Synthesis and antitumor activity of several new analogues of penclomedine and its metabolites

Article abstract of DOI:10.1021/jm010334e

Analogues of penclomedine (PEN, 3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)pyridine) and its metabolites have been synthesized and evaluated as potential antitumor agents. PEN and 4-DMPEN (3,5-dichloro-4-hydroxy-6-methoxy2-(trichloromethyl)pyridine (3a

Full text of DOI:10.1021/jm010334e

J . Med. Chem. 2002, 45, 1079-1085
1079
Syn th esis a n d An titu m or Activity of Sever a l New An a logu es of P en clom ed in e
a n d Its Meta bolites
Anita Tiwari,* J ames M. Riordan, William R. Waud, and Robert F. Struck
Drug Discovery and Drug Development Divisions, Southern Research Institute, 2000 Ninth Avenue South,
Birmingham, Alabama 35255-5305
Received J uly 23, 2001
Analogues of penclomedine (PEN, 3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)pyridine) and
its metabolites have been synthesized and evaluated as potential antitumor agents. PEN and
4-DMPEN (3,5-dichloro-4-hydroxy-6-methoxy2-(trichloromethyl)pyridine (3a )), the major plasma
metabolite in patients, were modified at 4- and 6-positions with different alkyl, aryl, and ester
groups. All of the analogues and many of the intermediates were evaluated against the PEN-
sensitive MX-1 human breast tumor xenograft in vivo, and several analogues of PEN and
4-DMPEN showed modest to curative activity.
In tr od u ction
of these agents are superior to PEN in their activity,
whereas previous studies from our laboratory had
demonstrated the absolute dependence on the trichlo-
romethyl substituent for antitumor activity in vivo.11
Since the antitumor activity of 4-DMPEN in vivo
indicated that it was on the metabolic activation path-
way, although it was inactive in vitro,11 we were
interested in determining if metabolic dealkylation or
dearylation of groups other than methyl at the 4-postion
might yield a more active agent than PEN. In earlier
studies, Reid et al.5 had observed demethylation of PEN
by liver microsomes but did not determine if demeth-
ylation occurred at the 4- or 6-position or both, while
later studies from our laboratory indicated that both
6-DM-PEN and DDM-PEN (3,5-dichoro-4,6-dihydroxy-
2-(trichloromethyl)pyridine) were inactive in vivo.11 All
of the analogues were evaluated against MX-1 human
breast carcinoma xenografts in vivo.
PEN1 (3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)-
pyridine) is a chemical alkylating agent as revealed by
its facile reaction with 4-(p-nitrobenzyl)pyridine in the
Epstein test2 (unpublished results). Plowman et al.3
reported that, of 28 agents active against breast tumor
lines in vivo, 23 of the 28 were known alkylating agents,
and PEN was one of the five other active agents,
suggesting that it may be functioning as a biological
alkylating agent. In addition, Harrison et al.4 observed
cross resistance in vivo to P388 leukemia lines resistant
to clinical DNA-DNA cross-linking agents but sensitiv-
ity to lines resistant to antimetabolites, topoisomerase
II inhibitors, and a mitotic inhibitor, consistent with
PEN functioning as a DNA cross-linking agent in vivo.
PEN is a lipophilic, highly substituted 2-methylpyridine
derivative synthesized by Dow Chemical Company as
an insecticide and was selected for clinical development
by the National Cancer Institute because of evidence
of good antitumor activity against mouse CD8F1 mam-
mary carcinoma and human MX-1 mammary tumor
xenograft.3,4 PEN is more active in vivo than in vitro,5,6
suggesting that it may be metabolized to a more potent
compound.
The antitumor activity of PEN against intracranially
implanted MX-1 xenografts was up to 4 times greater
than that of carmustine, the most commonly used
clinical agent for the treatment of primary brain tumors,
but in all clinical trials, dose limiting neurotoxicity7-9
was observed after both iv and oral administration.
4-DMPEN, as reported10,11 earlier, was shown to be an
excellent antitumor active metabolite of PEN in vivo
when evaluated against the PEN-sensitive MX-1 human
tumor xenograft without neurotoxicity. Synthesis and
analytical data for 4-DMPEN are reported here in
detail, and studies on 4-DMPEN are still in progress.
On the basis of these considerations, we have under-
taken the synthesis of analogues of PEN and 4-DMPEN
with modifications in both 4- and 6-positions with
different alkyl or aryl groups to determine whether any
Resu lts a n d Discu ssion
Ch em ist r y. The pyridine 112,13 was prepared by a
sequence of reactions starting from pentachloropyridine,
and PEN (2a ) was prepared by a reported method.1
Compounds 2b-d (Scheme 1) were prepared by the
same method as PEN by refluxing with two or more
molar equivalents of sodium hydroxide and the corre-
sponding alcohols, while 2e and 2f were prepared from
sodium hydride and trifluoroethanol in dioxane and
from sodium phenoxide in dioxane, respectively. Re-
moval of the alkyl or aryl groups in the 4-position of
compounds 2a -f was attempted with DMSO at 150 °C
as reported for 4-DMPEN,10 but only compounds 3a -c
were prepared, the others failing to react. For this
reason 2e was first converted to 6 and then demethy-
lated to give 7. Compounds 3b and 3c were methylated
by trimethylsilyldiazomethane to produce 4a and 4b in
good overall yields. Acetylation of 3a and 3b with acetic
anhydride or methoxyacetyl chloride, respectively, yielded
5a -c. Treatment of 2b with AlCl3 in CH2Cl2 afforded
the 4,6-dihydroxy compound (DDM-PEN) 8 which on
methylation gave 3d and on acetylation gave 9. Treat-
ment of 1 with sodium hydroxide and ethanol at room
temperature yielded 10b (Scheme 2). On methylation
* To whom correspondence should be addressed. Tel: (205) 581-
2427. Fax: (205) 581-2877. E-mail: a.tiwari@sri.org.
10.1021/jm010334e CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/31/2002

1080 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Tiwari et al.
