Hydrophilic, pro-drug analogues of T138067 are efficacious in controlling tumor growth in vivo and show a decreased ability to cross the blood brain barrier

Article abstract of DOI:10.1021/jm000478d

The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization. Amides 3-6 were synthesized using standard methodologies and determined to be significantly less lipophilic than T138067 based on logP calculations. Tubulin poly

Full text of DOI:10.1021/jm000478d

J . Med. Chem. 2001, 44, 3599-3605
3599
Hyd r op h ilic, P r o-Dr u g An a logu es of T138067 Ar e Effica ciou s in Con tr ollin g
Tu m or Gr ow th In Vivo a n d Sh ow a Decr ea sed Ability To Cr oss th e Blood Br a in
Ba r r ier
Steven M. Rubenstein,* Vijay Baichwal, Holger Beckmann, David L. Clark, Walter Frankmoelle, Daniel Roche,
Edit Santha, Susan Schwender, Martin Thoolen, Qiuping Ye, and J uan C. J aen
Tularik Inc., Two Corporate Drive, South San Francisco, California 94080
Received November 8, 2000
The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization.
Amides 3-6 were synthesized using standard methodologies and determined to be significantly
less lipophilic than T138067 based on logP calculations. Tubulin polymerization and [3H]-
T138067 competition assays revealed that these amides are pro-drugs for parent aniline 2.
Amides 3-5 showed no detectable signs of crossing the blood brain barrier, while amide 6 was
found in extremely small amounts (12 ng/g of brain tissue). Aniline 2, which was formed in
vivo from these amides, was found in significantly smaller amounts (approximately 20 to >5000
times) in the brain than when 2 was administered directly. The in vivo efficacy of amide 6
approached that of T138067 and was better tolerated when administered to athymic nude mice
bearing MX-1 human mammary tumor xenografts.
In tr od u ction
Antimitotic drugs have emerged as an effective treat-
ment for a variety of cancers.1 Our group has recently
described the discovery of a new antimitotic agent,
T138067 (Figure 1), which is currently in phase II
clinical trials.2-4 This compound has been shown to
F igu r e 1. Structure of T138067.
covalently bind to cysteine-239 on â-tubulin isoforms 1,
2, and 4 by way of a nucleophilic aromatic substitution
reaction.2 The covalent modification of â-tubulin inhibits
the polymerization of the R,â-tubulin heterodimers into
microtubules. This leads to cell arrest at the G2/M cell
cycle boundary followed by apoptosis.2 This compound
and its analogues are effective against a variety of
tumors, including those that express the multidrug
resistant (MDR) phenotype (IC50 ) 11-165 nM).2-4
Although T138067 has the ability to penetrate the
blood brain barrier (BBB), to date, clinical studies have
not identified central nervous system (CNS) toxicity as
the main dose-limiting side effect.5 However, dose-
limiting CNS effects have been observed with other
lipophilic drugs.6 While compounds that have the ability
to penetrate the BBB are promising candidates for
treating brain tumors, we felt that an analogue of
T138067 that does not penetrate the BBB would possess
fewer potential problems with central neurotoxicity and
an increased therapeutic window relative to other
members of this class of compounds.
F igu r e 2. AC-7739 and AC-7700 are hydrophillic analogues
of Combretastatin A4.
Ch em istr y
Combretastatin A4 is a potent antimitotic agent in
vitro (Figure 2). However, this compound showed no
antineoplastic effects when tested in vivo.7 The authors
hypothesized that the lack of efficacy observed was due
to a poor pharmacokinetic profile originating from the
compound’s intrinsically low aqueous solubility. To
increase its aqueous solubility, they replaced the C-3
hydroxyl group in ring B with an amine substituent and
formed the HCl salt (AC-7739) as well as the L-serine
amide HCl salt (AC-7700) (Figure 2). Both compounds
showed enhanced aqueous solubility over Combretasta-
tin A4 and were efficacious in vivo.7 On the basis of
The ability of T138067 to penetrate the BBB could
be due, at least in part, to its lipophilicity (ClogP ) 3.26),
and thus we were interested in identifying an analogue
that was significantly less lipophilic. This report de-
scribes our results toward developing such compounds
while retaining the same mechanism of action as
T138067.
* To whom correspondence should be addressed. Phone: (650)-825-
7401. Fax: (650)-825-7433. E-mail: rubenstein@tularik.com.
10.1021/jm000478d CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/14/2001

3600 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Sch em e 1. General Synthesis of Amide Analoguesa
Rubenstein et al.
Ta ble 1. ClogP and Cell Based Assay Results
a
Reagents: (a) NaOMe, MeOH; (b) pentafluorobenzenesulfonyl
chloride, MeOH; (c) 10% Pd/C, H2(g), EtOAc; (d) N-methyl mor-
pholine, 60 °C, protected amino acid, coupling agents used: HBTU/
HOBT, HATU/HOAT, or EDCI/HOBT; (e) if R1 ) BOC then (1)
TFA, CH2Cl2, (2) Ac2O, 2,6-lutidine.
these results, we decided to synthesize a variety of
amide analogues of T138067.
Compound 2 was previously synthesized by us and
exhibited potent cytotoxic effects in vitro.8 Scheme 1
outlines the synthesis of 2 and the reaction conditions
used to synthesize amide analogues of T138067. 4-Fluoro-
3-nitroaniline 1 was allowed to react with sodium
methoxide followed by pentafluorobenzenesulfonyl chlo-
ride. The resulting product was then reduced over
palladium on carbon to furnish aniline 2. Due to the
unreactive nature of 2, the more active coupling re-
agents HBTU/HOBT and HATU/HOAT were frequently
used.9 In addition, elevated temperatures were required
to form the amide bond in a reasonable amount of time
and yield.10 The unacetylated amides 3A-5A were
synthesized using N-BOC protected L-alanine, glycine,
and L-serine as starting materials, respectively, followed
by acidic deprotection, Table 1. The synthesis of serine
amide 5A required a hydrogenolysis of the benzyl group
before BOC deprotection. Amides 3-5 were synthesized
using N-acetylated L-alanine, glycine, and L-serine,
respectively, Table 1. The synthesis of serine amide 5
required the deprotection of a tert-butyl group under
acidic conditions, following the initial coupling reaction.
