheme 2. Synthesis of Hydrolytically Robust Doxaz Carbamates 1, 2, and 3 and Proposed Chair-Twist-Boat Conformational
Equilibrium of the Daunosamine Amino Sugar Ring Showing the Change in Dihedral Angles between the Hydrogens at the 1′- and
2′-Positions
desired alkyl chloroformate buffered by 1.1 equiv of dimethy-
laminopyridine (DMAP) or to the desired alkyl p-nitrophenyl
carbonate. The crude Doxaz carbamates were purified directly
by radial chromatography and obtained in good yield (com-
pounds 1, 2, and 3; Scheme 2). The structures were established
from one and two-dimensional 1H NMR spectra and mass
spectral molecular ions. Two-dimensional homonuclear NMR
experimental data facilitated the assignment of proton NMR
resonances. Resonances in the ambient temperature H NMR
1
spectra showed line broadening, indicating conformational
exchange at a rate similar to the NMR time scale.
Variable temperature NMR experiments with the ethyl
carbamate (1) confirmed the presence of conformational dynam-
ics. Exchange rate constants determined at multiple temperatures

7004 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Burkhart et al.
Figure 1. Variable-temperature 1H NMR of Doxaz ethyl carbamate (1) to resolve the oxazolidine methylene (4.9-5.1 ppm) resonances and determine
the energy of interconversion between conformations.
(via spectral simulation) revealed the thermodynamic param-
eters, ∆Hq, ∆Sq, and ∆Gq, associated with the barrier between
the two conformers. Experimental and simulated NMR spectra
of the oxazolidine methylene resonances at all temperatures are
shown in Figure 1. At the low-temperature limit, the oxazolidine
methylene protons appear as two AB patterns that collapse into
one AB pattern at the high-temperature limit. Integration of the
AB patterns at the low-temperature limit establishes that the
conformational populations are approximately equal. To achieve
quality dynamic simulations, the AB patterns were first simu-
lated under static conditions (using the NUMARIT algorithm),
after which dynamic simulations (DNMR3) were performed
using the previously simulated peak frequencies. The free energy
barrier (∆Gq) for the interconversion between the conformations
as determined from the dynamic NMR results is 61.8 ( 1.6
kJ/mol at 293 K. Analysis of the slope and Y-intercept of an
Erying plot yields ∆Hq ) 49.0 ( 1.3 kJ/mol and ∆Sq ) -45
of the protons at the 2′-position and one of the protons at the
10-position (see Chart 1 for the numbering scheme). A possible
conformational change in the daunosamine sugar is a chair to
twist-boat interconversion, as shown with energy-minimized
molecular models in Figure 2. This conformational change
would have a much larger effect on the chemical shift of one
of the protons at the 2′-position than the other (see Scheme 1).
The dihedral angle defined by the 1′-C-O bond to the anomeric
oxygen and one of the 2′-C-H bonds changes from ap-
proximately 180° to 60° in going from twist-boat to chair. The
corresponding dihedral angle relating the other proton at the
2′-position is approximately 60° in both conformations. The
NMR spectra at the lowest temperature did not show sufficient
resolution for determination of the respective coupling constants.
The entropy of activation for this chair-twist boat intercon-
version is likely to be large because molecular models suggest
that the transition state will be relatively rigid. For the chair-
twist boat interconversion of cyclohexane itself, ∆Hq ) 42.5
( 5 J/mol-1K-1
.
( 0.4 kJ/mol and ∆Sq ) -11.71 ( 2.1 J/mol-1K-1 16
Also
.
One possible explanation for the conformational exchange
is bond rotation about the C-N bond of the carbamate functional
group. Although not required by symmetry, the conformational
populations might be approximately equal as observed. How-
ever, careful earlier measurements on simpler carbamates report
a ∆Hq in deuteriochloroform of 75 ( 0.5 kJ/mol and a ∆Sq of
only 1 ( 1 J/mol-1K-1 for this rotational barrier.15 The ∆Sq
observed for the conformational dynamics of Doxaz ethyl
carbamate is clearly inconsistent with a simple carbamate bond
rotation. Further, the conformational change appears to be
complex, involving the oxazolidine ring and the daunosamine
sugar, as well as the tetracycline A-ring, because proton
resonances in all three rings show coalescence as the temperature
is increased. Additional proton resonances that show more
dramatic temperature dependence of chemical shift include one
consistent with the proposal is the recent observation of a twist-
boat conformation for the daunosamine sugars in the crystal
structure of Doxf.2 The NMR spectra, however, of both Doxf
and Doxaz in chloroform solvent at ambient temperature are
consistent with a predominant chair conformation for their
respective daunosamine sugars, with no conformational ex-
change at a rate similar to the NMR time scale.2 The difference
between the energy of chair and the energy of twist-boat
conformations of Doxaz versus Doxaz carbamates likely results
from the difference in the hybridization of their respective
oxazolidine nitrogens, sp3 versus sp.2 A twist-boat conformation
should alleviate some of the oxazolidine ring strain associated
with an sp2 nitrogen in a five-member ring fused to a
six-member ring. The reduction in ring strain is needed to

Doxazolidine Carbamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7005
Figure 2. Molecular models showing the proposed chair and twist-boat conformations of the daunosamine sugar of Doxaz ethyl carbamate (1).
Framework structures of the A-ring, daunosamine, and oxazolidine with daunosamine in chair and twist-boat conformations were created in ChemDraw
and energy minimized in Chem3D. The numbering system is the anthracycline numbering system shown in Chart 1.
balance the increased torsional strain and loss of some of the
anomeric effect associated with a twist-boat conformation.
Biological Activity of Simple Doxazolidine Carbamates.
Because the prodrug carbamates were designed to be activated
by CES1 and/or CES2, cell lines of interest were measured for
expression of the respective mRNAs using reverse transcription
(RT), followed by PCR (polymerase chain reaction). Cancer
cell lines investigated included SHP-77 resistant small cell lung,
DU-145 prostate, MCF-7 sensitive breast, MCF-7/Adr resistant
breast, SK-HEP-1 liver, and Hep G2 liver cells. As a measure
of possible cardiotoxicity, H9c2(2-1) rat cardiomyocytes were
also investigated. Cardiotoxicity is relevant because the ultimate
product of metabolism and subsequent hydrolysis of these
carbamates is doxorubicin, which is cardiotoxic.4 Primers
corresponding to the 5′, middle, and 3′ regions of human or rat
CES1 and CES2 were identified by Primer Express (Applied
Biosystems, Foster City, CA). Each primer set produced a single,
compact band by agarose gel electrophoresis at the predicted
size, indicating that PCR with the primer sequences amplified
Figure 3. Relative expression of mRNAs for carboxylesterases CES1
and CES2 by SHP-77 small cell lung, DU-145 prostate, MCF-7
sensitive breast, MCF-7/Adr resistant breast, SK-HEP-1 liver, and Hep
G2 liver cancer cells, and rat cardiomyocytes (H9c2(2-1)). mRNAs
only those regions that were targeted. Sequences for all primers
are provided in Supporting Information. The results of the RT-
PCR (Figure 3) show that both liver cancer cell lines, SK-HEP-1
and Hep G2, strongly express the mRNA for CES2, however,
only the Hep G2 cell line strongly expresses the mRNA for
CES1. DU-145, MCF-7, and MCF-7/Adr cells express more
CES2 than CES1, and SHP-77 cells express both enzymes, but
in lesser amounts than Hep G2 cells. Additionally, the rat
cardiomyocytes, H9c2(2-1), also express more CES2 than CES1.