Sch em e 1
Sch em e 2
with 1 molar equiv of NaOH at room temperature, 1
gave 10a , which on demethylation and acetylation
yielded 11 and 12, but when methylation was ac-
complished with 1 mol of NaOH at 90 °C, isomers 13a
and 14a were obtained. Similarly, equimolar amounts
of sodium phenoxide and 1 in dioxane at 100 °C
produced isomers 13b and 14b. Compounds 14a and
14b were treated with NaOH and different alcohols to
yield 15a -e. Compound 1612 (Scheme 3), prepared
through a series of reactions as described,12 was chlo-
rinated to give 17 that on methylation afforded 18.
Distinguishing the 4- and 6-isomeric forms of mono
DMPEN was solved by NMR studies on PEN, on each
monodemethylated PEN derivative and on a 13C-methyl-
labeled, methylated derivative of the synthetic mono-
demethylated product that was identical to the major
plasma metabolite in patients, using 13C-diazomethane
1
prepared from N-methyl-13C-diazald (Aldrich). H and
13C chemical shifts and coupling constants are shown
Sch em e 3

Synthesis and Activity of Penclomedine Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1081
Ta ble 1. Summary of the In Vivo Antitumor Activity of
Penclomedine and Its Metabolites and Analogues against
Subcutaneously Implanted Human Mammary Tumor Xenograft
MX-1 on a Five-Successive Day Schedule
optimal ip dosage
(<LD10
)
T - C
tumor-free
(survivors/total)
compd
(mg/kg/dose/day)
(days)
1
F igu r e 1. 1H NMR (CDCl3) δ 4.02 ppm (d, 3, 4-OCH3, J C,H
) 147.0 Hz); 4.10 (s, 3, 6-OCH3). 13C NMR (CDCl3) δ 55.24
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
4a
4b
5a
5b
5c
6
7
90
135
135
135
135
135
90
>49.0
>41.0
-0.7
-0.6
0.4
5/5
4/5
0/5
0/5
0/5
0/5
3/5
0/5
0/5
0/5
2/5
0/5
5/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
1
1
ppm (s, 6-OCH3, J C,H ) 147.5 Hz); 61.13 (br s, 4-OCH3, J C,H
) 147.0 Hz); 96.07 (s, -CCl3); 114.27 (d, C-5 or C-3, 3J C,C ) 1.5
3
Hz); 118.23 (d, C-3 or C-5, J C,C ) 1.1 Hz); 147.66 (s, C-2);
3
2
155.86 (s, C-6, J C,H ) 3.8 Hz); 162.40 (d, C-4, J C,C ) 3.6 Hz,
3J C,H ) 3.7 Hz). The multiplicities shown are from the
hydrogen decoupled spectrum. The J C,H’s are from the hydro-
gen coupled spectrum.
0.2
>37.2
60
5.2
135
135
200
135
90
90
135
90
90
40
40
50
toxic
50
135
90
60
60
90
135
135
-0.9
0.8
>38.6
-0.8
>41.0
1.4
17.8
1.2
-0.2
-0.5
-0.5
2.0
in Figure 1 for 13C-PEN along with the description of
how the structure for the major plasma metabolite was
assigned.
The 13C labeling of one of the methyl groups allowed
the determination of the structure of monodemethyl-
penclomedine corresponding to the major plasma me-
tabolite and also allowed the assignments of the methyl
groups in the 1H spectrum of PEN. It is well-known that
ortho and para carbons of the pyridine ring will be the
most downfield of the pyridine carbon atoms (larger
chemical shifts). The meta carbons of the pyridine ring,
the ones with attached chlorine, are the most upfield.
13C labeling of one of the methyl groups with a 13C
carbon atom allowed the direct observation of long-range
8
9
11
12
13a
14a
15a
15b
15c
15d
15e
18
4.3
-1.4
>42.7
>35.2
5.4
20.5
2.3
-0.8
0/5
0/5
5/5
2/5
0/5
1/5
0/5
0/5
n
carbon-carbon coupling constants, J c,c. Four and five
4
5
bond carbon-carbon couplings, J c,c and J c,c, are very
3
2
rare. J c,c and J c,c are the most commonly observed
4
couplings, and both are generally larger than J c,c and
for the diethyl analogue 2b but not for 2c-f. In four
separate experiments, 2b produced either modest to
high noncurative activity in two experiments and cura-
tive activity in the other two, yielding 60% and 80%
tumor-free survivors, respectively.
5J c,c values. The above 13C NMR data show that both
3
meta carbons of pyridine have J c,c values of 1.1 and
1.5 Hz and para carbon has corresponding J c,c of 3.6
Hz. Therefore, the methyl with the label is in the para
position of the pyridine ring.
2
Of interest was the observation that the 4-hydroxy-
6-alkoxy analogue 7 was inactive and the 4-hydroxy-6-
ethoxy analogue 3b was only modestly active. Our
proposed mechanism of action of PEN shown in Figure
2 includes metabolic demethylation at the 4-position in
the liver to generate 4-DMPEN, followed by metabolic
activation of the trichloromethyl group to an intermedi-
ate capable of alkylating tumor cell DNA to produce a
DNA adduct. The adduct is then capable of keto-enol
tautomerism at the 4,5-positions to produce an R-chlo-
roketone moiety capable of completing a DNA-DNA
cross-link, which is presumably the cytotoxic lesion. The
proposed mechanism includes conversion of the trichlo-
romethyl group to a free radical, based on studies by
Reid et al.5 in which the major product of incubation of
PEN with liver microsomes resulted from free radical
coupling of two PEN molecules through their trichlo-
romethyl groups and also based on studies by Ben-
venuto et al.6 in which incubation of [14C] PEN with rat
liver S-9 fraction in the presence of calf thymus DNA
resulted in the stable transfer of radioactivity to DNA.
