Synthesis, pharmacokinetics, efficacy, and rat retinal toxicity of a novel mitomycin C-triamcinolone acetonide conjugate

Article abstract of DOI:10.1021/jm010511b

A novel conjugate of mitomycin C (MMC) and triamcinolone acetonide (TA) was synthesized using glutaric acid as a linker molecule. To determine the rate of hydrolysis, the conjugate was dissolved in aqueous solution and the rate of appearance of free MMC and TA was determined by high-performance liquid chromatography analysis. Antiproliferative activity of the MMC-TA conjugate and parent compounds was assessed using an NIH 3T3 fibroblast cell line. Cell growth was quantified using the MTT assay. Kinetic analysis of the hydrolysis rate demonstrated that the conjugate had a half-life of 23.6 h in aqueous solutions. The antiproliferative activities of the MMC-TA conjugate and MMC were both concentration dependent, with similar IC₅₀ values of 2.4 and 1.7 μM, respectively. However, individual responses at concentrations above 3 μM showed that the conjugate was less active than MMC alone. TA alone showed only limited inhibition of cell growth. Studies evaluating intravitreal injection of the conjugate demonstrate that this agent produced no measurable toxicity. Our data provide evidence that the MMC-TA conjugate could be used as a slow-release drug delivery system. This could in turn be used to modulate a posttreatment wound healing process or to treat various proliferative diseases.

Full text of DOI:10.1021/jm010511b

1
122
J . Med. Chem. 2002, 45, 1122-1127
Syn th esis, P h a r m a cok in etics, Effica cy, a n d Ra t Retin a l Toxicity of a Novel
Mitom ycin C-Tr ia m cin olon e Aceton id e Con ju ga te
Tamer A. Macky,†,‡,| Carsten Oelkers, Uwe Rix, Martin L. Heredia, Eva K u¨ nzel, Mark Wimberly,
§,‡
§
†
§
†
†
†
,§
Baerbel Rohrer, Craig E. Crosson, and J u¨ rgen Rohr*
Department of Ophthalmology and Department of Pharmaceutical Sciences, Medical University of South Carolina,
2
80 Calhoun Street, Charleston, South Carolina 29425-2301
Received November 5, 2001
A novel conjugate of mitomycin C (MMC) and triamcinolone acetonide (TA) was synthesized
using glutaric acid as a linker molecule. To determine the rate of hydrolysis, the conjugate
was dissolved in aqueous solution and the rate of appearance of free MMC and TA was
determined by high-performance liquid chromatography analysis. Antiproliferative activity of
the MMC-TA conjugate and parent compounds was assessed using an NIH 3T3 fibroblast
cell line. Cell growth was quantified using the MTT assay. Kinetic analysis of the hydrolysis
rate demonstrated that the conjugate had a half-life of 23.6 h in aqueous solutions. The
antiproliferative activities of the MMC-TA conjugate and MMC were both concentration
dependent, with similar IC50 values of 2.4 and 1.7 µM, respectively. However, individual
responses at concentrations above 3 µM showed that the conjugate was less active than MMC
alone. TA alone showed only limited inhibition of cell growth. Studies evaluating intravitreal
injection of the conjugate demonstrate that this agent produced no measurable toxicity. Our
data provide evidence that the MMC-TA conjugate could be used as a slow-release drug delivery
system. This could in turn be used to modulate a posttreatment wound healing process or to
treat various proliferative diseases.
In tr od u ction
retinopathy (PVR), which is scar tissue formation in
eyes with rhematogenous retinal detachment.1
0,11
Also,
Mitomycin C (1; MMC) is an antitumor antibiotic with
an unusual tetracyclic mitosan ring system, including
an aziridine ring, a pyrrolizidine ring, a pyrrolo-(1,2a)-
elevated levels of certain cytokines, such as tumor
1
2
13
necrosis factor-R (TNF-R) and interleukin-1 (IL-1),
1
-3
have been found in the vitreous of patients with
indole, and a substituted benzoquinone moiety.
Each
proliferative disorders. Glucocorticoids inhibit TNF-R
of these structural elements participates in the unique
mechanism-of-action of mitomycin, which is initiated by
a reduction of the benzoquinone system leading to a
quinone methide covalently binding to DNA and ulti-
mately causing DNA cross-linking and cell death. An
initial reduction reaction occurs preferentially in hy-
poxic cells, which is important for selective antitumor
1
4,15
and IL-1 expression
and can inhibit T-lymphocyte
1
6
proliferation. Despite the advantages of corticosteroids
in the suppression of the inflammatory arm of the
wound-healing response, they are limited in their clini-
cal effectiveness because of their weak antiproliferative
properties.