Cell growth inhibition experiments initially focused on MCF-
7, MCF-7/Adr, SK-HEP-1, and Hep G2 cancer cells, as well as
rat cardiomyocytes, as a measure of cardiotoxicity and Vero
cells (green monkey kidney cells) as an additional measure of
normal cell toxicity. The butyl and pentyl carbamates inhibited
the growth of MCF-7, MCF-7/Adr, and SK-HEP-1 cell lines
with a 24 h drug treatment period, with some selectivity for
cancer cells over cardiomyocytes relative to growth inhibition
by Dox, as shown in Table 1. The ethyl carbamate with the
least complex structure exhibited poor cancer cell growth
inhibition. The relative inactivity of the ethyl carbamate may
indicate that a substantial lipophilic interaction is required at
the active site of the carboxylesterases to hold the Doxaz
substrate. For all measurements with the pentyl carbamate, the
IC50 values were dependent on drug treatment time. With a 3 h
drug treatment period, the log IC50 values for MCF-7, SK-HEP-
1, and Hep G2 cells were greater than -6 compared to -6.5 to
-7 for identical treatments with doxorubicin. This is consistent
with only a small fraction of the butyl 2 and pentyl 3 carbamates
were reverse transcribed, and DNA was amplified by the PCR using
three different primers specific for the 5′, middle, and 3′ regions of the
genes. The DNA was separated by agarose gel electrophoresis and
detected with ethidium bromide staining. (a) The vertical bars indicate
nonadjacent gel lanes.
being hydrolyzed during the 24 h drug treatment because the
log IC50 for Doxaz with SK-HEP-1 cells is -8.4 and with Hep
G2, -8.0, and over many cancer cell lines, -8.5 to -8.0, all
with 3 h drug treatment (a few of these are reported in Table
1). A control experiment showed that the pentyl carbamate is
stable to hydrolysis in pH 7.4 buffer in the absence of cells or
growth media over 24 h at ambient temperature, monitoring
the reaction by HPLC.
Uptake of the pentyl carbamate 3 relative to uptake of Dox
was measured by flow cytometry, monitoring fluorescence of
the Dox fluorophore as a measure of the drug in cells. The
measurements were performed over a period of 3 h in the
presence and absence of fetal bovine serum, and the results are
shown in Figure 4. Clearly, the pentyl carbamate was taken up
at a higher level and more rapidly than Dox. Further, the
presence of 10% fetal bovine serum decreased the uptake of
doxorubicin and the pentyl carbamate by about 25%. The more
rapid uptake of the pentyl carbamate in the presence and absence
of serum probably reflects its increased hydrophobicity relative
to that of doxorubicin, which is a cation at physiological pH.

7006 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Burkhart et al.
Figure 4. Uptake of Dox or the Doxaz pentyl carbamate (3) by SK-HEP-1 liver cancer cells, as determined by flow cytometry, measuring relative
fluorescence of the Dox fluorophore as a function of treatment time in RPMI media containing 10% fetal bovine serum (+) and RPMI media with
no serum (-). Drug concentration in media was 0.5 µM.
Table 1. Growth Inhibition and IC50 Valuesa
1
2
3
drug/cell line,
treatment time
Doxaz
ethyl carbamate
Doxaz
butyl carbamate
Doxaz
pentyl carbamate
Dox
-6.5b
Doxaz
MCF-7, 3 h
24 h
MCF-7/Adr, 3 h
24 h
SK-HEP-1, 3 h
24 h
Hep G2, 3 h
24 h
H9c2(2-1), 3 h
24 h
Vero, 3 h
24 h
> -6
> -6
> -6
-6.1 ( 0.03
-8.5 ( 0.03c
-7.9 ( 0.04
-5.2 ( 0.1
-5.6 ( 0.1
-7.0 ( 0.1
-7.3 ( 0.2
-6.7 ( 0.09
-7.5 ( 0.1
-7.5 ( 0.2
-7.7 ( 0.3
-6.0 ( 0.02
-6.3 ( 0.08
-6.1 ( 0.04
-6.2 ( 0.3
-6.4 ( 0.2
-6.7 ( 0.3
-5.8 ( 0.2
-6.3 ( 0.1
-5.3 ( 0.04
> -5
-8.5 ( 0.03c
-8.4 ( 0.1
-8.0 ( 0.08
-7.5 ( 0.2
-6.0 ( 0.09
> -6
-6.7 ( 0.1
> -6
-6.5 ( 0.3
> -6
> -6
> -6
> -6
-5.8 ( 0.2
-6.6 ( 0.1
-5.4 ( 0.1
-5.3 ( 0.01
-8.2 ( 0.04
-8.1 ( 0.3
a IC50 values are reported as the log of the molar concentration of sensitive breast (MCF-7), resistant breast (MCF-7/Adr), and liver (SK-HEP-1 and Hep
G2) cancer cells, as well as noncancerous rat cardiomyocytes (H9c2(2-1)) and green monkey kidney cells (Vero) with 3 and/or 24 h drug treatments with
Doxaz alkyl carbamates (1, 2, and 3) versus treatment with Dox or Doxaz. Viability of cells was assayed with crystal violet, except viability of H9c2(2-1)
cells treated for 3 h and of Vero cells treated for 3 or 24 h, which were assayed with MTT. b Reference 1. c Reference 2.
Increased hydrophobicity may also be the explanation for the
effect of serum on the uptake of the carbamate; namely, the
carbamate binds to serum proteins, reducing drug uptake.