Addition of butylated hydroxytoluene, a free radical
scavenger, to the incubation mixture inhibited the
binding of drug to DNA, implicating free radicals in the
formation of the reactive species. These data suggest
that PEN can be metabolized to free radical, DNA-
reactive products. Analogues 3b and 7 would similarly
be able to activate the 5-chloro group, but their modest
Biologica l Eva lu a tion . All of the analogues and
some of the intermediates were evaluated against
staged, subcutaneously implanted human breast tumor
xenograft MX-1 in vivo with intraperitoneal treatment
on a successive 5 day schedule, which was shown
previously to be optimal for PEN and 4-DMPEN11 (Table
1). While most of the analogues were inactive, the
limited SAR study revealed that only minor variation
from the parent structure was permissible at the 4- and
6-positions for retention of high or even modest activity.
Previous investigations had established the absolute
necessity of the 2-trichloromethyl moiety and allowed
demethylation of the 4- but not the 6-position.11
In the present study, the 4-methyl group could be
replaced with ethyl 15a , isopropyl 15b, or butyl 15d for
curative activity, although the percentage of tumor-free
survivors decreased with increasing number of carbons.
The ethyl analogue 15a was comparably active to PEN
itself, yielding 100% tumor-free survivors. Replacement
of the 6-methyl group was more restrictive, since activity
was observed only for the ethyl analogue 4a but not for
the trifluoroethyl 6, butyl 4b, and phenyl 15e analogues.
Three separate experiments with the ethyl analogue 4a
indicated modest activity in two experiments and some
curative activity in the third experiment (40% tumor-
free survivors). Replacement of both methyl groups was
similarly restrictive, with activity being observed only

1082 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Tiwari et al.
dryness. The compound was extracted with CH2Cl2 (2 × 20
mL) and washed with water. The organic layer was dried over
MgSO4, filtered, and concentrated to dryness. The crude
product was purified by column chromatography (silica gel
230-400 mesh, elution with hexanes). The desired fractions
were combined, concentrated, and dried in vacuo over P2O5:
yield 1.53 g (78.86%); mp 72-74 °C; MS m/z 324 (M + H)+; 1H
NMR (CDCl3) δ 4.02 (s, 3H, 4-CH3), 4.09 (s, 3H, 6-CH3).
3,5-Dich lor o-4,6-d ie t h oxy-2-(t r ich lor om e t h yl)p yr i-
d in e (2b). Compound 2b was prepared from compound 1 (450
mg, 1.34 mmol), sodium hydroxide (216 mg, 5.4 mmol), and
ethanol (4.5 mL) using the same procedure described for the
preparation of 2a : yield after column chromatography (97:3
hexanes:methylene chloride) 286 mg (60.2%); mp 27-28 °C;
MS m/z 352 (M + H)+; 1H NMR (CDCl3) δ 1.46 (t, 3H, 4-CH3),
1.51 (t, 3H, 6-CH3), 4.24 (q, 2H, 4-CH2), 4.53(q, 2H, 6-CH2);
Anal. (C10H10Cl5NO2) C, H, N.
3,5-Dich lor o-4,6-d iisop r op oxy-2-(tr ich lor om eth yl)p y-
r id in e (2c). The same procedure as described for 2a was used
to prepare 2c from compound 1 (500 mg, 1.49 mmol), sodium
hydroxide (220 mg, 5.5 mmol), and 2-propanol (15 mL): yield
after column chromatography (hexanes) 326 mg, liquid (57.2%);
MS m/z 380 (M + H)+; 1H NMR (CDCl3) δ 1.42 (d, 6H, 4-CH3,
J ) 6.3 Hz), 1.43 (d, 6H, 6-CH3, J ) 6.3 Hz), 4.83 (m, 1H, 4-CH),
5.37 (m, 1H, 6-CH); Anal. (C12H14Cl5NO2) C, H, N.
3,5-Dich lor o-4,6-d ib u t oxy-2-(t r ich lor om e t h yl)p yr i-
d in e (2d ). The same procedure previously described for 2a
was used to prepare 2d from compound 1 (800 mg, 2.39 mmol),
sodium hydroxide (352 mg, 8.8 mmol), and n-butanol (25
mL): yield after column chromatography (hexanes) 511 mg,
liquid (52.14%); MS m/z 408 (M + H)+; 1H NMR (CDCl3) δ 0.98
(t, 3H, 4-CH3), 1.00 (t, 3H, 6-CH3), 1.43-1.63 (m, 4H, CH2-
CH3), 1.77-1.90 (m, 4H, CH2-CH2), 4.15 (t, 2H, 4-OCH2), 4.46
(t, 2H, 6-OCH2); Anal. (C14H18Cl5NO2) C, H, N.
3,5-Dich lor o-4,6-ditr iflu or oeth oxy-2-(tr ich lor om eth yl)-
p yr id in e (2e). To a stirred suspension of NaH (287 mg, 11.95
mmol) in 10 mL of anhydrous dioxane was added compound 1
(1.00.g, 2.99 mmol). After being stirred for 20 min, the solution
was treated with 2,2,2-trifluoroethanol (1.21 g, 0.87 mL, 11.95
mmol) and heated at 110 °C for 1.5 h. The reaction mixture
was cooled and concentrated to dryness. The compound was
extracted with CH2Cl2 (2 × 20 mL) and washed with water.
The organic layer was dried over MgSO4, filtered, and con-
centrated to dryness. The resulting oil was purified by column
chromatography (silica gel 230-400 mesh) using hexanes. The
desired fractions were combined, concentrated, and dried in
vacuo over P2O5 to give an oil that solidified in the freezer:
yield 315 mg (22.82%); mp 50-52 °C; MS m/z 460 (M + H)+;
1H NMR (CDCl3) δ 4.58 (q, 2H, 4-OCH2), 4.88 (q, 2H, 6-OCH2);
Anal. (C10H4F6Cl5NO2) C, H, N.