Amide 6 was synthesized from N-BOC-O-benzyl-L-
threonine. Acidic deprotection of the BOC group fol-
lowed by acetylation and hydrogenolysis of the benzyl
group gave the desired product.
a
Calculated using the LSW data analysis software, MDL
Information Systems, 14600 Catalina St., San Leandro, CA, 94577.
b
Calculated using Crippen’s method as found in CS Chem Draw
Ultra Version 5.0, Cambridge Soft Corporation, 100 Cambridge
Park Drive, Cambridge, MA, 02140. c The standard deviation is
(0.47. Human cervical carcinoma cell line. e Human mammary
d
carcinoma cell line. f Human mammary carcinoma cell line that
g
exhibits the MDR phenotype. Not determined.
expressing the MDR phenotype.11 The unacetylated
amides 3A-5A were also tested and were observed to
be significantly less active, Table 1. Also, the unnatural
antipode of amides 3 and 5 were also synthesized and
observed to be less cytotoxic.12 This lack of efficacy and
potency led to suspicions regarding the in vivo stability
of these compounds and whether their cytotoxic effects
were originating from parent aniline 2.13 To directly
address this question, glycine amide 4 was synthesized
in radiolabeled form (by introducing tritium via reduc-
tive dehalogenation on the nonfluorinated aromatic
ring) and incubated with HepG2 cells in vitro. The t1/2
for disappearance of radiolabeled amide 4 was ap-
proximately 150 min. Aniline 2 was the main degrada-
tion product as determined by radioisotope HPLC
analysis.12 To further test the pro-drug hypothesis,
aniline 2 and amide 4 were tested for their ability to
inhibit tubulin polymerization in vitro. Aniline 2 and
T138067 exhibited a concentration-dependent inhibition
of tubulin polymerization based on turbidometric analy-
sis (panels C and A of Figure 3, respectively). In con-
Biologica l Resu lts a n d Discu ssion
A variety of amides were synthesized and tested for
their cytotoxicty against three tumor human cell lines.
The biological activity and ClogP values of the most
potent amides synthesized are summarized in Table 1.
The N-acetylated amino acid amides 3-6 are 100 to 550
times less lipophilic than T138067 as predicted based
on Ohsumi’s results.7 In addition, these amides were
observed to be 2 to 7 times more potent than T138067
in all three cell lines tested, suggesting that these
compounds would also be effective in treating tumors

Hydrophilic, Pro-Drug Analogues of T138067
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3601
F igu r e 4. Amides 4 and 5 do not compete with 3H-T138067
for â-tubulin binding. (A-D) Purified brain tubulin (0.5 µM)
was incubated in the absence of 3H-T138067 (lane 1) or in the
presence of 500 nM of 3H-T138067 (lanes 2-24) and the
indicated concentration of T138067 (panel A, lanes 2-7),
aniline 2 (panel B, lanes 8-12), amide 4 (panel C, lanes 13-
17), and amide 5 (panel D, lanes 20-24). After a 2 h incubation
at 37 °C, â-tubulin-modification was analyzed by SDS-PAGE.
Shown is autoradiogram of SDS-PAGE.
Ta ble 2. Maximum Tolerated Dose (MTD) and Convulsion
Threshold
convulsionb
threshold (mg/kg)
compd
MTD (mg/kg)a
2
3
4
5
6
30
75
40
125
100
35
>100
100
>100
>100
a
The number represents the maximum dose administered to
CD-1 male mice that resulted in one or two deaths per cohort.
This dose would be administered to an appropriate mouse model
to test for in vivo efficacy. CD-1 male mice were administered
the compound by bolus iv injection. The number represents the
minimum dose required to induce convulsions.
F igu r e 3. In vitro effect of aniline 2 and amide 4 on
microtubule formation. (A-C) Polymerization reactions (40
mM tubulin) were performed at 37 °C in the absence of drug
(no Drug) or at the indicated concentrations (1, 3, and 10 mM)
of T138067 (panel A), amide 4 (panel B), and aniline 2 (panel
C). Shown is a graphical representation of changes in the
optical density at 340 nm over time (min) in the absence (no
drug) or presence of the indicated compounds.
b
formed that support the hypothesis that aniline 2, with
a ClogP comparable to that of T138067, was responsible
for the cytotoxicity observed in the whole cell assay. In
fact, convulsions were observed when aniline 2 was
administered to male CD-1 mice via bolus injection,
Table 2. We suspected that the central neurotoxicity
observed was a result of the compound’s ability to enter
the brain. Surprisingly, bolus injections of amides 3, 5,
and 6 were well tolerated at doses 2 to 4 times above
those of aniline 2, Table 2. Convulsions were only
observed when administering amide 4 to CD-1 mice at
three times (100 mg/Kg) the level of aniline 2, while the
other amides (3, 5, 6) did not induce convulsions even
at the highest dose administered (g100 mg/kg). Table
3 outlines the amount of compound that was found in
the brain (ng/g of brain tissue) and plasma (ng/mL of
plasma) of CD-1 mice exposed for 5 min to 30-40 mg/
kg of T138067, aniline 2, and amides 3-6 via bolus
injection. None of the amides, except 6, showed any
detectable levels in the brain while aniline 2 was found
in comparable amounts to T138067. More importantly,
the aniline 2 formed from the in vivo hydrolysis of the
various amides was found in significantly less amounts
(approximately 20 to >5000 times) than when aniline
2 itself was directly administered. This was most likely
the main reason amides 3-6 were better tolerated than
aniline 2 by the CD-1 mice.