Synthesis and Characterization of Doxazolidine Carbam-
ates with Self-Eliminating Spacer. The growth inhibition data
in Table 1 together with our experience and that of others with
prodrugs suggested drug efficacy might be improved by adding
a self-eliminating spacer between the alkyl carbamate function-
ality and the anthracycline.17-19 This could present the lipophilic
carbamate to the enzyme with significantly less steric bulk and
increase the rate of enzymatic hydrolysis. A Katzenellenbogen-
type spacer from p-aminobenzyl alcohol (PABA) often works
well in this capacity.20 The spacer was incorporated by reacting
PABA with the desired chloroformate, followed by conversion
to the p-nitrophenyl carbonate ester and reaction with Doxaz,
as shown in Scheme 3. The structures again were established
supplied in the Supporting Information, and chemical shift
assignments for all the protons and carbons at 55 °C are reported
in the Experimental Section, with the numbering scheme given
in Chart 2. Proton and carbon chemical shifts were established
from the combination of high-resolution 1D proton, homonuclear
COSY, HSQC, and HMBC spectra. Activation of these car-
bamates is predicted to occur by enzymatic hydrolysis of the
alkyl carbamate, followed by rapid decarboxylation at the anilino
carbamic acid and 1,6-elimination of imino quinone methide,
as shown in Scheme 4. A second spontaneous decarboxylation
step releases Doxaz.
Biological Activity of Doxazolidine Carbamates with Self-
Eliminating Spacer. Growth inhibition experiments with butyl
and pentyl carbamates bearing the PABA self-eliminating spacer
(compounds 4 and 5) focused on Hep G2 liver cancer cells that
strongly express both CES1 and CES2 and SK HEP-1 cells that
express significantly more CES2 than CES1. SHP-77 small cell
lung cancer cells were also of interest because they express
modest amounts of both enzymes. Inhibition of the growth of
cardiomyocytes was used as a measure of cardiotoxicity, and
inhibition of the growth of Vero cells, green monkey kidney
1
from NMR spectra and mass spectral molecular ions. The H
NMR spectra at ambient and elevated temperatures also showed
evidence of conformational dynamics. Spectra of the pentyl
PABC-Doxaz, pentyl 4-(N-doxazolidinylcarbonyloxymethyl)-
phenylcarbamate (5), at ambient and elevated temperature, are

Doxazolidine Carbamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7007
Scheme 3. Synthesis of Doxaz Carbamates 4 and 5 with Self-Eliminating PABC Spacer
Chart 2. Numbering Scheme for Doxaz Alkyl Carbamates and Alkyl PABC-Doxaz Derivatives for the Assignment of Proton and
Carbon Chemical Shifts in the Experimental Section
Scheme 4. Proposed Enzymatic Activation of Alkyl PABC-Doxaz Prodrugs (4 and 5)
cells, was again used as another measure of toxicity to normal
cells. Clearly, both the butyl and the pentyl carbamates with
PABA spacer are more active than the simple carbamates, as
shown in Table 2. They also show more activity against the
cancer cells that express higher levels of CES1. Of particular
note is the higher activity against Hep G2 cells than against
SK-HEP-1 cells. This suggests that CES1 is more active than
CES2. Pentyl PABC-Doxaz (5) also shows more than 1 order
of magnitude lower toxicity to cardiomyocytes and Vero cells
than to Hep G2 and SHP-77 cells. The low toxicity of pentyl
PABC-Doxaz to cardiomyocytes relative to the toxicity of Dox
is encouraging and consistent with the low level of expression
of CES1 in cardiomyocytes, as shown in Figure 3.
Uptake of pentyl PABC-Doxaz (5) and Dox by multidrug

7008 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Burkhart et al.
Figure 5. Uptake of Dox or pentyl PABC-Doxaz (5) by MCF-7/Adr breast and SK-HEP-1 liver cancer cells, as determined by flow cytometry,
measuring relative fluorescence of the Dox fluorophore as a function of treatment time in RPMI media containing 10% fetal bovine serum. Drug
concentration in media was 0.5 µM.
Table 2. Cell Growth Inhibition Values (IC50) Expressed in Log of Molar Concentrations for Doxaz Alkyl Carbamates (2 and 3) and Alkyl
PABC-Doxaz Compounds (4 and 5) Compared with Dox for 24 h Drug Treatmenta
2
4
3
5
Doxaz
butyl
Doxaz
pentyl
drug/cell line
Dox
butyl carbamate
PABC-Doxaz
pentyl carbamate
PABC-Doxaz
MCF-7
-7.9 ( 0.04
-5.6 ( 0.1
-7.5 ( 0.08
-6.9 ( 0.05
-7.5 ( 0.1
-7.3 ( 0.2
-7.7 ( 0.3
-6.3 ( 0.08
-6.1 ( 0.04
-6.2 ( 0.3
-6.4 ( 0.02
-6.6 ( 0.3
-6.7 ( 0.3
-6.4 ( 0.2
-5.8 ( 0.2
-6.3 ( 0.1
-4.1 ( 0.3
-5.9 ( 0.06
-6.3 ( 0.3
-7.1 ( 0.1
-7.7 ( 0.03
-5.8 ( 0.04
-7.2 ( 0.2
-6.1 ( 0.08
-6.1 ( 0.03
-6.0 ( 0.09
-6.0 ( 0.04
-6.3 ( 0.3
-6.5 ( 0.3
-6.7 ( 0.1
-6.6 ( 0.1
-5.3 ( 0.01
-5.6 ( 0.3
-6.5 ( 0.06
-6.7 ( 0.07
-7.3 ( 0.1
-7.3 ( 0.2
-6.0 ( 0.06
-6.2 ( 0.2
-6.0 ( 0.04
MCF-7/Adr
DU-145
SHP-77
HepG2
SK-HEP-1
H9c2(2-1)
Vero
a Cancer cells are defined in the legends to Table 1 and Figure 3. Growth inhibition of cardiomyocytes (H9c2(2-1)) is a measure of cardiotoxicity, and
growth inhibition of Vero cells (green monkey kidney cells) is a second measure of toxicity to normal cells. Viability of cells was assayed with crystal violet,
except the viability of Vero cells which were assayed with MTT.
resistant MCF-7/Adr breast cancer cells and SK-HEP-1 liver
cancer cells in the presence of fetal bovine serum is compared
in Figure 5, as determined by flow cytometry using drug
fluorescence as a measure of the drug in cells. Pentyl PABC-
Doxaz is taken up much better by both cell lines than Dox.