F igu r e 2. Proposed mechanism of action of penclomedine
(PEN).
activity to inactivity demonstrates a pharmacologic role
for the 6-substituent in these two analogues, although
analogues 2b and 4a are presumably metabolized to 3b
in vivo. Didemethyl-PEN 8 was previously reported to
be inactive,11 and its diacetyl derivative 9 was similarly
inactive. In addition, the analogues in which a chloro
group replaced the 4- or 6-methoxy group (13a , 14a )
were inactive. Finally, a single analogue 18 in which
the 6-methoxy group was replaced with a trichloro-
methyl group was also inactive.
Synthesis and evaluation of a series of 4-acyl deriva-
tives of 4-DMPEN illustrated by 5a revealed consistent
and high activity14 whereas the 6-ethyl analogues 5b
and 5c were inactive or only modestly active, respec-
tively.
Exp er im en ta l Section
Melting points were determined on a Mel-Temp apparatus
and are uncorrected. TLC was carried out on Analtech pre-
coated (250 µm) silica gel (GF) plates. All chromatographic
separations were carried out by flash chromatography using
230-400 mesh silica gel from E. Merck. Analytical results
indicated by element symbols were within (0.4% of the
theoretical values, and where solvents were indicated in the
formula, their presence was confirmed by 1H NMR. The 1H
NMR spectra and 13C NMR spectra in Me2SO-d6 or CDCl3 with
tetramethylsilane as an internal reference were determined
with a Nicolet NT 300 NB spectrometer at 300.635 MHz for
1H NMR spectra and at 75.6 MHz for 13C NMR spectra.
Chemical shifts (δ) quoted for multiplets were measured from
the approximate centers, and relative integrals of peak areas
agreed with those expected for the assigned structures. Mass
spectra were recorded on a Varian/MAT 311A mass spectrom-
eter in the fast atom bombardment (FAB) mode.
3,4,5,6-Tetr ach lor o-2-(tr ich lor om eth yl)pyr idin e (1). This
compound was prepared by a reported method as described
in refs 12a-c and 13.
3,5-Dich lor o-4,6-d im et h oxy-2-(t r ich lor om et h yl)p yr i-
d in e (P EN) (2a ). This compound was prepared by a reported
method.1 To a solution of NaOH (598 mg, 14.95 mmol) in
anhydrous methanol (25 mL) was added compound 1 (2.00 g,
5.98 mmol), and the reaction mixture was heated to reflux for
2 h. The reaction mixture was cooled and concentrated to
3,5-Dich lor o-4,6-d ip h en oxy-2-(t r ich lor om et h yl)p yr i-
d in e (2f). Compound 1 (800 mg, 2.39 mmol) was dissolved in
1 mL of anhydrous dioxane. Sodium phenoxide trihydrate (1.22
g, 7.17 mmol) was added, and the solution was heated to reflux
for 3 h. The reaction mixture was cooled and concentrated to
dryness. The compound was extracted with CH2Cl2 (2 × 30
mL) and washed with water. The organic layer was dried over
MgSO4, filtered, and concentrated to dryness. The residue was
purified by column chromatography (silica gel 230-400 mesh,
elution with hexanes). The desired fractions were collected,
concentrated, and dried in vacuo over P2O5 to give a white
solid: yield 778 mg (72.71%); mp 72-74 °C; MS m/z 448 (M +
1
H)+; H NMR (CDCl3) δ 6.91 (bd, 2H, 4-C6H5 (2,6)), 7.14 (bt,
1H, 4-C6H5 (4)), 7.31-7.23 (m, 3H, 6-C6H5 (2,4,6)), 7.37 (m,
2H, 4-C6H5 (3,5)), 7.43 (bt, 2H, 6-C6H5 (3,5)); Anal. (C18H10Cl5-
NO2) C, H, N.
3,5-Dich lor o-4-h ydr oxy-6-m eth oxy-2-(tr ich lor om eth yl)-
p yr id in e (4-DMP EN) (3a ). Compound 2a (6.00 g, 18.43
mmol) was dissolved in 20 mL of anhydrous DMSO and heated
at 150 °C for 45 min. The reaction mixture was cooled and
lyophilized. The residue was treated with 50 mL of ether with
stirring. The ether layer was decanted from an insoluble syrup
and treated with 100 mL of hexanes, allowed to stand until
the supernate was clear, and decanted from a precipitated oil.

Synthesis and Activity of Penclomedine Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1083
The supernate was evaporated to dryness, and the compound
stirred at room temperature for 2 days and concentrated to
dryness. The reaction did not go to completion. The product
was purified by column chromatography (silica gel 230-400
mesh, elution with CHCl3). The desired fractions were col-
lected, concentrated, and dried in vacuo over P2O5: yield 477
was dried in vacuo over P2O5: yield 3.00 g (52.26%); mp 52-
1
54 °C; MS m/z 310 (M + H)+; H NMR (CDCl3) δ 4.09 (s, 3H,
6-OCH3), 7.00 (bs, 1H, OH); Anal. (C7H4Cl5NO2) C, H, N.
3,5-Dich lor o-6-eth oxy-4-h yd r oxy-2-(tr ich lor om eth yl)-
p yr id in e (3b). The general procedure previously described for
3a was used to prepare 3b using 2b (1.48 g, 4.18 mmol) and
DMSO (15 mL), and the reaction mixture was heated for 2.5
h. Starting material remained but since the solution was
getting darker, heating was stopped. The product was purified
by column chromatography (95:5 chloroform:methanol) to give
an oil that solidified upon freezing: yield 371 mg (27.28%);
mp 57-59 °C; MS m/z 324 (M + H)+; 1H NMR (CDCl3) δ 1.45
(t, 3H, CH3), 4.54 (q, 2H, CH2), 6.65 (s, 1H, OH); Anal. (C8H6-
Cl5NO2‚0.15C2H5OH) C, H, N.
3,5-Dich lor o-6-bu toxy-4-h yd r oxy-2-(tr ich lor om eth yl)-
p yr id in e (3c). The same procedure as described for 3a was
used to prepare 3c from 2d (2.00 g, 4.88 mmol) and DMSO
(18 mL), and the reaction mixture was heated for 5.5 h.
