Structure-activity relationships of phenylcyclohexene and biphenyl antitubulin compounds against plant and mammalian cells

Article abstract of DOI:10.1016/S0960-894X(01)00246-3

Phenylcyclohexenes (PCHs) [e.g., trans-4-nitro-5-(2,3,4-trimethoxyphenyl)cyclohexene, 2d] were found to bind weakly to the colchicine site of bovine tubulin, but are the first mimics of colchicine found to have high activity towards plant cells. Structure

Full text of DOI:10.1016/S0960-894X(01)00246-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1393–1396
Structure–Activity Relationships of Phenylcyclohexene
and Biphenyl Antitubulin Compounds against Plant
and Mammalian Cells
David H. Young,* Colin M. Tice, Enrique L. Michelotti, Renee C. Roemmele,
Richard A. Slawecki, Fernando M. Rubio and Judith A. Rolling
Rohm and Haas Company, 727 Norristown Road, Spring House, PA 19477-0904, USA
Received 12 February 2001; accepted 28 March 2001
Abstract—Phenylcyclohexenes (PCHs) [e.g., trans-4-nitro-5-(2,3,4-trimethoxyphenyl)cyclohexene, 2d] were found to bind weakly to
the colchicine site of bovine tubulin, but are the first mimics of colchicine found to have high activity towards plant cells. Structure–
activity relationships for PCHs and biphenyl AC-ring analogues of colchicine (e.g., 2,3,4,40-tetramethoxy-20-methyl-1,10-biphenyl,
3e) are discussed. # 2001 Elsevier Science Ltd. All rights reserved.
Tubulin is the major component of microtubules and is
the target of various anticancer drugs1 (paclitaxel, Vinca
alkaloids and compounds interacting at the colchicine
binding site) and herbicides2 (dinitroanilines, N-phe-
nylcarbamates, amiprofos-methyl, pronamide, and
dithiopyr) that inhibit mitosis. Although tubulin is
highly conserved between organisms, mammalian and
plant tubulins differ greatly in their interaction with
antitubulin agents.3 Colchicine (1, Fig. 1), one of the
best studied antitubulin compounds, is known to be
much more potent toward mammalian cells than
toward plant cells.3
prevent seed germination but strongly inhibited root
elongation and produced swollen root tips. These
symptoms are typical of an antimitotic herbicide.2
Compound 2d (EC50=0.27 mM) was more potent than
the commercial herbicides pronamide (EC50=0.72 mM)
and trifluralin (EC50=1.32 mM) in the tobacco root
assay. Cytological staining of chromosomes in tobacco
suspension-cultured cells6 treated with 2d showed an
increase in the mitotic index (Fig. 2) resulting from an
accumulation of cells in arrested metaphase, confirming
the inhibition of mitosis. The increase in mitotic index
produced by 2d was of similar magnitude to that
obtained with 1, however the concentration of 2d
required to arrest mitosis in tobacco was more than
three orders of magnitude lower than the concentration
needed for 1 (Fig. 2).
Random screening identified 2a (Fig. 1) as a weakher-
bicide lead with pre-emergence grass activity. Synth-
esis4,5 of several dozen analogues of 2a, including 2b–e,
led to the identification of 2d and the cyano analogue 2e
as substantially more potent herbicides than 2a in
greenhouse tests.5
By contrast, colchicine was >4000-fold more active
than 2d and 2e towards HCT-116 tumor cells,7 and in
experiments using tubulin isolated from bovine brain 2d
and 2e weakly inhibited microtubule assembly8 at 200
mM (Fig. 3), whereas colchicine strongly inhibited
assembly at 2 mM. Although much less potent than col-
chicine towards mammalian tubulin, 2d was found to be
The symptoms exhibited by plants treated in the green-
house with 2d and other phenylcyclohexenes (PCHs)
were similar to those seen in plants treated with the
herbicides pronamide and trifluralin, suggesting that the
PCHs function by disrupting mitosis. In an in vitro
assay for inhibition of tobacco root growth,6 2d did not
3
a competitive inhibitor of the binding of H-colchicine
to bovine brain tubulin9 (Fig. 4) indicating a common
binding site.