Better uptake probably results from increased hydrophobicity
and the absence of positive charge; Dox is a cation at
physiological pH, whereas the pentyl PABC-Doxaz is un-
charged. Release of pentyl PABC-Doxaz and Dox from MCF-
7/Adr and SK-HEP-1 cells was also measured by flow cytom-
etry, measuring drug fluorescence as a function of time after 3
h drug treatment, as shown in Figure 6. The Dox fluorophore
is released from both cell lines treated with pentyl PABC-Doxaz
at approximately an equal rate, with apparent faster and then
slower first-order kinetics. The half-lives are 0.3 and 30 h,
respectively. Disappearance of the Dox fluorophore from Dox-
treated cells is faster and appears to follow simple first-order
kinetics from MCF-7/Adr cells with a half-life of approximately
0.1 h. With Dox-treated SK-HEP-1 cells, the initial release is
similar but appears to plateau after the faster release, as observed
with pentyl PABC-Doxaz-treated cells, but at an order of
magnitude lower level. MCF-7/Adr cells are known to over-
express P-170 glycoprotein efflux pump. Substrates for this
efflux pump commonly bear hydrophobic regions as well as a
positive charge, characteristic of Dox but not of pentyl PABC-
Doxaz.21 So, we conclude that pentyl PABC-Doxaz is taken up
better and retained better than Dox in SK-HEP-1 liver cancer
cells and the MDR expressing MCF-7/Adr cancer cells, mostly
because of its increased hydrophobicity.
The cell growth inhibition data together with the mRNA
expression data implicate CES1 as the primary enzyme respon-
sible for activation of Doxaz carbamates, and this activation
likely occurs inside cells where the enzyme resides. Further
evidence comes from measurement of the lack of hydrolysis of
the prodrug in phosphate-buffered saline (PBS) or phosphate-
buffered fetal bovine serum (FBS). HPLC analysis of the
hydrolysis reactions after 24 h at 37 °C showed an intact drug,
no formation of doxorubicin, and no formation of the product
from loss of formaldehyde from the carbamate, namely, the
pentyl carbamate of PABC-Dox. The fraction of carbamates
hydrolyzed in Hep G2 liver cancer cells is still estimated to be
low (in the range of 10% over 24 h), because the IC50 values
are high relative to the values for Doxaz. Similar inefficiency
is observed for carboxylesterase hydrolysis of capecitabine and
irinotecan, and inefficient hydrolysis is a desirable property for
a carboxylesterase-activated prodrug to minimize side effects.22
The hydrolysis experiment required a higher concentration
of the pentyl PABC-Doxaz (5), 50 µM, than the cell experiments
because of the sensitivity of the HPLC analytical method. This
higher concentration necessitated a method of formulation,
because pentyl PABC-Doxaz is not soluble in PBS or FBS at
50 µM concentration. Several excipients were explored, includ-
ing various cyclodextrins, cremophor ELP, Tween 80, and
bovine albumin in combination with DMSO. Cremophor ELP/

Doxazolidine Carbamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7009
cytes and Vero cells than doxorubicin. In fact, doxorubicin is
more toxic to cardiomyocytes than to Hep G2 cells, but 5 is at
least 10-fold more toxic to Hep G2 cells than to cardiomyocytes.
These properties together with the favorable drug uptake and
retention by the MDR expressing MCF-7/Adr cells and stability
in plasma indicate that 5 should be explored further using in
vivo models of liver cancer or other cancers whose cells
overexpress CES1 and CES2 or possibly other carboxylesterase
enzymes. Primary liver cancer, or hepatocellular carcinoma
(HCC), is diagnosed in over 14 000 patients per year and leads
to approximately 9500 deaths in the United States each year.24
Despite its low incidence in western society, HCC is almost
invariably fatal, and it is the third highest cancer-related cause
of death in men and the seventh in women worldwide.25-27
Further, its incidence in the U.S. is predicted to accelerate in
the next two to three decades.28
Figure 6. Release of Dox or pentyl PABC-Doxaz (5) by MCF-7/Adr
breast and SK-HEP-1 liver cancer cells, as determined by flow
cytometry, measuring relative fluorescence of the Dox fluorophore as
a function of time after 0.5 µM drug treatment for 3 h in RPMI media
containing 10% fetal bovine serum. The y-scale was expanded at low
fluorescence to allow presentation of data for both drugs in the same
graph. Release of pentyl PABC-Doxaz was fit with a double exponential
rate law, giving half-lives of 3 × 10-1 h and 3 × 101 h for both cell
lines with R2 ) 0.9998 with SK-HEP-1 cells and 0.9769 with MCF-
7/Adr cells. Insufficient signal was obtained for similar analysis of Dox
release, but the half-life for the faster release was approximately 1 ×
10-1 h.
Experimental Section
1. General Remarks. Doxorubicin hydrochloride clinical samples
(formulated with lactose) were received as a gift from FeRx (Aurora,
CO). Pharmaceutical grade Tween 80 was a gift from Tapestry
Pharmaceuticals, Inc., Boulder, CO. Doxazolidine was synthesized
as previously described.2 DMSO and chloroform were prepared by
addition of activated 4 Å molecular sieves for at least 48 h prior to
use. Dichloromethane was distilled from calcium hydride prior to
use. NMR solvents were purchased from Cambridge Isotope
Laboratories, Inc (Andover, MA). All other chemicals were
purchased from Aldrich (Milwaukee, WI) and used without further
purification. Analytical HPLC was performed on a Hewlett-Packard/
Agilent 1050/1100 chromatograph equipped with auto injector,
diode array UV-vis absorption detector, and fluorescence detector
interfaced to an Agilent ChemStation data system (Palo Alto, CA).
Analytical HPLC injections were onto an Agilent Zorbax 5 µm
reverse-phase octadecylsilyl (ODS) column, 4.6 mm i.d. × 150 mm,
eluting at 1.0 mL/min with method 1, a gradient of acetonitrile/20
mM triethylammonium acetate buffer, pH 7.4 (80% buffer to 70%
buffer at 5 min to 30% buffer at 15 min, then isocratic to 25 min)
or with method 2, a gradient of acetonitrile/20 mM triethylammo-
nium acetate buffer, pH 7.6 (60% buffer to 0% buffer at 10 min,
isocratic until 12 min, and to 60% buffer at 15 min. The eluent
was monitored for absorption at 280 and 480 nm and/or for
fluorescence at 550 nm with excitation at 480 nm. All NMR spectra
were performed using Varian Unity INOVA 400 and 500 MHz
spectrometers (Palo Alto, CA). All spin simulations and dynamic
NMR simulations were performed using SpinWorks 2.4 software
by Kirk Marat (SpinWorks software is available at ftp://davin-
ci.chem.umanitoba.ca/pub/marat/SpinWorks). Sample temperatures
for the dynamic NMR experiments were calibrated using the
separation between hydroxyl resonance and CHn resonances in
methanol (below ambient) and ethylene glycol (above ambient).
Standard calibration formulas included in the Varian VNMR 6.1C
software were used to calculate the sample temperature. Electro-
spray mass spectra were measured with Sciex API III+ (now
Toronto, Canada) or ABI Pulsar QqT of high resolution instrument
(Foster City, CA), equipped with an ion-spray source, at atmo-
spheric pressure. UV-vis spectrometry was performed with a
Hewlett-Packard/Agilent 8452A diode array spectrophotometer
interfaced to an Agilent ChemStation data system (Palo Alto, CA).