Starting material remained but since the solution was getting
darker, heating was stopped. The product was purified by
column chromatography (chloroform) to give an oil: yield 442
mg (25.7%); MS m/z 352 (M + H)+; 1H NMR (CDCl3) δ 0.93 (t,
3H, CH3), 1.42 (m, 2H, CH2CH3), 1.73 (m, 2H, CH2CH2), 4.41
(t, 2H, OCH2); Anal. (C10H10Cl5NO2‚0.3C2H5OH) C, H, N.
3,5-Dich lor o-6-h ydr oxy-4-m eth oxy-2-(tr ich lor om eth yl)-
p yr id in e (6-DMP EN) (3d ). DidemethylPEN 8 (2.00 g, 6.72
mmol) in 10 mL of methanol was treated dropwise with
stirring at room temperature with 30 mL of ethereal di-
azomethane prepared from Diazald as reported. The solution
was stirred 10 min and evaporated in vacuo. The residue was
separated on an 8 in. 2 mm silica gel plate (Analtech) in
dichloromethane:methanol (9:1), and elution of the UV-visible
band at Rf 0.7 with methanol and evaporation gave a solid
(345 mg); bands at Rf 0.9 and Rf 0.5 were extracted and
identified by co-TLC as PEN and 4-DMPEN, respectively. The
solid was separated again by preparative TLC in the same
solvent, and elution with methanol and evaporation gave a
white solid (250 mg). The solid was dissolved in 5 mL of ether
followed by 20 mL of hexanes. Concentration in vacuo to 5 mL
and filtration gave a white crystalline solid, which was
homogeneous upon analytical TLC and whose Rf was different
from the Rfs of PEN and 4-DMPEN upon co-TLC in CH2Cl2:
MeOH (9:1): yield 170 mg (8.13%); MS m/z 310 (M + H)+; 1H
NMR (CDCl3) δ 4.02 (s, 3H, 4-OCH3), 2.50 (vbs, 1H, OH).
3,5-Dich lor o-6-eth oxy-4-m eth oxy-2-(tr ich lor om eth yl)-
p yr id in e (4a ). To a solution of compound 3b (824 mg, 2.53
mmol) in 20 mL of anhydrous acetone was added trimethyl-
silyldiazomethane solution (2 M solution in hexanes, 2.53 mL),
and the reaction mixture was stirred for 45 min at room
temperature. The reaction mixture was evaporated to dryness.
The residual product was purified by column chromatography
(silica gel 230-400 mesh) and was eluted with hexanes. The
desired fractions were combined, concentrated, and dried in
vacuo over P2O5 to give an oil that solidified in the freezer:
yield 764 mg (88.94%); mp 42-44 °C; MS m/z 338 (M + H)+;
1H NMR (CDCl3) δ 1.46 (t, 3H, 6-CH3), 4.02 (s, 3H, 4-CH3),
4.54 (q, 2H, CH2); 13C NMR (CDCl3) δ 14.38 (CH3), 61.10
(OCH3), 64.21 (OCH2), 96.21 (CCl3), 114.29, 117.83 (3-C, 5-C),
147.62 (2-C), 155.61 (6-C), 162.41 (4-C); Anal. (C9H8Cl5NO2)
C, H, N.
1
mg (42.21%); MS m/z 352 (M + H)+; H NMR (CDCl3) δ 2.45
(s, 3H, 4-CH3), 4.18 (s, 3H, 6-CH3); 13C NMR (CDCl3) δ 20.20
(s, CH3 of Ac), 55.44 (OCH3), 95.69 (CCl3), 114.78, 117.40
(3-C, 5-C), 147.68 (2-C), 154.39 (4-C), 155.60 (6-C), 165.86
(CdO); Anal. (C9H6Cl5NO3‚0.1CHCl3.) C, H, N.
3,5-Dich lor o-6-et h oxy-4-a cet oxy-2-(t r ich lor om et h yl)-
p yr id in e (5b). Compound 5b was prepared from 3b (400 mg,
1.22 mmol), DMAP (148 mg), and Ac2O (137 mg, 1.34 mmol).
After column chromatography (9:1 hexanes:CH2Cl2), a solid
was obtained: yield 220 mg (48.78%); mp 63-65 °C; MS m/z
1
366 (M + H)+; H NMR (CDCl3) δ 1.47 (t, 3H, CH2CH3), 2.45
(s, 3H, COCH3), 4.56 (q, 2H, OCH2); Anal. (C10H8Cl5NO3) C,
H, N.
3,5-Dich lor o-6-eth oxy-4-m eth oxya cetoxy-2-(tr ich lor o-
m eth yl)p yr id in e (5c). The same procedure as described for
5a was used to prepare 5c from 3b (240 mg, 0.73 mmol),
DMAP (90 mg), and methoxyacetyl chloride (96 mg, 0.88
mmol). After column chromatography (6:4 hexanes:dichlo-
romethane), an oil was obtained: yield 220 mg (75.08%); MS
m/z 396 (M + H)+; 1H NMR (CDCl3) δ 1.48 (t, 3H, 6-CH2CH3),
3.58 (s, 3H, 4-OCH3), 4.46 (s, 2H, 4-COCH2), 4.57 (q, 2H,
6-OCH2); Anal. (C11H10Cl5NO4) C, H, N.
3,5-Dich lor o-4-m et h oxy-6-t r iflu or oet h oxy-2-(t r ich lo-
r om eth yl)p yr id in e (6). A mixture of compound 2e (545 mg,
1.18 mmol), NaOH (54 mg, 1.35 mmol), and anhydrous
methanol was stirred at room temperature for 4 h. The
reaction mixture was concentrated to dryness. The compound
was extracted with CH2Cl2 (2 × 20 mL) and washed with
water. The organic layer was dried over MgSO4, filtered, and
concentrated to dryness. The residue was purified by column
chromatography (silica gel 230-400 mesh, elution with hex-
anes). The desired fractions were collected, concentrated, and
dried in vacuo over P2O5 to give an oil that solidified in the
freezer: yield 444 mg (95.68%); mp 48-50 °C; MS m/z 392(M
1
+ H)+; H NMR (CDCl3) δ 4.06 (s, 3H, 4-OCH3), 4.87 (q, 2H,
OCH2); Anal. (C9H5F3Cl5NO2) C, H, N.