The efficacy of antimetabolites and corticosteroids is
limited by their short half-life in cavitary organs (e.g.,
vitreous cavity of the eye). A new slow-delivery system
toxicity since many tumors and proliferative disorders
are associated with hypoxia.3b In addition, mitomycin
can alkylate the DNA at the 2-amino group of guanine
nucleosides and cause single-strand breakage of DNA
as well as chromosomal breaks. MMC (1) is used in
combination with other antitumor drugs to treat pan-
creatic carcinoma as well as lymphatic leukemia.
addition, MMC has applications in ophthalmology, such
1
7
is discussed by Berger and colleagues, utilizing codrug
technology. They investigated the use of a 5-fluorouracil
and TA conjugate to inhibit experimental PVR in an
animal model. In our study, we have investigated a
conjugate consisting of MMC and TA. The MMC-TA
conjugate (5) was synthesized by linking TA and MMC
via a glutaric acid unit (Figure 1). In vivo, dissolution
of the conjugate followed by hydrolysis allows both
active components to be released in an equimolar ratio.
We describe the synthesis of the MMC-TA conjugate
4
-7
In
as postoperative treatment of the pterygium and ad-
junctive treatment in glaucoma surgery.8
Corticosteroids, such as triamcinolone acetonide (2;
TA), have only limited antiproliferative properties;
however, they may play an important role by preventing
the access of inflammatory mediators to a disease site.
T-lymphocytes have been, for example, identified in
excised membranes from cases of proliferative vitreo-
9
(5), its kinetics, in vitro efficacy, and toxicity to rat
retina.
Resu lts
Ch em istr y. The conjugate of MMC and TA was
*
To whom correspondence should be addressed. Tel.: (843)876-
synthesized in reasonable yields via the route shown
in Figure 1. This involved three steps: (i) the catalyzed
ketal formation of the secondary (16-OH) and tertiary
alcohol (17-OH) groups of triamcinolone with acetone;
(ii) the esterification of the primary alcohol (21-OH) of
5
091. Fax: (843)792-0759. E-mail rohrj@musc.edu.
†
Department of Ophthalmology.
These authors contributed equally to this work.
Department of Pharmaceutical Sciences.
New address: Department of Ophthalmology, Cairo University,
‡
§
|
Cairo, Egypt.
1
0.1021/jm010511b CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/05/2002

Mitomycin C-Triamcinolone Acetonide Conjugate
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1123
F igu r e 2. Hydrolysis of the MMC-TA conjugate in 0.1 mol/L
phosphate buffer (pH 7.4) and propylene glycol (1:1) is graphi-
cally presented.
F igu r e 1. MMC-TA (5) was synthesized from the reaction
of TA (2), the TA glutaric acid-linker molecule (4), and MMC
(1). For the hydrolysis studies, the MMC glutaric acid-linker
molecule (3) was synthesized from the reaction of MMC (1)
with glutaric acid.
the resulting TA with the glutaric acid linker using
glutaric anhydride and pyridine; and (iii) the amide
bond formation between the remaining free, activated
F igu r e 3. Dose response curve for MMC, TA, and the MMC-
TA conjugate, showing different percentages of fibroblast cell
growth inhibition (88, 25, and 67%, respectively) when com-
pared to the DMSO similarly treated control.
(with carbonyl diimidazole) carboxyl group of the glu-
taric acid linker and the secondary amine group of the
aziridine ring of MMC. The resulting MMC-TA conju-
gate (5) was characterized by mass spectrometry and
by 1D and 2D nuclear magnetic resonance (NMR)
spectroscopy. All obtained data, in particular NMR long-
range couplings (see Discussion), verified the conjugate’s
structure (5). For comparative reasons, in context with
the hydrolysis studies (see later), MMC-glutaric acid
amide (3), subsequently called MMC-linker complex,
was synthesized from MMC and glutaric anhydride.
Structure 3 was verified by its NMR data.
Ta ble 1. Antiproliferative Assay Results Showing the Maximal
Percentage of Fibroblast Cell Growth Inhibition (Response
Maximum) and the 50% Inhibitory Concentration (IC50) of
MMC, TA, MMC plus TA (1:1, Mixture), and the Conjugate
MMC-TA
IC50a
response maximum
(% inhibition)
(µM)
MMC
MMC-TA conjugate
TA
1.7
2.4
2.5
1.6
88
67
25
83
MMC+TA mixture
a
Concentration required to induce 50% of response maximum.
Kin etics a n d Hyd r olysis Stu d ies. In aqueous solu-
tions at pH 7.4, the MMC-TA conjugate was hydrolyzed
by cleavage of either the amide bond linking mitomycin
and position 5′ of the glutaric acid linker or the ester
bond linking TA to the 1′ position of the linker.
Therefore, four different compounds were released, (i)
MMC (1), (ii) TA (2), (iii) TA-linker complex (4), and
the evolution of TA and the TA-linker complex (9.73
and 5.71 min, respectively) confirmed the half-life of the
conjugate (data not shown).