Preparative scale purification was performed by radial chromatog-
raphy with a Harrison Research Model 7924T Chromatotron (Palo
Alto, CA). Human plasma, pooled from 40 normal donors ages 16
to 65, was a gift from SomaLogic, Inc., Boulder, CO. 96-Well cell
culture plates were read using a PowerWave X plate reader from
BIO-TEK Instruments, Inc. (Winooski, VT), using their Kineticalc
software. Flow cytometry was performed with a Becton Dickinson
Biosciences FACScan (San Jose, CA) flow cytometer, using Becton
Dickinson Biosciences CellQuest software. All tissue culture
materials were obtained from Gibco Life Technologies (Grand
Island, NY) unless otherwise noted. Cancer cells were obtained
DMSO, polysorbate 80 (Tween 80)/DMSO, and bovine albumin/
DMSO emerged as successful candidates. Pentyl PABC-Doxaz
is completely soluble at 50 µM when a 100× solution in 1:1
DMSO/cremaphor ELP (v/v) or 1:1 DMSO/Tween 80 (v/v) is
diluted 100-fold with either PBS or FBS. Bovine albumin/
DMSO was also successful, except a small fraction of the red
color from the drug appeared at the bottom of the Eppendorf
tube upon centrifugation of the solution at 16 600 × g. Any of
these formulations should be suitable for future iv exploratory
animal experiments. Cremophor ELP is used in the formulation
of paclitaxel, and Tween 80 is used in the formulation of
docetaxel, but not without some side effects with each excipi-
ent.23
As a further predictor of survival of the pentyl PABC-Doxaz
in the vascular system, prodrug concentration was measured
after 24 h at 37 °C in pooled human plasma. Pentyl PABC-
Doxaz was quantitated in three separate experiments at times 0
and 24 h by HPLC, detecting prodrug both by absorption at
480 nm and by fluorescence at 550 nm. The average survival
of prodrug over all six measurements was 82 ( 8%.
Conclusions
The very active but hydrolytically unstable derivative of
doxorubicin, doxazolidine, is stabilized to hydrolysis by car-
bamylation of its oxazolidine nitrogen. The ethyl carbamate is
inactive, and the butyl and pentyl carbamates show modest
inhibition of the growth of some cancer cell lines. RT-PCR
measurements suggest both carboxylesterase CES1 and CES2
as possible activating enzymes. More favorable cancer cell
growth inhibition was achieved by including a PABA self-
eliminating spacer in the design to separate the alkyl carbamate
functional group from the bulky anthracycline. The lead
compound, pentyl PABC-Doxaz (5), shows similar growth
inhibition of Hep G2 primary liver cancer cells and better growth
inhibition of SHP-77 small cell lung cancer cells than doxoru-
bicin. Further, pentyl PABC-Doxaz shows better drug uptake
and retention and much lower growth inhibition of cardiomyo-

7010 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Burkhart et al.
from American Type Culture Collection (Rockville, MD), except
as noted in the acknowledgments.
8 Hz, 1). ESI-MS m/z observed, 729.2 (M + HN+Et3); calculated,
729.4 (M + HN+Et3).
3.2. N-(Butyloxycarbonyl)doxazolidine (Doxaz Butyl Car-
2. General Procedure for the Synthesis of p-Nitrophenyl
Carbonates of p-Aminobenzyl Alcohol Alkyl Carbamates.
p-Aminobenzyl alcohol (PABA, 1.0 mmol) was added to a dry 25
mL round-bottom flask equipped with a magnetic stir bar. Dry
methylene chloride (5 mL) was added under an argon atmosphere.
4-Diisopropylethylamine (1.5 equiv) was added to the flask with
stirring, followed by 1.1 equiv of the desired alkyl chloroformate
by dry syringe addition. The reaction was monitored by HPLC and
run until complete (typically 3 h). An additional 1.5 equiv of
diisopropylethylamine was added, followed by 1.5 equiv of
p-nitrophenyl chloroformate. The reaction was monitored by HPLC
and run until complete (typically 24-48 h). After rotary evapora-
tion, the product was purified by radial chromatography of the crude
reaction mixture on a 2-mm silica gel Chromatotron plate, using a
30:1 chloroform/methanol eluent. p-Nitrophenyl carbonates of
p-aminobenzyl alcohol alkyl carbamates, pure as indicated by HPLC
1
bamate, 2). H NMR (ambient temperature, CDCl3): δ 0.91 (b,
3H, δ-CH3), 1.34 (d, 3H, J ) 7 Hz, 5′-Me), 1.34 (b, 2H, γ-CH2),
1.60 (p, 2H, J ) 7 Hz, â-CH2), 1.74 (bd, 1H, J ) 15 Hz, 2′), 2.13
(dd, 1H, J ) 4, 14 Hz, 8), 2.25 (br m, 1H, 2′), 2.42 (br d, 1H, J )
14 Hz, 8), 3.99 (t, 1H, J ) 5 Hz, 14-OH), 3.01 (d, J ) 18 Hz, 10),
3.25 (dd, 1H, J ) 18, 2 Hz, 10), 4.04 (m, 1H, 4′), 4.07 (s, 3H,
4-OCH3), 4.09 (m, 4, 3′, 5′, R-CH2), 4.74 (d, 2H, J ) 5 Hz, 14),
4.76 (s, 1H, 9-OH), 4.85-5.06 (br m, 2H, O-CH2-N), 5.30 (br s,
1H, 7), 5.45 (t, 1H, J ) 5 Hz, 1′), 7.38 (d, 1H, J ) 8 Hz, 3), 7.77
(t, 1H, J ) 8 Hz, 2), 8.02 (d, 1H, J ) 8 Hz, 1); ESI-MS m/z
observed, 678.2175; calculated, 678.2157 (M + Na+).
3.3. N-(Pentyloxycarbonyl)doxazolidine (Doxaz Pentyl Car-
1
bamate, 3). H NMR (ambient temperature, CDCl3): δ 0.8-0.9
(m, 3H, ꢀ-CH3), 1.24-1.38 (m, 4H, γ-CH2 and δ-CH2), 1.35 (d,
3H, J ) 6 Hz, 5′-Me), 1.62 (bt, J ) 7 Hz, 2H, â-CH2), 1.74 (dt,
1H, J ) 15, 5 Hz, 2′), 2.13 (dd, 1H, J ) 15, 5 Hz, 8), 2.2-2.3 (br
m, 1H, 2′), 2.42 (br d, 1H, J ) 15 Hz, 8), 2.99 (t, 1H, J ) 5 Hz,
14-OH), 3.00 (d, 1H, J ) 19 Hz, 10), 3.25 (dd, 1H, J ) 19, 2 Hz,
10), 4.03 (m, 1H, 4′), 4.07 (s, 3H, 4-OCH3), 4.09 (m, 4H, R-CH2,
3′ and 5′), 4.74 (d, 2H, 5 Hz, 14), 4.76 (s, 1H, 9-OH), 4.85-5.1
(m, 2H, O-CH2-N), 5.30 (m, 1H, 7), 5.45 (t, 1H, J ) 5 Hz, 1′),
7.38 (d, 1H, J ) 8 Hz, 3), 7.77 (t, 1H, J ) 8 Hz, 2), 8.02 (d, 1H,
J ) 8 Hz, 1). ESI-MS m/z observed, 771.2 (M + HN+Et3);
calculated, 771.4 (M + HN+Et3).