3,5-Dich lor o-4-h yd r oxy-6-t r iflu or oet h oxy-2-(t r ich lo-
r om et h yl)p yr id in e (7). The same procedure previously
described for 3a was used to prepare 7 from compound 6 (540
mg, 1.37 mmol) and DMSO (18 mL) by heating for 1.5 h at
150 °C. After column chromatography (7:1 chloroform:metha-
nol), an oil was obtained: yield 555 mg (99.46%); MS m/z 378
1
(M + H)+; H NMR (CDCl3) δ 1.8 (bs, 1H, OH), 4.86 (q, 2H,
OCH2); Anal. (C8H3F3Cl5NO2‚0.1H2O) C, H, N.
3,5-Dich lor o-4,6-d ih yd r oxy-2-(t r ich lor om et h yl)p yr i-
d in e (8). Compound 2b (1.00 g, 2.8 mmol) was dissolved in
20 mL of anhydrous dichloromethane. To this solution was
added AlCl3 (1.00 g). The reaction mixture was stirred for 1 h
at room temperature and evaporated to dryness. The crude
product was purified by column chromatography (silica gel
230-400 mesh, elution with 5:1 CHCl3:MeOH). The desired
fractions were collected, concentrated, and dried in vacuo over
P2O5: yield 818 mg (97%); MS m/z 295 (M + H)+; 1H NMR
(CDCl3) δ 3.42 (bs, 2H, OH); 13C NMR (CDCl3) δ 92.71 (CCl3),
107.07, 109.24 (3-C, 5-C), 142.54 (2-C), 155.93 (6-C), 158.67
(4-C).
3,5-Dich lor o-6-bu toxy-4-m eth oxy-2-(tr ich lor om eth yl)-
p yr id in e (4b). The same procedure as described for 4a was
used to prepare 4b from 3c (798 mg, 2.25 mmol) and tri-
methylsilyldiazomethane solution (2 M solution in hexanes,
2.24 mL). After column chromatography (hexanes), an oil was
obtained: yield 718 mg (86.6%); MS m/z 366 (M + H)+; 1H
NMR (CDCl3) δ 0.98 (t, 3H, 6-CH3), 1.49 (m, 2H, CH2CH3),
1.82 (m, 2H, CH2CH2), 4.02 (s, 3H, 4-CH3), 4.47 (t, 2H, OCH2);
Anal. (C11H12Cl5NO2) C, H, N.
3,5-Dich lor o-6-m eth oxy-4-a cetoxy-2-(tr ich lor om eth yl)-
p yr id in e (5a ). To a solution of compound 3a (1.00 g, 3.21
mmol) in 25 mL of anhydrous CH2Cl2 was added DMAP (375
mg) and Ac2O (327 mg, 3.21 mmol). The reaction mixture was
3,5-Dich lor o-4,6-d ia cet oxy-2-(t r ich lor om et h yl)p yr i-
d in e (9). Compound 8 (950 mg, 3.19 mmol) in 2 mL of Ac2O
and 2 drops of pyridine was heated at 80 °C for 15 min, allowed
to stand 30 min at room temperature, and stored at -20 °C
overnight. The solvent was removed, and the residue was dried
in vacuo. The product was purified by column chromatography
(silica gel 230-400 mesh, elution with CH2Cl2). The desired
fractions were collected, concentrated, and dried in vacuo over
P2O5: yield 600 mg (49.58%); mp 122-124 °C; MS m/z 380 (M
1
+ H)+; H NMR (CDCl3) δ 2.42 (s, 3H, 4-OCH3), 2.47 (s, 3H,
6-OCH3); 13C NMR (CDCl3) δ 20.13 (CH3 of 4-Ac), 20.57 (CH3
of 6-Ac), 94.45 (CCl3), 121.42, 123.72 (3-C, 5-C), 149.36, 149.90

1084 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Tiwari et al.
(2-C, 6-C), 155.58 (4-C), 165.39 (CdO of 4-Ac), 166.98 (CdO
of 6-Ac); Anal. (C10H6Cl5NO4) C, H, N.
3,5-Dich lor o-4-eth oxy-6-m eth oxy-2-(tr ich lor om eth yl)-
p yr id in e (15a ). Sodium hydroxide (88 mg, 2.2 mmol) was
dissolved in 12 mL of anhydrous ethanol. After 20 min, 14a
(730 mg, 2.2 mmol) was added. The reaction mixture was
stirred at room temperature for 7 days and concentrated to
dryness. The compound was extracted with CH2Cl2 (2 × 20
mL), and the extract was washed with water. The organic layer
was dried over MgSO4, filtered, and concentrated to dryness.
The resulting oil was purified by column chromatography
(hexanes) to give an oil: yield 525 mg (69.9%); MS m/z 338
(M + H)+; 1H NMR (CDCl3) δ 1.51 (t, 3H, 4-CH3), 4.09 (s, 3H,
6-CH3), 4.24 (q, 2H, CH2); 13C NMR (CDCl3) δ 15.58 (CH3),
55.22 (OCH3), 70.50 (OCH2), 96.20 (CCl3), 114.40, 118.48 (3-
C, 5-C), 147.61 (2-C), 155.85 (6-C), 161.86 (4-C); Anal. (C9H8-
Cl6NO2) C, H, N.
4-Me t h oxy-3,5,6-t r ich lor o-2-(t r ich lor om e t h yl)p yr i-
d in e (10a ). Compound 10a is the same as 13a but it was
prepared under different reaction conditions. To a solution of
NaOH (90 mg, 2.25 mmol) in anhydrous methanol (10 mL)
was added compound 1 (749 mg, 2.24 mmol), and the reaction
mixture was stirred at room temperature overnight. No isomer
was obtained. Isolation of 10a was the same as described for
13a : yield 497 mg (67.25%).