trast, amide 4 (Figure 3B) showed no significant inhibi-
tion. Amides 3, 5, and 6 showed a profile similar to
amide 4 (see Supporting Information). Figure 4 shows
the results of a competition experiment that also sup-
ports the pro-drug hypothesis. The competition between
radiolabeled and cold T138067 for covalently modifying
â-tubulin is shown by SDS-PAGE analysis in Figure
4A. High concentrations of unlabeled T138067 effec-
tively compete with radiolabeled T138067. Aniline 2 also
has the ability to compete with radiolabeled T138067
(Figure 4B). However, unlabeled amides 4 and 5 did not
compete with radiolabeled T138067 in covalently modi-
fying â-tubulin (panels C and D of Figure 4, respec-
tively). The tubulin polymerization results, in addition
to the competition experiments, as well as the HePG2
cell incubation experiment support the hypothesis that
these amides are behaving as prodrugs and their
cytotoxic effects are originating from aniline 2 in the
whole cell assay.
As stated earlier, we wanted to synthesize analogues
that were significantly less lipophilic than T138067, and
on the basis of the ClogP values outlined in Table 1,
this was achieved. However, experiments were per-

3602 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Rubenstein et al.
Ta ble 3. Brain-Plasma Comparison Using CD-1 Micea
reduce tumor size to levels similar to T138067 at day
13 (Figure 5). Amides 4 and 5 were less efficacious than
amides 3 and 6 at suppressing tumor growth (Figure
5). A certain percentage of animals from each of these
groups had to be euthanized during the course of the
experiments due to excessive weight loss, g20% of their
original weight, or ulcerating tumors. In addition, the
last of the control animals for T138067, amide 4, and
amide 5 either died or were euthanized (due to an
ulcerating tumor or excessive tumor size) on day 13, 11,
and 12, respectively. The lack of control animals in these
experiments precluded the ratio of mean tumor volume
from being calculated.
concentration
compd
brain (ng/g)
plasma (ng/mL)
T138067
2
864 ( 210b
1169 ( 836
0
0.2 ( 0.2
0
13.2 ( 5.6
0
54.4 ( 14.85
12.6 ( 0.67
1 ( 0.4
6272 ( 1425
8926 ( 2194
62 ( 34
156 ( 26.8
112.8 ( 7
729 ( 232
4.2 ( 1.5
695 ( 120
52.2 ( 15.7
113 ( 38
3
3 f 2c
4
4 f 2c
5
5 f 2c
6
6 f 2c
a
CD-1 mice (n ) 5) were exposed for 5 min to the desired
b
compound by volus injection. The standard deviation was cal-
culated using the STDEVP program in Microsoft Excel 5.0. c This
line represents the amount of aniline 2 formed by the in vivo
hydrolysis of the parent amide.
Con clu sion s
Amides 3-6 can be readily synthesized using stan-
dard methodologies. The calculated logP values suggest
that these amides are significantly less lipophilic than
T138067, and this is manifested in their inability to
penetrate the BBB, Table 3. It is possible that the
amides cytotoxic effects are originating from another
mechanism(s). However, the HEpG2 cell and polymer-
ization experiments (Figures 3 and 4) support the
hypothesis that the less lipophilic amides are not the
constitutively active agents but rather are acting as
prodrugs for the more lipophilic aniline 2. Due to the
acute toxic nature of 2, this compound was never
evaluated for its efficacy in vivo. However, when amides
3-6 are dosed at their maximally tolerated doses, these
compounds are efficacious at suppressing tumor growth
with no signs of CNS toxicity, especially amides 3 and
6. This lack of CNS toxicity is understandable given that
significantly smaller amounts of aniline 2 enter the
brain via the in vivo hydrolysis of the amide bond, Table
3. The data described supports the idea that the amide
side chains are actively or passively facilitating the
distribution of efficacious quantities of the pro-drugs to
the intracellular site of action before a significant
amount of amide hydrolysis can take place in the
plasma. Once inside the cell, amide cleavage takes place
releasing the active component, aniline 2.
Exp er im en ta l Section
1H NMR spectra were obtained on a Bruker DRX 400 MHz
or a Varian Gemini 400 MHz spectrometer. Chemical shifts
were reported in parts per million (ppm) with DMSO-d6 as
the internal standard. The abbreviations s ) singlet, d )
doublet, t ) triplet, q ) quartet, quin ) quintet, m ) multiplet,
and b ) broad are used throughout. Melting points were
determined in open-ended capillary tubes using a FP62 Mettler
Toledo melting point apparatus and are uncorrected. Elemen-
tal analysis was performed by Atlantic Microlab Inc, Norcross,
GA, and are within (0.4% of the calculated values. Low-
resolution mass spectra were determined at Tularik Inc. using
a Hewlett-Packard Series 1100 MSD ES-mass spectrometer.
Thin-layer chromatography (TLC) was performed on 0.25 mm
Analtech Inc. precoated silica gel glass plates. Visualization
on TLC was achieved using ultraviolet light, an I2 developing
chamber, and/or heating of TLC plates previously submerged
in a 7% solution of phosphomolybdic acid in 95% ethanol. Flash
column chromatography was performed using silica gel 60
(230-400 mesh) obtained from EM Science. Reagents and
solvents were commercial grades and were used as supplied,
unless otherwise stated. All moisture-sensitive reactions were
carried out in glassware that was flame dried under high
vacuum (0.5-2.0 mmHg) and then purged with nitrogen. The
term “concentrated” refers to the removal of volatile solvents
F igu r e 5. Effect of amides 3-6 and T138067 on tumor growth
of mice bearing MX-1 tumor xenografts. The maximum toler-
ated dose was administered for each test compound: amide 3
(75 mg/kg), amide 4 (40 mg/kg), amide 5 (125 mg/kg), amide 6
(100 mg/kg). T138067 was infused at a dose of 30 mg/kg/h for
4 h. Results are expressed as the daily ratio of the mean tumor
volume of treated animals to saline treated control mice (n )
7-12 for each treatment group). Arrows indicate treatment.