1
(method 1) and H NMR, were obtained as yellow solids in 43-
65% yield over two steps.
2.1. p-Nitrophenyl Carbonate of Butyl p-(Hydroxymethyl)-
1
phenylcarbamate. H NMR (CDCl3): δ 0.98 (t, 3H, J ) 8 Hz,
CH3), 1.46 (sex, 2H, J ) 8 Hz, CH2), 1.69 (pent, 2H, J ) 7.5 Hz,
CH2), 4.21 (t, 2H, J ) 7 Hz, OCH2), 5.27 (s, 2H, benzyl CH2),
6.89 (s, 1H, NH), 7.39-7.45 (m, 6H, benzyl and aromatic adjacent
to carbonate), 8.30 (d, 2H, J ) 10 Hz, aromatic adjacent to nitro).
ESI-MS m/z observed, 490.6 (M + HN+Et3); calculated, 490.3 (M
+ HN+Et3).
3.4. Butyl 4-(N-Doxazolidinylcarbonyoxymethyl)phenyl-
carbamate (Butyl PABC-Doxaz, 4). 1H NMR (assignments at 55
°C from 1D proton and homonuclear COSY spectra; see Chart 2
for the numbering scheme; CDCl3) δ 0.92 (t, 3H, J ) 7 Hz, δ-CH3),
1.33 (d, 3H, J ) 6 Hz, 5′-Me), 1.38 (p, 2H, J ) 7 Hz, δ-CH2),
1.61 (p, 2H, J ) 7 Hz, â-CH2), 1.70 (dt, 1H, J ) 15, 6 Hz, 2′),
2.12 (dd, 1H, J ) 15, 4 Hz, 8), 2.26 (br m, 1H, 2′), 2.44 (br d, 1H,
J ) 15 Hz, 8), 2.91 (t, 1H, J ) 5 Hz, 14-OH), 3.02 (d, 1H, J ) 19
Hz, 10), 3.25 (dd, 1H, J ) 19, 2 Hz, 10), 4.04 (dd, 1H, J ) 7, 2
Hz, 4′), 4.08 (s, 3H, 4-OMe), 4.1-4.15 (m, 2H, 3′ and 5′), 4.12 (t,
1H, J ) 7 Hz, R-CH2), 4.66 (s, 1H, 9-OH), 4.72 (d, 2H, J ) 5 Hz,
14), 4.92 (d, 1H, J ) 4 Hz, O-CH2-N), 5.01 (b, 1H, O-CH2-
N), 5.05 (b, 1H, Bn), 5.15 (b, 1H, Bn), 5.26 (br s, 1H, 7), 5.38 (t,
1H, J ) 6 Hz, 1′), 6.84 (br s, 1H, NH), 7.29 (d, 2H, J ) 8 Hz,
PABC, 3′′), 7.38 (d, 1H, J ) 8 Hz, 3), 7.39 (d, 2H, J ) 8 Hz,
PABC 2′′), 7.76 (t, 1H, J ) 8 Hz, 2), 8.03 (d, 1H, J ) 8 Hz, 1),
13.18 (s, 1H, phenolic-OH), 13.78 ppm (s, 1H, phenolic-OH). ESI-
MS m/z observed, 906.8 (M + HN+Et3); calculated, 906.6 (M +
HN+Et3). ESI-MS exact mass measurement with buffer-free sample
showed m/z ) 827.2616 (M + Na+); calculated, 827.2633 (M +
Na+).
2.2. p-Nitrophenyl Carbonate of Pentyl p-(Hydroxymethyl)-
phenylcarbamate. 1H NMR (CDCl3): δ 0.95 (m, 3H, CH3), 1.38
(m, 4H, CH2CH2), 1.71 (m, 2H, CH2), 4.20 (t, 2H, J ) 7 Hz, OCH2),
5.27 (s, 2H, benzyl CH2), 6.70 (s, 1H, NH), 7.39-7.42 (m, 6H,
benzyl and aromatic adjacent to carbonate), 8.29 (d, 2H, J ) 8 Hz,
aromatic adjacent to nitro). ESI-MS m/z observed, 504.4 (M + HN+-
Et3); calculated, 504.3 (M + HN+Et3).
3. General Procedure for the Synthesis of Doxazolidine
Carbamates. Either doxazolidine or doxoform, or a mixture of the
two, may be used for this synthesis, but the stoichiometry must be
corrected if doxoform is present because it is dimeric in doxazo-
lidine. The syntheses of doxoform and doxazolidine were described
earlier.2 For the synthesis of carbamates without PABA spacer,
doxazolidine (31 mg, 0.056 mmol) was added to a dry 25 mL round-
bottom flask equipped with a magnetic stir bar. Dry chloroform (5
mL) was added under an argon atmosphere. 4-Dimethylaminopy-
ridine (DMAP, 1.5 equiv, 10.2 mg) was added to the flask with
stirring, followed by 0.90 equiv of the desired chloroformate by
dry syringe addition. The respective p-nitrophenyl carbonate can
be used in place of the chloroformate. With this reagent, DMAP is
not used. For the synthesis of PABC-Doxaz alkyl carbamates, 1.0
equiv of the respective p-nitrophenyl carbonate of the p-aminoben-
zyl alkyl carbamate was added to doxazolidine in 1.0 mL of dry
DMSO. The reaction was monitored by HPLC (Method 1) and run
until complete (typically 2 days). After high vacuum (2 × 10-2
Torr) rotary evaporation of the DMSO, the product was purified
by radial chromatography of the crude reaction mixture on a 2 mm
silica gel plate using a 30:1 chloroform/methanol eluent. Doxazo-
lidine carbamates, pure as indicated by HPLC and 1H NMR, were
obtained as red solids in 51-83% yield, based on chloroformate
or p-nitrophenyl carbonate as the limiting reagent.