4-E t h oxy-3,5,6-t r ich lor o-2-(t r ich lor om et h yl)p yr id in e
(10b). The procedure was the same as reported above for 10a
using 1 (1.5 g, 4.48 mmol), NaOH (179 mg, 4.48 mmol), and
anhydrous ethanol (24 mL). In this case, a small amount of
the 6-ethoxy isomer was also obtained. After column chroma-
tography (hexanes), a solid was obtained: yield 1.21 g (78.5%);
mp 42-44 °C; MS m/z 342 (M + H)+; 1H NMR (CDCl3) δ 1.53
(t, 3H, 4-CH3), 4.28 (q, 2H, 4-OCH2); Anal. (C8H5Cl6NO) C, H,
N.
4-H yd r oxy-3,5,6-t r ich lor o-2-(t r ich lor om e t h yl)p yr i-
d in e (11). Compound 11 was prepared by the same procedure
as described for the preparation of 3a using 10a (786 mg, 2.38
mmol) and DMSO (15 mL). After column chromatography (7:3
chloroform:methanol), a foamy solid was obtained: yield 678
mg (90.15%); MS m/z 314 (M + H)+; 1H NMR (Me2SO-d6) δ
7.8 (bs, 1H, OH); Anal. (C6H1Cl6NO‚1.0CH3OH) C, H, N.
4-Ace t oxy-3,5,6-t r ich lor o-2-(t r ich lor om e t h yl)p yr i-
d in e (12). Compound 11 (656 mg, 2.07 mmol) in 2 mL of Ac2O
and a few drops of pyridine was heated at 80 °C overnight.
The reaction mixture was cooled and evaporated to dryness.
The residual product was purified by column chromatography
(silica gel 230-400 mesh) by elution with 97:3 hexanes:
dichloromethane. The desired fractions were combined, con-
centrated, and dried in vacuo over P2O5 to give a solid: yield
332 mg (44.6%); mp 96-98 °C; MS m/z 356 (M + H)+; 1H NMR
(CDCl3) δ 2.48 (s, 3H, CH3); Anal. (C8H3Cl6NO2) C, H, N.
3,5-Dich lor o-4-isop r op oxy-6-m e t h oxy-2-(t r ich lor o-
m eth yl)p yr id in e (15b). The same procedure used to prepare
15a was used to prepare 15b except in this case, after 1 day
of stirring at room temperature, the reaction mixture was
heated at 65 °C for 2 days using 14a (600 mg, 1.81 mmol),
NaOH (72 mg, 1.8 mmol), and 2-propanol (20 mL). After
column chromatography (hexanes), an oil was obtained that
solidified upon freezing: yield 358 mg (55.67%); mp 26-28 °C;
1
MS m/z 352 (M + H)+; H NMR (CDCl3) δ 1.42 (d, 6H, CH-
(CH3)2, J ) 6.2 Hz), 4.09 (s, 3H, 6-OCH3), 4.85 (m, 1H, CH);
Anal. (C10H10Cl5NO2) C, H, N.
3,5-Dich lor o-4-pr opoxy-6-m eth oxy-2-(tr ich lor om eth yl)-
p yr id in e (15c). Compound 15c was prepared by the same
procedure as reported for 15b except the temperature was kept
at 55 °C using 14a (600 mg, 1.81 mmol), NaOH (72 mg, 1.8
mmol), and n-propanol (20 mL). After column chromatography
(hexanes), an oil was obtained: yield 428 mg (66.56%); MS
1
m/z 352 (M + H)+; H NMR (CDCl3) δ 1.10 (t, 3H, CH2CH3),
1.92 (m, 2H, CH2CH3), 4.09 (s, 3H, 6-OCH3), 4.12 (t, 2H,
4-OCH2); Anal. (C10H10Cl5NO2) C, H, N.
3,5-Dich lor o-4-bu toxy-6-m eth oxy-2-(tr ich lor om eth yl)-
p yr id in e (15d ). The procedure described for 15b was used to
prepare 15d from 14a (500 mg, 1.51 mmol), NaOH (60 mg,
1.5 mmol), and n-butanol (15 mL). After column chromatog-
raphy (hexanes), an oil was obtained that solidified upon
freezing: yield 217 mg (39%); mp 33-35 °C; MS m/z 366 (M
+ H)+; 1H NMR (CDCl3) δ 1.00 (t, 3H, CH2CH3), 1.56 (m, 2H,
CH2CH3), 1.87 (m, 2H, CH2CH2), 4.09 (s, 3H, 6-OCH3), 4.16
(t, 2H, 4-OCH2); Anal. (C11H12Cl5NO2) C, H, N.
3,5-Dich lor o-4-m eth oxy-6-ph en oxy-2-(tr ich lor om eth yl)-
p yr id in e (15e). Compound 15e was prepared from 14b (510
mg, 1.3 mmol), NaOH (62 mg, 1.55 mmol), and methanol (15
mL) by refluxing for 2.5 h using the procedure described for
the preparation of 6. After column chromatography (hexanes),
a white solid was obtained: yield 358 mg (71%); mp 91-93
°C; MS m/z 386 (M + H)+; 1H NMR (CDCl3) δ 4.09 (s, 3H,
4-CH3), 7.20-7.26 (m, 3H, C6H5 (2,4,6)), 7.38-7.43 (m, 2H,
C6H5 (3,5)); 13C NMR (CDCl3) δ 61.25 (OCH3), 95.58 (CCl3),
115.11 (3-C), 119.59 (5-C), 121.54 (C2, C6, Ph), 125.34 (C4,
Ph), 129.24 (C3, C5, Ph), 147.82 (C1, Ph), 152.69 (2-C), 154.94
(6-C), 163.11 (4-C); Anal. (C13H8Cl5NO2) C, H, N.