The in vivo efficacies of T138067 and amides 3-6 are
outlined in Figure 5.14 The athymic nude mice were
treated intravenously (iv) with maximum tolerated
doses of each compound on days 1, 8, and 15. Amides
3-6 were administered as bolus iv injections while
T138067 was infused over 4 h.15 Suppression of tumor
growth by T138067 was significant (Figure 5). Both
amides 3 and 6 were able to suppress tumor growth
effectively, particularly amide 6 which was able to

Hydrophilic, Pro-Drug Analogues of T138067
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3603
using a rotary evaporator connected to a KNF Neurberger
vacuum pump.
for amide 3. HBTU/HOBT were used as coupling agents. BOC
deprotection was performed using the procedure described for
amide 5. The crude oil was purified by column chromatography
(2-10% MeOH in CH2Cl2) to yield 33 mg (59%) of product as
Abbr evia tion s: N-hydroxybenzotriazole (HOBT), 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HBTU), N-methylmorpholine (NMM), 1-hydroxy-
7-azabenzotriazole (HOAT), O-(7-azabenzotriazole-1-yl)-N,N,-
N′,N′-tetramethyluronium hexafluorophosphate (HATU), 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCI), trifluoracetic acid (TFA), acetic acid (AcOH).
1
a pale yellow solid. H NMR (400 MHz, DMSO-d6) δ 11.0 (s,
1H), 9.75 (s, 1H), 8.1 (s, 1H), 7.85 (d, J ) 2.6 Hz, 1H), 7.03 (d,
J ) 8.8 Hz, 1H), 6.9 (dd, J ) 8.8, 2.6 Hz, 1H), 4.15 (m, 1H),
3.8 (s, 3H),1.4 (d, J ) 6.9 Hz, 3H). MS(EI) m/z 438 (100, M-H).
2-Acet yla m in o-N-(2-m et h oxy-5-p en t a flu or o-b en zen e-
su lfon yla m in o-p h en yl) a ceta m id e (4) was synthesized in
90% yield from aniline 2 (500 mg, 1.36 mmol) and N-acetyl-
glycine (207 mg, 1.77 mmol) using the procedure described for
amide 3. HBTU/HOBT were used as coupling agents. The final
product was purified by trituration with MeOH/CH2Cl2, mp
209-210 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.9 (s, 1H), 9.1
(s, 1H), 8.29 (t, J ) 5.7 Hz, 1H), 7.88 (s, 1H), 6.99 (d, J ) 8.9
Hz, 1H), 6.88 (dd, J ) 8.8, 2.6 Hz, 1H), 3.85 (d, J ) 5.9 Hz,
2H), 3.79 (s, 3H), 1.89 (s, 3H). Anal. Calcd for C17H14F5N3O5S:
C, 43.68; H, 3.0; N, 8.99. Found: C, 43.43; H, 3.14; N, 8.79.
MS(EI) m/z 467 (20, M+), 466 (100, M-H).
2-Am in o-N-(2-m et h oxy-5-p en t a flu or o-b en zen esu lfo-
n yla m in o-p h en yl) a ceta m id e (4A) was synthesized from
aniline 2 (60 mg, 0.16 mmol) and tert-butoxycarbonylamino-
acetic acid (29 mg, 0.163 mmol) using the two-step procedure
described for amide 3A. EDCI/HOBT were used as coupling
agents. The crude oil was purified by column chromatography
(1-5% MeOH in CH2Cl2) to yield 33 mg (61%) of product as a
pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.7 (s, 1H),
7.75 (s, 1H), 6.85 (d, J ) 8.9 Hz, 1H), 6.72 (d, J ) 8.8 Hz, 1H),
3.75 (s, 3H), 3.5 (s, 2H). MS(EI): m/z 369 (100, M-glycine),
426 (25, M+H).
(2S)-2-Ace t yla m in o-3-h yd r oxy-N -(2-m e t h oxy-5-p e n -
ta flu or oben zen esu lfon -yla m in o-p h en yl)-p r op ion a m id e
(5) was synthesized in two steps from aniline 2 (500 mg, 1.36
mmol) and N-acetyl-O-tert-butyl-L-serine (360 mg, 1.77 mmol)
using the procedure for amide 3. HBTU/HOBT were used as
coupling reagents. To a 0.16 M solution of (2S)-2-acetylamino-
3-tert-butoxy-N-(2-methoxy-5-pentafluorobenzenesulfonyl-amino-
phenyl)-propionamide (700 mg, 1.26 mmol) at 0 °C was added
TFA (8.4 mL, 100 mmol), and the resulting solution was
allowed to stir for 8.5 h at 0 °C followed by warming to ambient
temperature for an additional 5 h. The crude reaction was
concentrated under vacuum with azeotropic removal of TFA
with hexane and purified by column chromatography (1-5%
MeOH in CH2Cl2). The resulting material was repurified using
reverse phase HPLC chromatography (5-95% CH3CN in a
0.1% TFA aqueous solution) to yield 507 mg of amide 5 (68%)
as a white solid, mp 98-99 °C. 1H NMR (400 MHz, DMSO-d6)
δ 10.9 (s, 1H), 9.09 (s, 1H), 8.2 (d, J ) 7.3 Hz, 1H), 7.99 (d, J
) 2.2 Hz, 1H), 7.0 (d, J ) 8.6 Hz, 1H), 6.88 (dd, J ) 8.7, 2.6
Hz, 1H), 5.1 (t, J ) 5.1 Hz, 1H), 4.4 (apparent q, J ) 6.1 Hz,
1H), 3.8 (s, 3H), 3.7-3.55 (m, 2H), 1.9 (s, 3H). Anal. Calcd for
4-Meth oxy-3-n itr oa n ilin e. To a 1 M solution of 3-nitro-
4-fluoroaniline (1) (16.7 g, 107 mmol) in anhydrous methanol
at ambient temperature was added sodium methoxide (23.1
g, 428 mmol), and the resulting solution was heated to reflux
with stirring for 21 h. The reaction mixture was then cooled
to 0 °C, and a 12 M solution of HCl (13.4 mL) was added
dropwise followed by water (250 mL). The crude mixture was
extracted three times with Et2O (200 mL). The organic layers
were combined, washed with brine (300 mL), dried over
Na2SO4, and concentrated under vacuum to yield 17.5 g (97%)
of product as a dark brown solid which was used without
1
further purification. H NMR (400 MHz, DMSO-d6) δ 7.09 (d,
J ) 9 Hz, 1H), 7.01 (dd, J ) 2.8, 1.3 Hz, 1H), 6.85 (ddd, J )
9, 2.8, 1.4 Hz, 1H), 5.2 (s, 2H), 3.75 (s, 3H).