3.1. N-(Ethoxycarbonyl)doxazolidine (Doxaz Ethyl Carbam-
ate, 1). 1H NMR (CDCl3): δ 1.30 (t, 3H, J ) 7 Hz, â-CH3), 1.40
(d, 3H, J ) 6 Hz, 5′-Me), 1.80 (dt, 1H, J ) 4.5 and 16 Hz, 2′),
2.16 (dd, J ) 4 and 15 Hz, 8), 2.1-2.4 (bm, 1H, 2′), 2.45 (bd, 1H,
J ) 14 Hz, 8), 3.03 (t, 1H, J ) 5 Hz, 14-OH), 3.09 (dd, 1H, J )
1 and 19 Hz, 10), 3.33 (dd, 1H, J ) 1 and 19 Hz, 10), 4.07 (dd,
1H, J ) 2 and 7 Hz, 3′), 4.12 (s, 3H, 4-OCH3), 4.15 (dd, 1H, J )
1 and 7 Hz, 5′), 4.17 (m, 1H, 4′), 4.21 (q, 2H, J ) 7 Hz, R-CH2),
4.79 (d, 2H, J ) 5 Hz, 14), 4.81 (s, 1H, 9-OH), 4.9-5.1 (br m,
2H, OCH2N), 5.35 (m, 1H, 7), 5.50 (t, 1H, J ) 6 Hz, 1′), 7.44 (d,
1H, J ) 9 Hz, 3), 7.82 (t, 1H, J ) 8 Hz, 2), 8.08 ppm (d, 1H, J )
3.5. Pentyl 4-(N-Doxazolidinylcarbonyoxymethyl)phenyl-
1
carbamate (Pentyl PABC-Doxaz, 5). H NMR (assignments at
55 °C from 1D proton, homonuclear COSY, HSQC, and HMBC
spectra; see Chart 2 for the numbering scheme; CDCl3) δ 0.90 (t,
3H, J ) 7 Hz, ꢀ-CH3), 1.34 (m, 2H, γ-CH2 + δ-CH2), 1.36 (d, 3H,
J ) 6 Hz, 5′-Me), 1.66 (p, 2H, J ) 7 Hz, â-CH2), 1.72 (dt, 1H, J
) 14, 5 Hz, 2′), 2.14 (dd, 1H, J ) 16, 4 Hz, 8), 2.27 (br m, 1H,
2′), 2.46 (br d, 1H, J ) 14 Hz, 8), 2.96 (t, 1H, J ) 5 Hz, 14-OH),
3.00 (d, 1H, J ) 18 Hz, 10), 3.24 (dd, 1H, J ) 18, 2 Hz, 10), 4.06
(dd, 1H, J ) 5, 2 Hz, 4′), 4.07 (s, 3H, 4-OMe), 4.09 (m, 1H, 5′),
4.13 (t, 2H, J ) 7 Hz, R-CH2), 4.16 (m, 1H, 3′), 4.67 (s, 1H, 9-OH),
4.74 (d, 2H, J ) 5 Hz, 14), 4.94 (d, 1H, J ) 4 Hz, O-CH2-N),
5.03 (b, 1H, O-CH2-N), 5.06 (b, 1H, Bn), 5.16 (b, 1H, Bn), 5.26
(br s, 1H, 7), 5.40 (t, 1H, J ) 6 Hz, 1′), 6.89 (br s, 1H, NH), 7.30
(d, 2H, J ) 8 Hz, PABC, 3′′), 7.39 (d, 1H, J ) 8 Hz, 3), 7.41 (d,
2H, J ) 8 Hz, PABC 2′′), 7.76 (t, 1H, J ) 8 Hz, 2), 8.02 (d, 1H,
J ) 8 Hz, 1), 13.15 (s, 1H, 11-OH), 13.78 ppm (s, 1H, 6-OH). 13
C
NMR chemical shift assignments for protonated carbons at 55 °C
from proton assignments and HSQC spectra: δ 14.1 (ꢀ), 16.1 (5′-
Me), 22.4 (δ), 28.0 (γ), 28.9 (â), 29.6 (2′), 34.2 (10), 36.0 (8),
56.9 (4-OMe), 65.6 (R), 65.6 (14), 65.9 (5′), 67.1 (Bn), 69.4 (7),
78.1 (4′), 79.5 (O-CH2-N), 100.3 (1′), 119.0 (PABC 3′′), 119.0
(3), 120.1 (1), 129.5 (PABC 2′′), 135.9 (2); assignment of

Doxazolidine Carbamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7011
unprotonated carbons from the HMBC spectrum, 76.9 (9), 111.7
(10a), 112.5 (5a), 121.5 (12a), 131.6 (PABC, 1′′), 133.4 (11a), 133.9
(6a), 135.8 (4a), 138.4 (PABC 4′′), 153.9 (PABC CO), 157.1 (6),
161.3 (4), 187.1 (5), 187.2 (12), 213.9 ppm (13). ESI-MS m/z
observed, 920.6 (M + HN+Et3); calculated, 920.5 (M + HNEt3).
ESI-MS exact mass measurement with buffer-free sample showed
m/z ) 841.2765 (M + Na+); calculated, 841,2790 (M + Na+).
3.6. Hydrolytic Stability. Bovine albumin (Sigma, A-7030) was
used together with DMSO to solubilize the pentyl PABC-Doxaz
(5) in aqueous media at higher concentrations of prodrug. Bovine
albumin (45 mg) was dissolved in 500 µL of HPLC grade water
and centrifuged at 16 600 × g for 5 min. The upper 200 µL was
removed and used in the formulation. For the first solution, 10 µL
of 1 mM pentyl PABC-Doxaz in DMSO was mixed with 90 µL of
the bovine albumin solution, and then the mixture was diluted with
90 µL of D-PBS. For the second solution, 10 µL of 1 mM pentyl
PABC-Doxaz in DMSO was mixed with 50 µL of the bovine
albumin solution and then diluted with 50 µL of D-PBS and 80
µL of fetal bovine serum. The solutions were then 53 µM pentyl
carbamate. A portion of each solution (100 µL) was heated at 37
°C for a period of 24 h, and the remainder of each solution (90
µL) was stored at -80 °C. The heated and stored solutions were
then each diluted with an equal volume of absolute ethanol to
precipitate the proteins and then centrifuged at 16 600 × g for 5
min at ambient temperature. Hydrolysis was monitored by HPLC
using method 1, described under General Remarks. The chromato-
grams showed only pentyl PABC-Doxaz (retention time ) 20 min),
no hydrolysis to doxorubicin, and no loss of formaldehyde to give
the corresponding pentyl PABC-Dox. The retention time of the
pentyl PABC-Dox (16 min) was established with a sample formed
as a byproduct of the synthesis of pentyl PABC-Doxaz and
characterized by UV/vis and ESI-MS of a pure sample collected
from the HPLC.