4-Me t h oxy-3,5,6-t r ich lor o-2-(t r ich lor om e t h yl)p yr i-
d in e (13a ) a n d 6-Met h oxy-3,4,5-t r ich lor o-2-(t r ich lor o-
m eth yl)p yr id in e (14a ). Compound 1 (5.00 g, 14.9 mmol) and
sodium hydroxide pellets (600 mg, 15 mmol) in 60 mL of
anhydrous methanol in a round-bottom flask were heated at
90 °C for 45 min. The reaction mixture was cooled and
concentrated to dryness. The compound was extracted with
CH2Cl2 (2 × 70 mL), and the extract was washed with water.
The organic layer was dried over MgSO4, filtered, and con-
centrated to dryness. The crude product was purified by
column chromatography. The column (packed with silica gel
230-400 mesh) was eluted with hexanes. The desired fractions
were collected, concentrated, and dried over P2O5 in vacuo
yielding two isomers. F1 (13a ): yield 4.17 g (84.5%); mp 69-
1
70 °C; MS m/z 328 (M + H)+; H NMR (CDCl3) δ 4.07 (s, 3H,
4-CH3); Anal. (C7H3Cl6NO) C, H, N. F2 (14a ): yield 462 mg
(9.37%); mp 78-80 °C; MS m/z 328 (M + H)+; 1H NMR (CDCl3)
δ 4.12 (s, 3H, 6-CH3); Anal. (C7H3Cl6NO) C, H, N.
4-P h e n oxy-3,5,6-t r ich lor o-2-(t r ich lor om e t h yl)p yr i-
d in e (13b) a n d 6-P h en oxy-3,4,5-t r ich lor o- 2-(t r ich lor o-
m eth yl)p yr id in e (14b). Compound 1 (1.6 g, 4.78 mmol) was
dissolved in 18 mL of anhydrous dioxane. To this solution was
added sodium phenoxide trihydrate (814 mg, 4.78 mmol). The
reaction mixture was slowly heated to 100 °C and kept at 100
°C for 1.5 h. The reaction mixture was cooled and concentrated
to dryness. The compound was extracted with CH2Cl2 (2 × 60
mL), and the extract was washed with water. The organic layer
was dried over MgSO4, filtered, and concentrated to dryness.
The residue was purified by column chromatography (silica
gel 230-400 mesh, elution with hexanes). The desired frac-
tions were collected, concentrated, and dried in vacuo over P2O5
to give two isomers and other products. F1(13b): yield 810
3,4,5-Tr ich lor o-2,6-d im eth ylp yr id in e (16). This com-
pound was prepared by a reported method.12
3,4,5-Tr ich lor o-2,6-bis(tr ich lor om eth yl)p yr id in e (17).
A slow stream of chlorine gas was passed through a refluxing
solution of 16 (2.00 g, 9.5 mmol) in CCl4 (100 mL) illuminated
with a 500 W incandescent lamp for 4 h. Evaporation of the
solvent afforded 17, which was purified by column chroma-
tography using hexanes for elution: yield 1.62 g (41%); mp
104-106 °C; MS m/z 414 (M + H)+; Anal. (C7Cl9N) C, H, N.
3,5-Dich lor o-4-m eth oxy-2,6-bis(tr ich lor om eth yl)p yr i-
d in e (18). The procedure was the same as reported above for
10a using 17 (500 mg, 1.19 mmol), NaOH (56 mg, 1.4 mmol),
and MeOH (15 mL). After column chromatography (hexanes),
a white solid was obtained: yield 450 mg (91.09%); mp 115-
117 °C; MS m/z 410 (M + H)+; 1H NMR (CDCl3) δ 4.09 (s, 3H,
CH3); Anal. (C8H3Cl8NO) C, H, N.
1
mg (43.31%); mp 93-95 °C; MS m/z 390 (M + H)+; H NMR
(CDCl3) δ 6.84 (bd, 2H, C6H5 (2,6)), 7.15 (bt, 1H, C6H5 (4)),
7.36 (bt, 2H, C6H5 (3,5)); Anal. (C12H5Cl6NO) C, H, N. F2
(14b): yield 370 mg (19.78%); mp 113-115 °C; MS m/z 390
(M + H)+; 1H NMR (CDCl3) δ 7.22-7.27 (m, 3H, C6H5 (2,4,6)),
7.39-7.44 (m, 2H, C6H5 (3,5)); Anal. (C12H5Cl6NO) C, H, N.
An titu m or Eva lu a tion in Vivo. Athymic NCr-nu/nu mice
were obtained from various suppliers under contract with NCI

Synthesis and Activity of Penclomedine Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1085
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and housed in sterile, filtered-capped microisolater cages in a
barrier facility. Human MX-1 breast tumor was obtained from
the NCI Tumor Repository (Frederick, MD). For intraperito-
neal (ip) injection into mice, the analogues were prepared as
a suspension in aqueous hydroxypropyl cellulose. For subcu-
taneous implants, tumor fragments (30-40 mg) from in vivo
passage were implanted into the axillary region of the mice.
Treatment of groups of five mice each was initiated when
the tumors reached approximately 300 mg in mass and was
continued for 5 days for all treatment groups. Each tumor was
measured by caliper in two dimensions twice weekly and
converted to tumor mass. Antitumor activity was assessed on
the basis of tumor growth delay in comparison to a vehicle-
treated control (T - C, i.e., the difference in the median time
poststaging for tumors of the treated (T) group to double twice
in mass compared to the median of the control (C) group),
tumor regression (partial and complete), and tumor-free
survivors, and experiments were terminated when the control
tumors attained a mass of 1 g, which was typically 57-61 days.
Ack n ow led gm en t . This investigation was sup-
ported by NIH grants PO1 CA34200 and R44 CA85021.
We thank Mark D. Richardson and J oan C. Bearden of
the Molecular Spectroscopy Section of Southern Re-
search Institute for spectroscopic and elemental analyti-
cal determinations.
(10) Hartman, N. R.; O’Reilly, S.; Rowinsky, E. K.; Collins, J . M.;
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Hartman, N. R.; O’Reilly, S.; Struck, R. F. 4-Demthylpenclom-
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N-Oxides from polyhalogenated N-Heteroaromatic Compounds.
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J M010334E