An tip r olifer a tive Assa ys. Concentration-response
curves for MMC, TA, and the conjugate-induced inhibi-
tion of NIH 3T3 cell growth are shown in Figure 3. The
calculated response maximums for MMC and the con-
jugate were 88 and 67%, respectively. The IC50 (the
concentration required to induce 50% of the response
maximum) was calculated to be 1.7 and 2.4 µM for MMC
and the conjugate, respectively (Table 1). The addition
of unconjugated MMC and TA together resulted in an
inhibitory response similar to that observed with MMC
alone (Table 1). The addition of TA alone to the cells
produced only limited inhibition of cell growth (25%) at
the highest concentration tested.
(
iv) MMC-linker complex (3). Peaks for MMC, TA,
MMC-linker complex, TA-linker complex, and the
MMC-TA conjugate were identified by high-perfor-
mance liquid chromatography (HPLC) through direct
comparison with these molecules, which were available
from the synthesis outlined above (Figure 1).
Regression analysis of the disappearance of MMC-
TA conjugate in aqueous solution demonstrated that the
conjugate has a half-life of approximately 23.6 h (Figure
2
). Evolution of MMC and the MMC-linker complex
(retention times of 3.07 and 3.26 min, respectively) and

1
124 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Macky et al.
Discu ssion
Our goal in this study was to link MMC (1) and TA
2) through a C5 dicarboxylic acid moiety, in which the
(
linker is connected by an ester bond with the primary
alcohol function of TA and by an amide bond with the
aziridine nitrogen of MMC. Both bonds were anticipated
to hydrolyze in aqueous solution at physiological pH.
Because the MMC-TA conjugate is a lipophilic mol-
ecule, it was expected to dissolve slowly, releasing both
active ingredients of this codrug (MMC (1) and TA (2))
over a period of time. The C5 linker was chosen because
it is long enough to avoid sterical hindrance between
the two principal building blocks. The glutaric acid
linker was first connected to TA. To avoid a reaction of
the secondary alcohol group in the 11-position with an
excess of glutaric anhydride, only 1.1 equiv of it (relative
to TA) was used. The final conversion of the TA linker
to the MMC-TA conjugate (5) was easily accomplished
by the addition of 3-fold excess of TA linker to MMC.
The overall yield (51%) of MMC-TA (relative to MMC)
was acceptable, and the procedure was not further
optimized. The alternative strategy of attaching the
glutaric linker first to MMC was less effective, produc-
ing low yields of the MMC-linker molecule (3) needed
for the hydrolysis studies.
F igu r e 4. Average amplitudes of the electroretinogram a- and
b-waves before (baseline) and 5 (3 rats) and 20 days (5 rats)
postintravitreal injection of the MMC-TA conjugate (right eye)
and the DMSO control (left eye).
The conjugation of MMC and TA was confirmed by
mass spectrometry and by 1D and 2D NMR spectros-
copy. The positive electrospray ionization (ESI) mass
spectrum revealed the molecular ion at m/z 887 [MH
+
+
Na] , which confirmed the molecular formula of
C44H53N4O13F. The proton NMR spectrum showed all
necessary signals, most of which were assigned with the
help of the HSQC, HMBC, and H,H-correlation spec-
1
3
troscopy spectra. Also, the C NMR spectrum showed
all expected signals, which were assigned through the
J C-H and J C-H couplings observed in the HSQC and
HMBC spectra. In the HMBC spectrum couplings
between C-5′ of the linker and the protons attached to
C-1′′ and C-2′′ of the mitomycin moiety on one hand and
between 21-H2 and C-1′ on the other hand clearly
indicated that the linkage of the two compounds oc-
curred in the desired fashion.
F igu r e 5. Histopathological sections of the neurosensory
retina stained with hematoxylin and eosin (×40 magnifica-
tion). (A) Left eye of control (R06) at day 5 postintravitreal
injection of 2 µL, 99.9% DMSO; and (B) right eye of same rat
at day 5 postintravitreal injection of 2 µL 0.39 mg/mL of
MMC-TA conjugate. Legend: NFL, nerve fiber layer; GCL,
ganglion cell layer; IP, inner plexiform layer; IN, inner nuclear
cell layer; OP, outer plexiform layer; ON, outer nuclear cell
layer; PhR, photoreceptor cell layer.
1
n
The conjugate of MMC and TA was hydrolyzed by
cleavage of either the amide bond linking mitomycin and
position 1′ of the glutaric moiety of the conjugate or the
ester bond linking TA and position 1′ of the glutaric
moiety of the MMC-TA conjugate (Figure 1). As a
result, five compounds were identified in these studies:
conjugate, TA linker, MMC linker, TA, and MMC.