Biphenyl colchicine mimics, including 3e and 3f (Fig. 1),
which are active towards mammalian tubulin and bear
*Corresponding author. Tel.:+1-215-641-7410; fax: +1-215-619-
1617; e-mail: rsadhy@rohmhaas.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00246-3

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D. H. Young et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1393–1396
Figure 1. Compounds.
The results given by colchicine, 2b–e, and 3a–f in three
assays are shown in Table 1. Colchicine and its known
AC-ring biphenyl mimic 3e10 showed much stronger
activity against HCT116 colon cancer cells and as inhi-
bitors of bovine microtubule assembly than against
tobacco. In contrast, PCH 2d was very active toward
tobacco but showed at best weakactivity against
HCT116 cells and as an inhibitor of bovine microtubule
assembly. The 2,3,4-trimethoxy substitution pattern in
the A ring appears to be critical for optimal activity in
colchicine based on structure–activity studies16 of ana-
logues of 5-(trimethoxyphenyl)tropolone methyl ether,
which has similar potency to colchicine17 but lacks the
B-ring. Similarly, the trimethoxy substitution on the
phenyl ring of the PCHs was important for activity
toward tobacco (2d>2c>2b) and also towards HCT116
cells, although to a lesser extent.
Figure 2. Arrest of mitosis in tobacco suspension-cultured cells.
Consistent with their potent herbicidal activity, 2d and
2e were highly active in the tobacco root assay. Against
HCT116 cells, 2d and 2e were weakly active and com-
parable in activity. To better characterize the activity of
2d and 2e, they were separated into their individual
enantiomers by preparative HPLC on a chiral column.18
The trans stereoisomers of 2e were much more active
than the cis stereoisomers towards tobacco. The faster
eluting enantiomer 2d-trans-I was almost 20-fold more
potent towards tobacco than the slower eluting enan-
tiomer 2d-trans-II, while they were equipotent against
HCT116 cells. The faster eluting trans enantiomer in the
cyano series 2e-trans-I was 10-fold more potent on
tobacco than its enantiomer 2e-trans-II; however, 2e-
trans-II was more potent than 2e-trans-I against
HCT116 cells. Furthermore, the slower eluting cis
enantiomer 2e-cis-II was about equipotent to 2e-trans-II
against HCT116 cells.
Figure 3. Inhibition of microtubule assembly.
an intriguing structural similarity to the PCHs have
been described in the literature.10ꢀ13 Compound 3c has
been described in the literature14 but its biological
activity has not been reported. Thus, it was of interest to
synthesize biphenyls 3a–f15 and compare the structure–
activity relationships of colchicine, 2b–e, and 3a–f on
both plant and mammalian cells.
Biphenyls 3a and 3b, the fully aromatic analogues of
PCHs 2d and 2e, were substantially less active towards
tobacco than the corresponding PCHs, but gained
potency against HCT116 cells.

D. H. Young et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1393–1396
1395
Itoh and co-workers reported that a methoxy group in
place of H at the 40-position in the biphenyl system (i.e.,
3, R2=OMe) increased activity towards mammalian
cells.10 Their most active compounds were 3e and 3f.
Hence, it was of interest to prepare 3c and 3d and com-
pare them with 3e and 3f. Against HCT116 cells, 3c was
found to be less active than 3e and 3f; however, 3d
showed increased potency. In the tobacco root assay,
the 40-methoxy group (3c) abolished all activity.
binding site differences. X-ray crystallography is in
progress to determine the absolute configuration of 2e-
trans-I.
Acknowledgements
We are grateful to Dr. Kebede Beshah for identifying
the cis and trans stereoisomers of 2d and 2e by NMR,
and Barbara Cressman for the chiral HPLC separa-
tions.