2,3,4,5,6-P en ta flu or o-N-(4-m eth oxy-3-n itr op h en yl)ben -
zen esu lfon a m id e. To a 0.4 M solution of 4-methoxy-3-
nitroaniline (17.5 g, 104 mmol) in anhydrous methanol was
added dropwise pentafluorobenzenesulfonyl chloride (7.7 mL,
52 mmol), and the resulting mixture was stirred at ambient
temperature for 1 h. The reaction mixture was concentrated
under vacuum and purified by column chromatography (10-
30% EtOAc in hexane) to yield 18.1 g (87%) of product as an
orange solid, mp 95-97 °C. 1H NMR (400 MHz, CDCl3) δ 7.64
(d, J ) 2.7 Hz, 1H), 7.51 (dd, J ) 9, 2.7 Hz, 1H), 7.09 (d, J )
9.0 Hz, 1H), 3.95 (s, 3H). MS (EI): m/z 817 (30, 2M+Na-2H),
398 (30, M+), 397 (100, M-H).
N-(3-Am in o-4-m et h oxy-p h en yl)-2,3,4,5,6-p en t a flu or o-
ben zen esu lfon a m id e (2). To a 0.15 M solution of 2,3,4,5,6-
pen t a flu or o-N-(4-m et h oxy-3-n it r oph en yl)ben zen esu lfon -
amide (18.1 g, 45.5 mmol) in 100% anhydrous ethanol was
added 10% Pd/C (4.84 g, 4.55 mmol). Hydrogen gas was
bubbled through the solution for 1 min, and the resulting
mixture was stirred for 24 h under 1 atm of hydrogen. The
crude reaction mixture was filtered through a pad of Celite,
and the Celite was washed with ethanol (500 mL). The filtrate
was concentrated under vacuum to yield 16.5 g (99%) of
product as an off white solid which was used without further
1
purification, mp 142-143 °C. H NMR (400 MHz, DMSO-d6)
δ 10.64 (s, 1H), 6.68 (d, J ) 8.4 Hz, 1H), 6.44 (d, J ) 2.1 Hz,
1H), 6.3 (dd, J ) 8.4, 2.1 Hz, 1H), 4.88 (bs, 2H), 3.69 (s, 3H).
MS(EI) m/z 369 (100, M+H).
(2S)-2-Acet yla m in o-N-(2-m et h oxy-5-p en t a flu or ob en -
zen esu lfon yla m in o-p h en yl)-p r op ion a m id e (3). To aniline
2 (500 mg, 1.36 mmol) was added N-acetyl-L-alanine (356 mg,
2.72 mmol), HOAT (370 mg, 2.72 mmol), and HATU (1.034 g,
2.72 mmol). DMF (7 mL) was then added, followed by NMM
(149 µL, 0.36 mmol), and the resulting mixture was heated to
65 °C with stirring for 22 h. The crude reaction mixture was
cooled, followed by addition of 1 M solution of HCl (50 mL)
and EtOAc (50 mL). The aqueous phase was extracted three
times with EtOAc (50 mL). The organic phase was washed
with brine (50 mL), dried over Na2SO4, and concentrated under
vacuum. The crude oil was purified by column chromatography
(1-5% MeOH in CH2Cl2) to yield 468 mg (72%) of product as
a pale yellow solid, mp 189-190 °C. 1H NMR (400 MHz,
DMSO-d6) δ 10.89 (s, 1H), 9.1 (s, 1H), 8.28 (d, J ) 6.5 Hz,
1H), 7.89 (s, 1H), 7.0 (d, J ) 8.9 Hz, 1H), 6.88 (dd, J ) 8.8, 2.7
Hz, 1H), 4.55-4.35 (m, 1H), 3.8 (s, 3H), 1.85 (s, 3H), 1.23 (d,
J ) 7.2 Hz, 3H). Anal. Calcd for C18H16F5N3O5S: C, 44.91; H,
3.35; N, 8.73. Found: C, 44.69; H, 3.29; N, 8.67. MS(EI) m/z
481 (25, M+), 480 (100, M-H).
C
18H16F5N3O6S‚0.5H2O: C, 42.69; H, 3.38; N, 8.30. Found: C,
42.55; H, 3.32; N, 8.19. MS(EI) m/z 497 (20, M+), 496 (100,
M-H).
2(S)-Am in o-3-h ydr oxy-N-(2-m eth oxy-5-pen taflu or o-ben -
zen esu lfon yla m in o-p h en yl)-p r op ion a m id e (5A) was syn-
thesized from aniline 2 (203 mg, 0.55 mmol) and 3-benzyloxy-
2(S)-tert-butoxycarbonylaminopropionic acid (195 mg, 0.66
mmol) using the two-step procedure described for amide 3A.