Analysis of the area under the curve (AUC)-time plots
demonstrated that the evolution of TA-linker complex
occurred faster than the evolution of TA indicating that
the amide bond between mitomycin and position 5′ of
the linker was more easily cleaved than the ester bond
between TA and position 1′ of the linker. Normally,
amide bonds are more stabile than ester bonds; how-
ever, this is not true for amide bonds whose nitrogen is
part of an aziridine ring. Because of the strain on the
aziridine ring, the free electron pair of the aziridine
nitrogen cannot delocalize freely with the 5′-carbonyl
and thus cannot contribute to a +M effect, which would
normally decrease the reactivity of the amide C-5′
In Vivo Toxicity. Electr or etin ogr a m . Rats were
injected intravitreally (2 µL) with 0.784 µg of conjugate
or vehicle, and ERG a- and b-wave amplitudes were
analyzed (Figure 4). Eyes that received intravitreal
injections of the MMC-TA conjugate (right eyes) were
compared to those that received dimethyl sulfoxide
(DMSO) only (control, left eyes). There were no statisti-
cally significant differences between the amplitudes of
the a- and b-waves at 5 or 20 days posttreatment. The
slight reduction in ERG amplitudes observed at 5 days
after treatment was attributed to the injection itself,
as it affected both eyes equally.
Histop a th ology. The retina, by light microscopic
examination, was normal in appearance at 5 and 20
days following the intravitreal injection of the MMC-
TA conjugate, when compared to the DMSO control
(Figure 5). No signs of retinal necrosis, photoreceptor
cell loss, cystic degeneration, inflammatory cell infiltra-
tion, or hypocellularity of nuclear layers were observed
microscopically in any rats.
1
8
carbonyl.

Mitomycin C-Triamcinolone Acetonide Conjugate
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1125
Once most of the conjugate had been hydrolyzed, the
amount of TA linker decreased slightly due to cleavage
of the ester bond between the TA and the linker. This
is in agreement with the observed steady increase in
the concentration of TA, even after most of the original
TA-MMC conjugate had been hydrolyzed. The cleavage
of this ester bond, however, appeared to be slower once
the amide bond had been broken, since changes in the
amounts of the compounds were only slight.
found to be effective in treating experimental prolifera-
1
9-21
tive ocular diseases,
thus taking advantage of the
best characteristics of these two agents while minimiz-
ing their individual liabilities. The drugs become rela-
tively insoluble when linked together. Hydrolysis of the
conjugate allows the MMC and TA to be released slowly
in equimolar concentrations. Ideally, a sustained drug
delivery system should release the drug over a pro-
longed period of time, corresponding to the duration of
disease activity. Our data suggest that the MMC-TA
conjugate pharmacokinetics profile would be advanta-
geous in the treatment of proliferative diseases affecting
cavitary organs, where the short half-life of these agents
has limited their clinical use.
Similar quantitative observations could not be made
for MMC and the mitomycin linker, since we were
unable to completely separate their peaks by HPLC. The
AUC of the combined peaks was fairly constant, once
most of the conjugate had been hydrolyzed; thus, one
can assume that mitomycin was reasonably stable under
these conditions (aside from the conversion of mitomy-
cin-linker complex to mitomycin). A control study
supported this view, in which pure mitomycin was
dissolved in phosphate buffer and propylene glycol to
analyze the behavior of the compound under the same
conditions as those used in the kinetic assay. Samples
were taken every 24 h and analyzed by HPLC showing
that no additional peaks (in addition to the mitomycin
peak) appeared and the AUC of the single peak was
fairly constant.
Exp er im en ta l Section
TA (2).22 Triamcinolone, 800 mg (2.03 mmol), was sus-
pended in 200 mL of acetone, and 20 drops of concentrated
hydrochloric acid were added. The mixture was stirred under
reflux for 1 h, during which triamcinolone was completely
dissolved. The solution was stirred at room temperature for
another 17 h. Thereafter, the mixture was poured into a
solution of sodium bicarbonate (1 g/100 mL) and extracted with
ethyl acetate. The combined organic layers were washed with
a saturated sodium chloride solution, dried over potassium
carbonate, and evaporated to dryness. Purification of the crude
product was accomplished by column chromatography (silica,
methylene chloride/methanol: 15/1) yielding TA (2), 458 mg
In the antiproliferative assay study, the MMC-TA
conjugate and MMC showed a concentration-dependent
antiproliferative activity at concentrations from 0.03 to
(
1.05 mmol; 52%), as a white solid; mp 275 °C (dec); R
f
0.42
1
(CH Cl /CH OH 15:1). H NMR (400 MHz, acetone-d ): δ 7.30
2
2
3
6
3
0 µM. The IC50 values for MMC and the MMC-TA
(d, J ) 10, 1H, 1-H), 6.21 (dd, J ) 10, J ) 2, 1H, 2-H), 6.02
dd, J ) 2, J ) 2, 1H, 4-H), 5.00 (dd, J ) 3, J ) 2, 1H, 16-H),
.65 (dd, J ) 9, J ) 4, 1H, 21-H), 4.51 (s, 1H, exchangeable by
O, 11-OH), 4.40 (dddd, J ) 11, J ) 4, J ) 3, J ) 2, 1H,
1-H), 4.16 (dd, J ) 9, J ) 3, 1H, 21-H), 3.89 (br s, 1H,
exchangeable by D
(
4
D
1
conjugate were 1.7 and 2.4 µM, respectively. Although
regression analysis demonstrated that the IC50 for MMC
and the conjugate was not significantly different, the
individual responses at concentrations above 3 µM, as
well as the response maximum, were significantly less
2
2
O, 21-OH), 2.74 (dddd, J ) 14, J ) 14, J )
6, J ) 2, 1H, 6-H), 2.60 (dddd, J ) 29, J ) 12, J ) 12, J ) 5,
1H, 8-H), 2.39 (ddd, J ) 14, J ) 5, J ) 2, 1H, 6-H), 2.21 (ddd,
(p < 0.05) for the conjugate when compared to the
J ) 14, J ) 4, J ) 4, 1H, 12-H
ddd, J ) 14, J ) 6, J ) 6, 1H, 7-H
H, 12-H ), 1.63 (m, 1H, 15-H), 1.62 (s, 1H, 19-H), 1.50 (dddd,
J ) 14, J ) 14, J ) 14, J ) 5, 1H, 7-H ), 1.39 (s, 3H, acetonide-
CH ), 1.13 (s, 3H, acetonide-CH ), 0.90 (s, 1H, 18-H). C NMR
e
), 2.08 (m, 1H, 14-H), 1.93
parent MMC compound at the same concentration. We
also compared the parent MMC response curve to the
coadministration of equal molar concentrations of MMC
and TA to determine if there were any interactions
between the two drugs (MMC and TA) that contributed
to the decreased efficacy of the conjugate. Although a
slight difference between MMC (88%) in comparison to
the MMC+TA mixture (83%) was observed, it could not
alone explain the decreased efficacy of the conjugate.