PCHs 2d and 2e are apparently the first examples of
antitubulin colchicine mimics with potent activity
toward plants. Our results suggest that the binding
pocket for the colchicine A-ring is conserved between
plant and mammalian tubulin while the tropolone C-
ring binding regions differ significantly. Molecular
modeling is underway to shed further light on the
References and Notes
1. Hamel, E. Med. Res. Rev. 1996, 16, 207.
2. Pallett, K. E. In Target Sites for Herbicide Action; Kirk-
wood, R. C., Ed.; Plenum: New York, 1991; pp 144–148.
3. Morejohn, L. C.; Foskett, D. E. In Cell and Molecular
Biology of the Cytoskeleton; Shay, J. W., Ed.; Plenum: New
York, 1986; pp 257–329.
4. Synthesis of 2b–d was accomplished by Diels–Alder reac-
tion of the appropriate trans-b-nitrostyrene with butadiene
sulfone yielding only the trans-4-nitro-5-phenylcyclohexene
derivatives following the literature procedure: Bryce, M. R.;
Gardiner, J. M. Tetrahedron 1988, 44, 599. Synthesis of 2e, as
a 1:1 mixture of the cis and trans cyclohexene isomers, was
accomplished by Diels–Alder reaction of ethyl 2-cyano-3-
(2,3,4-trimethoxyphenyl)propenoate with butadiene sulfone
followed by saponification and decarboxylation. Spectral data
for the new compounds 2d and 2e are reported below in ref 19.
5. Michelotti, E. L.; Roemmele, R.; Swithenbank, C.; Tice, C.
M.; Young, D. H. US Patent 5,866,513, 1999; Chem. Abstr.
1999, 130, 149865.
6. Young, D. H.; Lewandowski, V. T. Plant Physiol. 2000,
124, 115.
7. Cytotoxicity towards HCT116 tumor cells was measured in
96-well microtiter plates using a sulforhodamine B assay:
Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon,
J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd,
M. R. J. Natl. Cancer Inst. 1990, 82, 1107.
Figure 4. Competitive inhibition of [3H]-colchicine binding to bovine
tubulin by 2d.
Table 1. Structure–activity relationships
8. Inhibition of microtubule assembly was assayed turbidime-
trically as described in ref 6 using a 1 h incubation at 37 ꢁC of
the test compound with bovine brain tubulin before initiation
of assembly.
Compd
Tobacco
root assay
EC50 (mM)
HCT116
tumor
cell assay
EC50 (mM)
Bovine
microtubule
assembly
EC50 (mM)
9. Colchicine binding assays were carried out by the DEAE-
cellulose filter procedure (Borisy, G. G. Anal. Biochem. 1972,
50, 373) using conditions described by Hamel, E.; Lin, C. M.
Biochem. Biophys. Res. Commun. 1982, 104, 929, with a 1 h
1
356
>100
3.78
0.020
174
143
85
113
79
71
142
45
0.75–1.25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50–100
6.25–12.5
6.25–12.5
6.25–12.5
6.25–12.5
2ba
2ca
2da
0.27
2.29
0.100
1.96
3
incubation of the assay mixtures. H-Colchicine was obtained
2eb
from NEN Life Science Products, Boston, MA, USA.
10. Itoh, Y.; Brossi, A.; Hamel, E.; Lin, C. M. Helv. Chim.
Acta 1988, 71, 1199.
11. Itoh, Y.; Brossi, A.; Hamel, E.; Flippen-Anderson, J. L.;
George, C. Helv. Chim. Acta 1989, 72, 196.
12. Itoh, Y.; Brossi, A. Helv. Chim. Acta 1989, 72, 1690.
13. Olszewski, J. D.; Marshalla, M.; Sabat, M.; Sundberg,
R. J. J. Org. Chem. 1994, 59, 4285.
14. Tarbell, D. S.; Frank, H. R.; Fanta, P. E. J. Am. Chem.
Soc. 1946, 68, 502.