HBTU/HOBT were used as coupling agents (note: before BOC
deprotection, the benzyl group was removed using the proce-
dure for amide 6). The crude solid was purified by column
chromatography (1-10% MeOH in CH2Cl2) to yield 89 mg (30%
1
for the three steps) of product as a pale yellow solid. H NMR
(400 MHz, DMSO-d6) δ 9.9 (bs, 1H), 7.95 (s, 1H), 6.95 (d, J )
8.5 Hz, 1H), 6.8 (d, J ) 8.6 Hz, 1H), 6.9 (dd, J ) 8.8, 2.6 Hz,
1H), 4.1 (bs, 1H), 3.8 (s, 3H), 3.7-3.5 (m, 3H). MS(EI) m/z 454
(100, M-H).
(1S,2R)-[2-Ben zyloxy-1-(2-m eth oxy-5-p en ta flu or oben -
zen esu lfon yla m in o-p h en ylca r b a m oyl)-p r op yl]ca r b a m -
ic a cid ter t-bu tyl ester was synthesized in 43% yield from
aniline 2 (3.0 g, 8.2 mmol) and N-BOC-O-benzyl-L-threonine
(2.3 g, 7.6 mmol) using the procedure described for amide 3.
2(S)-Am in o-N-(2-m eth oxy-5-p en ta flu or o-ben zen esu lfo-
n yla m in o-p h en yl) p r op ion a m id e (3A) was synthesized
from aniline 2 (210 mg, 0.57 mmol) and tert-butoxycarbonyl-
L-alanine (140 mg, 0.74 mmol) using the procedure described

3604 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Rubenstein et al.
1H NMR (400 MHz, CD3OD) δ 8.01 (d, J ) 2.4 Hz, 1H), 7.28-
7.11 (m, 5H), 7.0 (dd, J ) 8.6, 2.6 Hz, 1H), 6.93 (d, J ) 8.8 Hz,
1H), 4.6 (1/2ABq, J ) 11.6 Hz, 1H), 4.46 (1/2ABq, J ) 11.4
Hz, 1H), 4.3 (bs, 1H), 4.09 (bs, 1H), 3.7 (s, 3H), 1.49 (s, 9H),
1.21 (d, J ) 3.2 Hz, 1H). MS(EI) m/z 682 (60, M+Na), 658
(100, M-H).
liquid nitrogen, and stored at -20 to -70 °C until further
analysis.
Br a in P r ep a r a tion . Each brain was weighed, followed by
washing with cold PBS (1 mL) and H2O (0.5 mL). The brain
was then transferred to a 5 mL homogenizer, and 40 µL (1
µg/mL) of an internal standard and 1% AcOH (1 mL) in 1:1
EtOH/CH3CN solution were then added. The brain was
homogenized and transferred to a high-speed tube. The
homogenizer was washed once with 1% AcOH (1 mL) in 1:1
EtOH/CH3CN solution and twice with CH3CN (0.5 mL) and
combined with the homogenized brain in the high-speed tube
and centrifuged at 20,000 rpm for 15 min at 4 °C. The extract
was transferred into a 5 mL glass tube, and the pellet was
washed once with 1% AcOH (1 mL) in 1:1 EtOH/CH3CN
solution and twice with CH3CN (0.5 mL) and added to the
extract. The homogenizer was washed as described above and
added to the extract. The extract was then concentrated to
100 µL in a speed vacuum chamber at ambient temperature.
The concentrated extract was transferred to a 1.5 mL tube,
and the original glass tube was washed once with a 1% AcOH
in a 1:2 H2O/CH3CN solution (400 µL) followed by 1% AcOH
in CH3CN (100 µL). The washes were combined with the
concentrated extract and centrifuged for 15 min at 4 °C. The
supernatant was transferred to another tube and dried using
a speed vacuum at ambient temperature. The resulting pellet
was suspended in 1% AcOH in MeOH (100 µL) and spun
briefly, and the supernatant was then analyzed.
(2S,3R)-2-Acetyla m in o-3-h yd r oxy-N-(2-m eth oxy-5-p en -
ta flu or oben zen e-su lfon yla m in op h en yl)bu tyr a m id e (6).
To a 0.08 M solution of fully protected amide 6 (2.14 g, 3.25
mmol) in CH2Cl2 at 0 °C was added TFA (20 mL), and the
resulting mixture was allowed to stir for 2 h at 0 °C. The crude
reaction was concentrated under vacuum with azeotropic
removal of TFA with hexane and used directly in the next
reaction. To a 0.06 M solution of crude amine (1.82 g, 3.25
mmol) at 0 °C was added 2,6-lutidine (760 µL, 6.5 mmol)
followed by acetic anhydride (336 µL, 3.58 mmol). The result-
ing mixture was allowed to stir for 1 h at 0 °C. The crude
reaction was acidified with 2 M aqueous HCl (100 mL) and
extracted 3 times with EtOAc (100 mL). The combined organic
layers were washed twice with saturated aqueous brine (150
mL), dried over Na2SO4, concentrated under vacuum, and used
directly in the next reaction. To a 0.03 M solution of crude
acetamide (1.95 g, 3.25 mmol) in AcOH was added 20% (50%
by wt) palladium hydroxide (3.46 g, 3.25 mmol) followed by
bubbling H2(g) through the solution for 1 min. The resulting
mixture was allowed to stir for 2 h under 1 atm of H2(g). The
crude reaction mixture was filtered through a pad of Celite,
and the Celite was then washed three times with EtOAc (50
mL). The filtrate was concentrated under vacuum with azeo-
tropic removal of AcOH with hexanes and purified by reverse
phase HPLC chromatography (15-45% CH3CN in a 0.1% TFA
aqueous solution) to yield 1.28 g of amide 6 (78%) as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ 10.9 (s, 1H), 9.1 (s, 1H),
8.05 (d, J ) 8.3 Hz, 1H), 7.99 (d, J ) 2.5 Hz, 1H), 7.0 (d, J )
8.8 Hz, 1H), 6.86 (dd, J ) 8.8, 2.6 Hz, 1H), 5.2 (d, J ) 4.8 Hz,
1H), 4.39 (dd, J ) 8.1, 3.6 Hz, 1H), 4.1-4.0 (m, 1H), 3.8 (s,
3H), 1.95 (s, 3H), 1.08 (d, J ) 6.3 Hz, 3H). Anal. Calcd for
P la sm a P r ep a r a tion . Plasma samples (50 µL) from each
animal in the cohort were combined. Proteins from the com-
bined aliquots were precipitated using 1% AcOH in CH3CN
(200 µL). The precipitated proteins were separated by cen-
trifugation at 10000g for 5 min, and the supernatant was then
analyzed.