Hence, this reduction in conjugate activity at high
concentrations is likely due to the conjugates limited
activity before hydrolyzing and releasing the parent
compounds, MMC and TA.
(
1
a
), 1.73 (dd, J ) 14, J ) 2,
a
e
13
3
3
(100.6 MHz, acetone-d ): δ 210.8 (s, 20-H), 185.9 (s, C-3), 166.5
6
(s, C-5), 152.5 (d, C-1), 130.2 (d, C-2), 125.5 (d, C-4), 111.7 (s,
C(CH
C-11), 67.4 (t, C-21), 48.9 (d, C-10), 45.9 (s, C-13), 44.1 (d, C-14),
7.4 (t, C-6), 34.1 (t, C-7), 33.9 (dd, C-8), 31.3 (t, C-12), 28.5 (t,
C-15), 26.7 (q, acetonide-CH ), 25.6 (q, acetonide-CH ), 23.6
q, C-19), 17.0 (q, C-18). UV (CH OH) λmax (ꢀ): 238 (18 400),
3 2
) ), 101.3 (d, C-9), 98.2 (d, C-17), 82.3 (d, C-16), 72.2 (dd,
3
3
3
(
2
3
+
01 (10 300), 192 (7400). MS (pos.-APCI) m/z 435 M (100).
TA-Glu ta r ic Acid Lin k er (4).22 TA, 260 mg (0.60 mmol),
and glutaric anhydride, 78.0 mg (0.68 mmol, 1.13 equiv
referred to TA), were placed into a 10 ml flask that had been
previously flushed with argon. Then, 2.5 mL of dried pyridine
was added, after which the mixture was stirred at room
temperature for 18 h. The mixture was poured into 50 mL of
ice water and acidified with 10% hydrochloric acid. After
extraction with methylene chloride, the combined organic
layers were evaporated to dryness, using toluene to remove
the remaining pyridine. Purification of the crude product was
accomplished by column chromatography (silica, methylene
chloride/methanol: 15/1) to yield 268 mg (0.49 mmol, 83%) of
Intravitreal administration of the conjugate revealed
no evidence of toxicity by ERG evaluation or histopatho-
logic examination. The total ocular dose administered
to these animals was approximately equivalent to the
maximum effective concentration identified in the cell
proliferation assay. These initial data support the idea
that this MMC-TA conjugate will be relatively safe for
various proliferative diseases including inflammatory
and neoplastic disorders.
TA linker (4) as a white solid; mp 220 °C (dec); R
Cl /CH OH 15:1). H NMR (400 MHz, acetone-d ): δ 7.31 (d,
2 3 6
f
0.23 (CH
2
-
1
In summary, the newly synthesized MMC-TA con-
jugate (5) appeared to have similar hydrolysis kinetics
as the previously described 5-FU-TA conjugate.
conjugate also showed sufficient antiproliferative activ-
ity of the released compounds (MMC and TA) after its
hydrolysis. The idea of using MMC in combination with
triamcinolone was suggested after both agents were
J ) 10, 1H, 1-H), 6.22 (dd, J ) 10, J ) 2, 1H, 2-H), 6.02 (s,
1H, 4-H), 5.15 (d, J ) 18, 1H, 21-H), 4.94 (m, 1H, 16-H), 4.81
1
0,17
The
(
1
2
d, J ) 18, 1H, 21-H), 4.66 (br s, 1H, exchangeable by D O,
1-OH), 4.44 (m, 1H, 11-H), 2.75 (dddd, J ) 14, J ) 14, J ) 8,
J ) 2, 1H, 6-H), 2.61 (dddd, J ) 29, J ) 12, J ) 12, J ) 5, 1H,
8-H), 2.42 (t, J ) 7, 2H, 2′- or 4′-H ), 2.52 (t, J ) 7, 2H, 2′- or
2
4′-H ), 2.39 (ddd, J ) 14, J ) 6, J ) 2, 1H, 6-H), 2.25 (ddd, J
2

1
126 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Macky et al.