15. Biphenyls 3a–f were synthesized by Suzuki coupling of
2,3,4-trimethoxybenzeneboronic acid with the appropriate
bromobenzene under standard conditions Fu, J.-M.; Zhao,
B. P.; Sharp, M. J.; Snieckus, V. Can. J. Chem. 1994, 72, 227.
Spectral data for new compounds 3a, 3b, and 3d are reported
below in ref 19.
2d-trans enantiomer Ic
2d-trans enantiomer IIc
2e-cis enantiomer Ic
>25
>25
0.667
7.17
52.0
>100
>100
>100
>100
>100
2e-cis enantiomer IIc
2e-trans enantiomer Ic
136
47
2e-trans enantiomer IIc
3a
3b
3c
3d
3e
3f
19.6
13.8
2.65
0.58
0.78
1.52
aRacemic mixture of the trans diastereomer.
bRacemic mixtures of both the cis and trans diastereomers.
cStereoisomers were separated by semi-preparative HPLC on a chiral
column. See ref 18.

1396
D. H. Young et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1393–1396
16. Banwell, M. G.; Cameron, J. M.; Collis, M. P.; Crisp,
G. T.; Gable, R. W.; Hamel, E.; Lambert, J. N.; Mackay,
M. F.; Reum, M. E.; Scoble, J. A. Aust. J. Chem. 1991, 44,
705.
17. Fitzgerald, T. J. Biochem. Pharmacol. 1976, 25, 1383.
18. A semi-preparative Chiralcel1 OC column from Chiral
Technologies, Inc., 250ꢂ4.6 mm was used with isooctane–
ethanol (95:5, by volume) as the mobile phase.
(m, 2H), 3.05 (m, 1H), 3.30 (m, 1H), 3.85 (s, 3H), 3.86 (s, 3H),
3.95 (s, 3H), 5.70 (m, 1H), 5.82 (m, 1H), 6.67 (d, J=8.7 Hz,
1H), 6.85 (d, J=8.7 Hz, 1H). The measured coupling constant
between C4HCN and C5HAr J4,5 was 10.3 Hz. The chemical
1
shift and multiplicity in the H NMR spectrum of this com-
pound matched those in 2d. 20-Nitꢁro-2,3,4-trimethoxy-1,10-
biphenyl (3a): yellow solid, mp 85–87 C; 1H NMR (300 MHz,
CDCl3) d 3.65 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 6.75 (d,
J=8.6 Hz, 1H), 6.98 (d, J=8.6 Hz, 1H), 7.39 (d, J=7.6 Hz,
1H), 7.45 (m, 1H), 7.60 (m, 1H), 7.92 (d, J=8.1 Hz, 1H); 13C
NMR (CDCl3) d 55.8, 60.4, 60.8, 104.9, 107.1, 123.5, 123.8,
124.3, 127.7, 132.4, 132.5, 141.5, 149.1, 150.3, 154.0; IR
(CDCl3) 1611, 1599, 1524, 1502, 1435, 1414, 1358 cmꢀ1. Anal.
calcd for C15H15NO5: C, 62.28; H, 5.23; N, 4.84. Found: C,
62.46; H, 5.35; N, 4.75. 20-Cyano-2,3,4-trimethoxy-1,10-biphe-
nyl (3b): white solid, mp 87–89 ꢁC; 1H NMR (300 MHz,
CDCl3) d 3.73 (s, 3H), 3.91 (s, 3H), 3.93 (s, 3H), 6.76 (d,
J=8.6 Hz, 1H), 7.01 (d, J=8.6 Hz, 1H), 7.38–7.52 (2H), 7.61
(m, 1H), 7.73 (d, J=8.0 Hz, 1H); 13C NMR (CDCl3) d 55.9,
60.9, 61.0, 107.0, 112.9, 118.5, 124.8, 125.0, 127.2, 130.9, 132.1,
132.7, 142.0, 142.1, 151.0, 154.2; IR (CDCl3) 2220 cmꢀ1; MS
(EI) m/z 270 (M+1)+, 254, 223, 140. Anal. calcd for
C16H15NO3: C, 71.36; H, 5.61; N, 5.20. Found: C, 71.16; H,
5.61; N, 5.08. 20-Cyano-2,3,4,40-tetramethoxy-1,10-biphenyl
(3d): white solid, mp 78–80 ꢁC; 1H NMR (300 MHz, CDCl3) d
3.71 (s, 3H), 3.85 (s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 6.75 (d,