The sample analyses were performed using a high perfor-
mance liquid chromatography mass spectrometer, Perkin-
Elmer Sciex (Toronto, Canada) API 365 triple quadruple mass
spectrometer. The mass spectrometer was interfaced via a
Sciex turbo ion spray probe to a liquid chromatograph,
consisting of a Shimadzu (Columbia, MD) LC-10AD pump
system and a DGU-14A degasser, and a Perkin-Elmer (Perkin-
Elmer, Norfolk, CT) series 200 autosampler. Separation of the
compounds and the interference was achieved using a YMC 5
µM C18 column (2 × 50 mm). Mass spectra were determined
with an ion source temperature at 300 °C and nitrogen gas
flow rate of 7 L/min. The ion source of the mass spectrometer
was operated in the negative ionization mode. The multiple
reaction monitoring (MRM) system, with a dwell time of 500
ms, was used for quantitation. The collision energy was set at
38 eV. Data were acquired by Sciex sample control and
MacQuan software.
In Vivo Effica cy Stu d ies. An im a ls. Male CD-1 outbred
mice, 8-10 weeks of age, were obtained from Charles River
(Hollister, CA, or Portage, MI). Female athymic nude mice,
5-8 weeks of age and weighing 18-20 g, were used (Simonsen
Laboratories, Gilroy, CA, or Harlan Sprague Dawley India-
napolis, IN). Animals were housed in groups on irradiated
corncob bedding in HEPA filtered ventilated rack housing.
Irradiated PMI Picolab 5058 rodent diet (PMI Mills, India-
napolis, IN) and autoclaved, hyperchlorinated water were
provided ad libitum.
C
19H18F5N3O6S‚0.5H2O: C, 43.81; H, 3.68; N, 8.07. Found: C,
43.87; H, 3.56; N, 7.89. MS(EI) m/z 511 (20, M+), 510 (100,
M-H).
Cytotoxicity An a lysis. The cytotoxicity analysis studies
were performed as previously described in ref 3.
In Vitr o Tu bu lin P olym er iza tion Rea ction . Ice-cold
bovine brain tubulin solution (400 µg in BRB80 buffer/10%
glycerol) was mixed with 49 µL of cold BRB80 buffer, 10 µL of
cold GTP solution (10 mM), and 1 µL of DMSO or 1 µL of
compound solution in DMSO. The mixture was immediately
transferred to a quartz cuvette equilibrated at 37 °C. Changes
in the optical density at 340 nm were monitored every 30 s in
a temperature-controlled photospectrometer (Hewlett-Packard)
at 37 °C.
In Vitr o Tu bu lin Bin d in g Com p etition Assa y. Ice-cold
bovine brain tubulin solution (0.5 µg in 8.9 µL BRB80 buffer
(80 mM PIPES (pH 6.8), 0.5 mM MgCl2, 1 mM EDTA, 10%
glycerol)) was mixed with 1 µL of DMSO or 1 µL of compound
solution in DMSO and incubated at 37 °C min in the presence
0.1 µL of 3H-T138067 (20 Ci/mmol; 1 mCi/ml) or 0.1 µL of
DMSO. Following a 90 min incubation, proteins were analyzed
by SDS-PAGE.
Br a in -P la sm a Con cen tr a tion Deter m in a tion Stu d ies.
T138067, aniline 2, and amides 3-6 were separately dissolved
in 0.4 M aqueous NaOH (5 mL) and titrated to a pH between
8 and 9 by dropwise addition of 1 M aqueous HCl. The final
volume was adjusted to 10 mL with the addition of saline (0.9%
NaCl) to furnish final concentrations between 3 and 4 mg/mL.
Five CD-1 male mice per cohort were administered between
30 and 40 mg/kg of desired compound by bolus injection into
the lateral tail vein within 10 s. The mice were then sacrificed
5-min post-dose administration. The animals were anesthe-
tized with carbon dioxide, and 0.5 mL of plasma was collected
from each animal by cardiac or vena cava puncture. The
plasma samples were placed in tubes containing EDTA, cooled
on ice until centrifugation, and stored at -20 to -70 °C until
final analysis. The brains were removed, flashed frozen in
Tu m or Cells. MX-1, a human mammary carcinoma, was
obtained from NCI, Frederick, MD. The tumor was maintained
as a solid tumor tissue harvested from donor mice. Tumor from
donor mice was minced thoroughly in sterile DMEM culture
medium (Mediatech, Herndon, VA) containing 1% penicillin/
streptomycin and 10% fetal bovine serum. A 200 µL aliquot of
the slurry was inoculated sc on the right flank of each mouse.
Tumor size was monitored beginning 2-3 days following
inoculation until it was an appropriate size.
Ma xim u m Toler a ted Dose Deter m in a tion Stu d ies.
Lethal dose assessment of test compounds was performed
using the “up-and-down” method.15
Tu m or Xen ogr a ft Effica cy Stu d ies. Animals bearing
tumors were randomly assigned to treatment and vehicle

Hydrophilic, Pro-Drug Analogues of T138067
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3605
(4) Medina, J . C.; Shan, B.; Beckmann, H.; Farrell, R. P.; Clark, D.