)
14, J ) 3, J ) 3, 1H, 12-H
obscured by 3′-H, 1H, 7-H ), 1.93 (t, J ) 7, 2H, 3′-H
), 1.68 (m, 1H, 15-H), 1.63 (s, 1H,
9-H), 1.50 (dddd, J ) 13, J ) 13, J ) 13, J ) 5, 1H, 7-H ),
.41 (s, 3H, acetonide-CH ), 1.20 (s, 3H, acetonide-CH ), 0.94
): δ 204.3 (s,
0-H), 186.1 (s, C-3), 174.2 (s, C-1′), 173.0 (s, C-5′), 166.7 (s,
C-5), 152.6 (d, C-1), 130.3 (d, C-2), 125.6 (d, C-4), 112.2 (s,
C(CH ), 101.5 (d, C-9), 98.5 (d, C-17), 82.6 (d, C-16), 72.3 (dd,
C-11), 68.1 (t, C-21), 49.0 (d, C-10), 46.4 (s, C-13), 44.2 (d, C-14),
7.4 (t, C-6), 34.4 (t, C-7), 34.1 (dd, C-8), 33.3 (t, C-2′), 33.1 (t,
C-4′), 31.5 (t, C-12), 28.6 (t, C-15), 26.9 (q, acetonide-CH ), 26.0
), 23.8 (q, C-19), 21.1 (t, C-3′), 16.9 (q, C-18).
OH) λmax (ꢀ): 238 (14 200), 200 (6800), 195 (6300),
e
), 2.08 (m, 1H, 14-H), 1.93 (m,
CH
3
OH) to yield 4 mg (8%) of MMC linker (3) as a purple solid.
1
a
2
), 1.84
H NMR (400 MHz, acetone-d
CD
CD
10-H), 4.43 (d, J ) 14, 1H, 3-H), 3.98 (dd, J ) 11, J ) 11, 1H,
10-H), 3.65 (dd, J ) 11, J ) 4, 1H, 9-H), 3.59 (d, J ) 5, 1H,
1-H), 3.48-3.53 (m, complex, 2H, 2-H and 3-H), 3.20 (s, 3H,
9a-OCH
1.82 (m, 2H, 3′-H
Kin etics a n d Hyd r olysis Stu d ies. To determine its hy-
6
): 6.39 (br s, exchangeable with
), 5.99 (br s, exchangeable with
), 4.95 (dd, J ) 11, J ) 4, 1H,
(
1
1
dd, J ) 14, J ) 2, 1H, 12-H
a
3
OD, 2H, 7-NH
OD, 2H, 7-NH
2
or CONH
or CONH
2
2
e
3
2
3
3
1
3
(s, 1H, 18-H). C NMR (100.6 MHz, acetone-d
6
2
3
), 2.42-2.62 (m, 2H, 2′-H
2
), 2.30 (t, J ) 7, 2H, 4′-H
2
),
), 1.80 (s, 3H, 6-CH
3
).
3
)
2
2
-
6
3
drolysis rate, 2.8 mg (3.3 × 10
mol) of the MMC-TA
3
conjugate was dissolved in 2 mL of 0.1 M phosphate buffer
(pH 7.4) and 2 mL of propylene glycol (PG).2
4-26
The addition
(q, acetonide-CH
3
UV (CH
3
of PG was necessary to increase the solubility of the MMC-
TA conjugate. The resulting solution was incubated at 37 °C.
Samples of 25 µL each were taken after 6 and 12 h and each
+
1
91 (6000). MS (pos.-APCI) m/z 549 M (100).
MMC-TA Con ju ga te (5).17,22,23 A total of 216 mg (0.39
1
2 h thereafter. The samples were immediately injected into
mmol) of TA linker and 65.0 mg (0.40 mmol, 1.02 equiv
referred to TA linker) of carbonyldiimidazole (CDI) were
dissolved in 3 mL of dried tetrahydrofuran (THF) (under
argon), and the solution was stirred at room temperature for
the HPLC to study the hydrolysis of the conjugate. To
determine absolute quantities of the hydrolysis fragments, four
calibration curves were made available using pure MMC,
MMC-TA conjugate, TA, TA linker, and MMC linker.
HP LC System . Column: M & W Chromatographietechnik
Kromasil 100 C18, 250 mm × 4.6 mm, 5 µm; flow: 1 mL/min;
mobile phase: methanol/water: 60/40 f methanol/water:
100/0 within 16 min, then methanol/water: 60/40 for 4 min;
detection of the compounds at wavelengths of λ ) 234 nm for
MMC, TA, and TA linker; and λ ) 356 nm for the MMC-TA
conjugate.