J=8.6 Hz, 1H), 6.99 (d, J=8.6 Hz, 1H), 7.15 (dd, J=8.6, 2.6
Hz, 1H), 7.22 (d, J=2.6 Hz, 1H), 7.37 (d, J=8.6 Hz); 13C
NMR (CDCl3) d 55.4, 55.8, 60.8, 60.9, 106.9, 113.3, 116.9,
118.4, 118.7, 124.5, 125.0, 132.1, 134.4, 142.0, 151.2, 153.9,
158.1; IR (CDCl3) 2225 cmꢀ1; MS (EI) m/z 300 (M+1)+, 285,
253, 170. Anal. calcd for C17H17NO4: C, 68.22; H, 5.72; N,
4.68. Found: C, 68.04; H, 5.76; N, 4.68.
19. trans-4-Nitro-5-(2,3,4-trimethoxyphenyl)cyclohexene (2d):
white solid, mp 99–100 ꢁC; 1H NMR (300 MHz, CDCl3) d 2.2–
2.5 (m, 2H), 2.7–2.9 (m, 2H), 3.70 (m, 1H), 3.81 (s, 3H), 3.84
(s, 3H), 3.95 (s, 3H), 5.12 (m, 1H), 5.65–5.85 (m, 2H), 6.72 (d,
J=8.6 Hz, 1H), 6.83 (d, J=8.6 Hz, 1H); 13C NMR (CDCl3) d
31.2, 32.5, 38.8, 55.7, 60.4, 61.0, 85.8, 107.1, 122.0, 122.3,
125.4, 126.7, 142.0, 151.8, 152.8; IR (CDCl3) 1603, 1548, 1498,
1421, 1368 cmꢀ1. Anal. calcd C15H19NO5: C, 61.42; H, 6.53;
N, 4.78. Found: C, 61.38; H, 6.47; N, 4.73. 4-Cyano-5-(3,4-
dimethoxyphenyl)cyclohexene (2, 1:1 mixture of diastereo-
mers): oily solid; 13C NMR (75 MHz, CDCl3) d 26.9, 29.2,
29.7, 29.8, 30.7, 31.7, 32.0, 33.1, 36.6, 55.9 (double height),
60.6, 60.7, 61.1, 61.2, 107.1, 107.4, 121.0, 121.2, 121.9, 122.7,
123.4, 127.2, 127.7, 141.9, 142.1, 151.3, 151.8, 152.9, 153.0; IR
(CHCl3) 3030, 2930, 2850, 2240, 1655, 1600 cmꢀ1; MS (ESI)
m/z 273 (M+). Anal. calcd for C16H19NO3: C, 70.31; H, 7.01;
N, 5.12. Found: C, 70.37; H, 6.87; N, 4.96. cis-4-Cyano-5-(3,4-
dimethoxyphenyl)cyclohexene (2e-cis-I, 2e-cis-II): 1H NMR
(300 MHz, CDCl3) d 2.22 (m, 1H), 2.30–2.45 (1H), 2.55–2.80
(2H), 3.14 (m, 1H), 3.38 (m, 1H), 3.87 (s, 3H), 3.88 (s, 3H),
3.90 (s, 3H), 5.70 (m, 1H), 5.94 (m, 1H), 6.70 (d, J=8.7 Hz,
1H), 7.13 (d, J=8.7 Hz, 1H). The measured coupling constant
between C4HCN and C5HAr J4,5 was 4 Hz. trans-4-Cyano-5-
1
(3,4-dimethoxyphenyl)cyclohexene (2e-trans-I, 2e-trans-II): H
NMR (300 MHz, CDCl3) d 2.25 (m, 1H), 2.40 (m, 1H), 250