L.; Learned, R. M.; Roche, D.; Li, A.; Baichwal, V.; Case, C.;
Baeurle, P. A.; Rosen, T.; J aen, J . C. Novel antineoplastic agents
with efficacy against multidrug resistant tumor cells. Biorg. Med.
Chem. Lett. 1998, 8, 2653.
(5) Abstract 563: A Phase I Study of a Novel Antimicrotubule
Agent: T138067. This poster was presented at the November
2000 NCI-EORTC-AACR Symposium on new drugs in cancer
therapy in Amsterdam.
(6) (a) J udson, I.; Briasoulis, E.; Raynaud, F.; Hanwell, J .; Berry,
C.; Lacey, H. Phase 1 trial and pharmacokinetics of tubulin
inhibitor 1069C85 - a synthetic agent binding at the colchicine
site designed to overcome multidrug resistance. Br. J . Cancer
1997, 75, 4, 608. (b) Toide, K,; Unemi, N.; Segawa, T. Effects of
fluorinated pyrimidinedione anti-cancer drug, 5-fluoro-1-(tet-
rahydro-2-furanyl)-2,4-pyrimidinedione (FT), and related com-
pounds on nigro-striatal dopaminergic neurons in the central
nervous system. Arch. Int. Pharmacodyn. 1985, 274, 111.
(7) (a) Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R,; Fukuda,
Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T.
Synthesis and antitumor activity of cis-restricted combreta-
statins: 5-membered heterocyclic analogues. Biorg. Med. Chem.
Lett. 1998, 8, 3153. (b) Ohsumi, K.; Nakagawa, R.; Fukuda, Y.;
Hatanaka, T.; Morinaga, Y.; Nihei, Y.; Ohishi, K.; Akiyama, Y.;
Tsuji, T. Novel combretastatin analogues effective against
murine solid tumors: design and structure - activity relation-
ships. J . Med. Chem. 1998, 41, 3022.
control groups. Test compounds were administered iv at the
maximum tolerated dose determined by the method described
above. Mice were dosed every 7 days for a total of 2 or 3 doses.
Saline treated control groups were run parallel to all treatment
groups. Animals receiving T138067 or saline controls had a
cannula surgically implanted in their jugular vein. All animals
received similar doses of anesthesia. T138067 was adminis-
tered by intravenous infusion at a dose of 30 mg/kg/h for 4 h
at a rate of 2.5 mg/kg/h. A subsequent intravenous infusion
was given via a lateral tail vein. Body weights and tumor
volumes were measured at regular intervals posttreatment.
Animals that became moribund due to drug toxicity, that is
demonstrating a body weight loss greater than 20%, were
euthanized. Animals with ulcerating tumors, or tumors larger
than 2000 mm3, were euthanized. Studies were terminated
when the tumor of the last control animal became too large.
Tumor volume was calculated by the following formula:
length (mm) × (width (mm))2
volume (mm3) )
2
Data are expressed as the mean ratio of the daily tumor
volume for the compound treated group to its saline treated
control group.
(8) Clark, D.; et al. Manuscript in preparation.
(9) Albericio, F.; Bofill, J . P.; El-faham, A.; Kates, S. A. Use of onium
salt-based coupling reagents in peptide synthesis. J . Org. Chem.
1998, 63, 26, 9678.
(10) Racemization was observed when coupling N-acetyl-L-threonine
to aniline 2 using HAOT, HATU. The racemization was signifi-
cantly reduced, less than 5% based on 1H NMR analysis, when
N-BOC-L-threonine was used instead. No attempt was made to
determine the enantiomeric excess of the amide coupling reac-
tions.
(11) Taxol (paclitaxel) has an IC50 > 15000 nM against the MCF-7/
ADR cell line while its IC50 ) 1.1 ( 0.2 nM against the MCF-7
cell line. Vincristine has an IC50 ) 350 ( 71 nM against the
MCF-7/ADR cell line while its IC50 ) 2.3 ( 0.5 nM against the
MCF-7 cell line. The MCF-7/ADR cell line expresses the MDR
phenotype; see refs 3 and 4.
Ack n ow led gm en t. The authors thank Laura Hoff-
man, Heather Knych, and Colleen Stein for their
technical assistance in carrying out the animal studies.
Su p p or tin g In for m a tion Ava ila ble: Tubulin polymeri-
zation assay results for amides 3, 5, and 6 (as outlined in
Figure 3) and experimentally determined ratios of mean body
weight for T138067 and amides 3-6 from the efficacy studies
outlined in Figure 5. This material is available free of charge
via the Internet at http://pubs.acs.org.
Refer en ces
(12) Data not shown.
(13) Ubiquitous peptidases found in cells are generally selective for
natural amino acids over nonnatural amino acids. Walsh, C.
Enzymatic Reaction Mechanisms; W. H. Freeman and Com-
pany: New York, 1979; Chapters 2 and 3.
(14) Efficacy studies involving aniline 2 were not performed due to
its acute toxicity.
(15) The optimal dosing protocol for T138067 requires infusion over
several hours.
(16) Cotruvo, J . A.; Lipnick, R. L. EPA/OPPTS activities, 1993. In
Refinement and Reduction in Animal Testing; Niemi, S. M.,
Willson, J . E., Eds.; Scientists Center for Animal Welfare;
Bethesda, MD, 1993; pp 111-117.
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T.-C.; Learned, R. M.; Narbut, M. R.; Stott, D.; Wu, P.; J aen, J .
C.; Rosen, T.; Timmermans, P. B. M. W. M. Beckmann, H.
Selective, covalent modification of â-tubulin residue Cys-239 by
T138067, an antitumor agent with in vivo efficacy against
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96, 5686.
(3) Medina, J . C.; Roche, D.; Shan, B.; Learned, R. M.; Frankmoelle,
W. P.; Clark, D. L.; Rosen, T.; J aen, J . C. Novel halogenated
sulfonamides inhibit the growth of multidrug resistant MCF-7/
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J M000478D