The areas for the observed peaks were determined and
plotted in an AUC-time plot. Because the two peaks for
mitomycin and mitomycin linker could not be separated by
HPLC, their AUCs were added and plotted in one plot. AUCs
for the evolution of MMC were therefore not converted to
absolute amounts (mg). Because of injection of the samples in
a different solvent than was used for the calibration curves,
the retention times changed somewhat but were verified by
injecting pure samples of the compound in the same solvent.
To study the behavior of pure mitomycin under the same
conditions, mitomycin was dissolved in phosphate buffer and
PG and also incubated at 37 °C. Samples were taken every 24
h and analyzed by HPLC.
4
h. Then, 37.0 mg (0.11 mmol, 0.29 equiv referred to TA
linker) of MMC (Bristol Myers Squibb Oncology, Princeton,
New J ersey) and 3 mg of (dimethylamino)pyridine (DMAP)
were added, and the mixture was stirred at room temperature
for 4 days. The reaction mixture was poured into 50 mL of
water and extracted 3 times with chloroform. The combined
organic layers were washed with water, dried over sodium
sulfate, and evaporated to dryness. Purification of the crude
product was accomplished by column chromatography (silica,
chloroform/methanol: 10/1) to yield 59 mg (0.07 mmol, 43%)
of the MMC-TA conjugate (5) as a purple solid. The purity of
the compound was verified by two different HPLC gradient
systems (system 1: Rrel 12.8 min, from H
2
O/CH
OH ) 50%:40%:10% within
O/CH CN/CH OH ) 45%:
OH ) 0%:83%:17% within 10
3 3
CN/CH OH
)
70%:24%:6% to H
min; system 2: Rrel 3.4 min, H
5%:10% to H O/CH CN/CH
2 3 3
O/CH CN/CH
5
2
3
3
4
2
3
3
min; Waters Nova-Pak C18 3.9 mm × 150 mm, flow rate for
1
both systems: 1.5 mL/min). R
f
0.31 (CHCl
3
/CH
3
OH 15:1). H
NMR (400 MHz, CDCl
J ) 10, J ) 2, 1H, 2-H), 6.12 (br s, 1H, 4-H), 5.38 (br s, 2H,
′′-NH or CONH ), 5.09 (br s, 2H, 7′′-NH or CONH ), 4.96
d, J ) 5, 1H, 16-H), 4.80-4.94 (m, complex, 3H, 10′′-H and
3
): δ 7.26 (d, J ) 10, 1H, 1-H), 6.35 (dd,
7
(
2
2
2
2
An tip r olifer a tive Assa y. The fibroblast cell line (NIH 3T3)
was cultured in Dulbecco’s modified Eagles’ medium contain-
ing 10% fetal bovine serum. Cells were plated at a density of
100 000 cells/well and allowed to grow for 24 h at 37 °C in 5%
carbon dioxide in air.
2
1
1
2
1-H ), 4.44 (m, obscured by 3′′-H, 1H, 11-H), 4.44 (d, J ) 13,
H, 3′′-H), 4.04 (dd, J ) 11, J ) 11, 1H, 10′′-H), 3.67 (dd, J )
1, J ) 5, 1H, 9′′-H), 3.56 (dd, J ) 13, J ) 2, 1H, 3′′-H), 3.51
(d, J ) 5, 1H, 1′′-H), 3.37 (dd, J ) 5, J ) 2, 1H, 2′′-H), 3.20 (s,
3
2
1
H, 9a′′-OCH
H, 4′-H ), 2.40 (m, 1H, 6-H), 2.35 (m,1H, 12-H), 2.10 (m, 1H,
4-H), 1.95 (ddd, J ) 7, J ) 7, J ) 7, 2H, 3′-H ), 1.85 (m,
), 1.75 (m, 1H, 12-H),
.65 (m, 1H, 8-H), 1.60 (m, 1H, 15-H), 1.60 (m, 1H, 7-H), 1.55
s, 1H, 19-H), 1.42 (s, 3H, acetonide-CH ), 1.19 (s, 3H,
), 0.94 (s, 1H, 18-H). C NMR (100.6 MHz,
3
), 2.65 (m, 6-H), 2.48 (m, 2H, 2′-H
2
), 2.46 (m,
The MMC-TA conjugate, MMC, and TA were dissolved in
DMSO and diluted with media to achieve a final concentration
2
-
4
2
2
between 3.0 × 10 and 3.0 × 10 µM. Each experiment was
performed in triplicate. Cell proliferation was determined by
means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra-
zolium bromide (MMT) assay (Roche Molecular Biochemical;
Mannheim, Germany). The assay was conducted according to
the manufacturer’s recommendations. MTT assay samples
were measured using a spectrophotometer at an absorbance
wavelength of 560 nm and a reference wavelength of 650 nm.
Toxicity to Ra t Retin a . Eight Wistar female rats, 4-weeks-
old and weighing 70 g each, were used in this part of the study.