3.7. Stability of Pentyl PABC-Doxaz in Human Plasma. An
aliquot of a 2 mM stock solution of pentyl PABC-Doxaz prodrug
in DMSO was diluted with an equal volume of pharmaceutical grade
Tween-80 and then further diluted to 50 µM with pooled serum
from 40 normal humans, ages 16-65 (Somalogic, Inc.). An aliquot
was removed as a 0 h time point, and the remaining solution was
incubated at 37 °C for 24 h, at which point another aliquot was
removed. Proteins present in the aliquots were precipitated by
dilution with an equivalent volume of absolute ethanol, followed
by centrifugation at 16 500 × g for 20 min at ambient temperature.
The supernatants were analyzed by HPLC, method 2, and the
reaction progress was observed by absorbance at 480 nm and
fluorescence at 550 nm. The reaction was performed in triplicate.
HPLC analysis indicated that, after 24 h, 85 ( 8% of pentyl PABC-
Doxaz remained intact, monitoring fluorescence at 550 nm and 79
( 8%, monitoring absorption at 480 nm with constant volume
injection. No identifiable hydrolysis products were observed in the
chromatograms.
media, and the cells were grown until the no-drug control lanes
reached ∼80% confluence (3-5 days). Cells were quantified by
measuring cellular metabolism of MTT, as indicated by optical
density at 554 nm or by uptake of crystal violet after fixing with
glutaraldehyde. Uptake of crystal violet was measured by optical
density at 588 nm after solublizing the dye in 70% ethanol. The
MTT and crystal violet assays are described in detail in the
literature.1,29-31 The results for the 3 h incubation showed that the
Doxaz carbamate prodrugs displayed detectable activity, but that
the conversion to the active form had not occurred to a significant
extent in 3 h. Therefore, the experiment was repeated as before,
but with a 24 h incubation time. Every experiment was performed
at least twice, and each condition of drug concentration was
performed six times.
4.2. Drug Uptake Measurements. To test the efficiency of the
cells for Doxaz carbamate uptake, both in the presence and in the
absence of fetal bovine serum, SK-HEP-1 cells in log phase growth
were dissociated with trypsin-EDTA, counted, suspended in media
at 6.7 × 104 cells/mL, plated into 6-well plates at 200 000 cells
per well, and allowed to attach overnight. The media was then
replaced with either fresh RPMI containing 10% fetal bovine serum
or serum-free RPMI. Either Dox or Doxaz carbamate was added
to the wells for a final concentration of 0.5 µM, and the cells were
incubated at 37 °C for five time points: 0.5, 1, 1.5, 2, and 3 h. The
cells at each time point were trypsinized and analyzed by FACS,
scanning for the presence of the doxorubicin fluorophore. The mean
fluorescence values were then divided by the untreated control
fluorescence values to calculate the relative fluorescence.
4.3. Drug Release Measurements. A total of 200 000 cells of
either SK-HEP-1 or MCF-7/Adr lines were seeded into 6-well plates
and allowed to adhere overnight in a humidified incubator at 37
°C. The media was then replaced with fresh, complete media
containing 1% DMSO and 500 nM of either pentyl PABC-Doxaz
or Dox. The cells were incubated at 37 °C in drug for 3 h, at which
point the media was changed to drug- and DMSO-free media and
incubated for time points of 0, 0.25, 0.5, 1, 2, or 3 h. All drug
additions and media changes were timed such that the final
incubation endpoints were reached simultaneously. The cells were
then washed in cold PBS, trypsinized, and resuspended in 1 mL of
ice-cold PBS and placed on ice. Observation of the Dox chro-
mophore (excitation at 480 nm and fluorescent emission at 550
nm) by flow cytometry was performed as described in the drug
uptake studies and compared to identical, untreated cells to establish
background cellular fluorescence levels. The data for release of
pentyl PABC-Doxaz were best fit to a double exponential rate law
and for release of Dox to a single-exponential rate law.
4.4. Measurement of mRNAs by RT-PCR Analysis. To
examine the mRNA expression of CES1 and CES2, RT-PCR was
utilized. Cells in log growth phase were trypsinized and counted,
and the mRNA was extracted from five million cells using the
RNAqueous-4PCR kit (Ambion, Austin, TX). Candidate primer
sequences were identified by Primer Express software (Applied
Biosystems, Foster City, CA). Primers were selected for the 5′,
middle, and 3′ regions of human or rat CES1 and CES2, and the
selected primer sets had Tm values between 55 and 60 °C. The
primer sequences given in Supporting Information were purchased
from IDT, Inc. (Coralville, IA). The RT-PCR reaction was
performed using the Access RT-PCR kit (Promega, Inc., Madison,
WI) and analyzed by agarose gel electrophoresis and ethidium
bromide staining.
4. Cell Experiments. The carcinoma cell lines SK-HEP-1, Hep
G2 (HCC), MCF-7, MCF-7/Adr (breast carcinoma), DU-145
(prostate carcinoma), and SHP-77 (small cell lung carcinoma) were
cultured in RPMI 1640 media containing 10% (v/v) fetal calf serum
and 1% (v/v) penicillin-streptomycin (100× solution from Gibco).
The noncancerous cell lines H9c2(2-1) (rat cardiomyocytes) and
Vero (green monkey kidney) were maintained in Dulbecco’s
modified Eagle media, supplemented with 10% (v/v) fetal calf
serum and 1% (v/v) penicillin-streptomycin. All cells were grown
in a humidified incubator at 37 °C under an atmosphere of 5%
CO2 and 95% air.
4.1. IC50 Measurements. Drug concentration in stock solutions
was established by optical density at 480 nm, assuming a molar
absorbtivity of 11 500 cm-1M-1 for the Dox chromophore. The
cells were seeded into 96-well plates at 1000 cells/well (500 cells/
well for H9c2(2-1) cells) and allowed to adhere overnight. The
media was then replaced with serum-free media, containing various
concentrations of Doxaz carbamate or Dox and placed into the
incubator. Initially, the cells were incubated with the drugs for 3
h. Following treatment, the media was replaced with complete, fresh
Acknowledgment. The authors thank the U.S. Army Prostate
Cancer Research Program (DAMD17-01-1-0046) and the
National Cancer Institute of the NIH (CA-92107) for financial
support, the National Science Foundation for help with the
purchase of NMR equipment (CHE-0131003), Stephane Houel
for exact mass measurements, William Wells for MCF-7/Adr
cells, Dan Chan for SHP-77 cells, Andrew Kraft for DU-145
cells, Tapestry Pharmaceuticals for Tween 80, and SomaLogic
for plasma.

7012 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
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Supporting Information Available: HPLC of Doxaz carbam-
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NMR spectra for Doxaz carbamates; Erying and Arrhenius plots
for the conformational change of Doxaz ethyl carbamate; and primer
sequences for RT-PCR. This material is available free of charge
via the Internet at http://pubs.acs.org.
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