Toxicity of the MMC-TA conjugate was assessed in response
to intravitreal injections, using both functional (scotopic ERG)
and anatomical (histopathology) analyses.
Our animal study was approved by the Institutional Animal
Care and Use Committee. Research animal care and use at
our institution meets all USDA and AAALAC approval re-
quirements. Our study protocol adhered to the Association for
Research in Vision & Ophthalmology Statement regarding the
Use of Animals in Ophthalmic and Vision Research.
obscured, 1H, 7-H), 1.78 (s, 3H, 6′′-CH
1
(
3
3
1
3
acetonide-CH
3
CDCl
75.2 (C-8′′), 172.4 (C-1′), 166.0 (C-5), 156.6 (CONH
C-4a′′), 152.0 (C-1), 147.2 (C-7′′), 130.0 (C-2), 125.0 (C-4), 111.6
C(CH ), 110.0 (C-8a′′), 105.7 (C-9a′′), 105.6 (C-6′′), 100.1 (d,
3
): δ 204.0 (20-H), 186.2 (C-3), 183.0 (C-5′), 178.2 (C-5′′),
1
(
(
2
), 154.0
3 2
)
C-9), 97.8 (C-17), 82.0 (C-16), 72.1 (d, C-11), 68.0 (C-21), 62.0
C-10′′), 52.0 (C-9′′), 50.0 (9a′′-OCH ), 49.0 (d, C-10), 49.0 (C-
′′), 45.8 (C-13), 43.4 (C-14), 42.8 (C-1′′), 39.8 (C-2′′), 37.6 (C-
2), 35.4 (C-4′), 34.1 (C-15), 33.8 (C-8), 33.5 (C-2′), 31.5 (C-6),
7.8 (C-7), 26.2 (acetonide-CH ), 26.0 (acetonide-CH ), 23.0 (C-
9), 19.8 (C-3′), 16.3 (C-18), 8.0 (6′′-CH ). UV (CH
(
3
3
1
2
1
3
3
3
3
OH) λmax
(ꢀ): 356 (16 600), 234 (19 700), 212 (22 400). MS (pos.-ESI) m/z
+
8
8
87 (40) [M - H + Na] , H
87.3491; found, 887.3489, 833 (100) [M - H - OCH
R
calcd for C44
H
53
N
4
O
3
13FNa,
] .
+
Mitom ycin -Glu ta r ic Acid Lin k er (3). A 37.1 mg (0.11
mmol) amount of MMC was dissolved in 4 mL of dry THF
under an argon atmosphere and treated with 14 mg (0.12
mmol, 1.1 equiv relative to MMC) of glutaric anhydride. After
the solution was stirred for 72 h at room temperature, the
solution was poured on ice, acidified with diluted hydrochloric
acid, and extracted with chloroform. Purification was achieved
In ta vitr ea l In jection s. Two microliters containing 0.784
µg of MMC-TA conjugate dissolved in absolute DMSO was
injected into the vitreous through the pars plana, under
visualization with a microscope, following a paracentesis. This
exposed the intraocular tissues to a concentration of ap-
proximately 30 µM of the MMC-TA conjugate, assuming a
rat vitreous volume of 30 µL. The right eye of each rat received
3
by chromatography ((1) silica, CH OH. (2) Sephadex LH 20,

Mitomycin C-Triamcinolone Acetonide Conjugate
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1127
the conjugate, while the fellow eye (left eye) received an
intravitreal injection of 2 µL of DMSO and served as the
control.
Electr or etin ogr a m . Animals were dark-adapted overnight
and anesthetized with ketamine (11 mg/kg) and xylazine (14
mg/kg), their pupils were dilated with 1% atropine, and their
corneas were anaesthetized with topical eye drops (0.5%
proparacaine hydrochloride).
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7
28
Pugh as described in Rohrer et al. A clear plastic holder
ending in a curved tip the shape of the cornea was used to
provide full-field light stimulation to the rat’s retina via a fiber
optic light guide. The diffuser element, which had a recording
electrode (0.2 mm platinum wire) glued to the inside, was
gently placed against the cornea and electrical contact was
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electrodes were placed in the neck and tail, to serve as
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and digitized with a 12 bit analogue to digital converter at 2
Hz. Data were stored, displayed, and analyzed with a PC
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was used for light stimulation.
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rats) and 20 days (5 rats) for histolopathological analyses. The
animals were deeply anesthetized, and their eyes were enucle-
ated and immediately immersed in a fixative consisting of 10%
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Ack n ow led gm en t. This work was supported by the
Medical University of South Carolina, Institutional
Research funds, 1999-2000; the South Carolina Com-
mission of Higher Education; the U.S. Department of
Defense; Research to Prevent Blindness; and NIH
EY09741 (C.E.C.). The clinical expertise of D. Virgil
Alfaro II, M.D., Farrow Counts, M.S., Todd Shearer,
Ph.D., and Enrique-Roig, M.D., is gratefully acknowl-
edged. The authors have no financial or proprietary
interest in the products or equipment herein.
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J M010511B