Tubulysin analogs incorporating desmethyl and dimethyl tubuphenylalanine derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.03.046

A series of tubulysin analogs in which one of the stereogenic centers of tubuphenylalanine was eliminated were synthesized. All compounds were tested for antiproliferative activity towards ovarian cancer cells and for inhibition of tubulin polymerization.

Full text of DOI:10.1016/j.bmcl.2008.03.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2996–2999
Tubulysin analogs incorporating desmethyl and dimethyl
tubuphenylalanine derivatives
Ranganathan Balasubramanian,a Bhooma Raghavan,a Jaeson C. Steele,a
Dan L. Sackettb and Robert A. Fecika,*
aDepartment of Medicinal Chemistry, 308 Harvard Street S.E., 8-101 Weaver–Densford Hall, University of Minnesota,
Minneapolis, MN 55455-0353, USA
bLaboratory of Integrative and Medical Biophysics, National Institute of Child Health and Human Development,
National Institutes of Health, 9 Memorial Drive, Building 9, Room 1E129, Bethesda, MD 20892-0924, USA
Received 15 February 2008; revised 12 March 2008; accepted 17 March 2008
Available online 20 March 2008
Abstract—A series of tubulysin analogs in which one of the stereogenic centers of tubuphenylalanine was eliminated were synthe-
sized. All compounds were tested for antiproliferative activity towards ovarian cancer cells and for inhibition of tubulin polymer-
ization. The dimethyl analogs were generally more active than the desmethyl analogs, and four analogs have tubulin polymerization
IC50 values similar to combretastatin A4 and the hemiasterlin analog HTI-286.
Ó 2008 Elsevier Ltd. All rights reserved.
The tubulysins, represented by tubulysins A and D (1
and 2), have emerged as important anticancer lead com-
pounds.1 They possess impressive activity against
multidrug-resistant cancer cells that overexpress P-gly-
coprotein.1b,2 Mechanistically, tubulysins inhibit tubulin
polymerization3 and also have antiangiogenic effects.2
Their potential for the treatment of drug-resistant can-
cers has stimulated a flurry of research into the chemis-
try and biology of these natural products.
synthesis and biological activity of tubulysin analogs 3
containing desmethyl and dimethyl tubuphenylalanine
derivatives. Both of these modifications result in the elim-
ination of a stereogenic center that further simplifies their
synthesis.
Limited availability of tubulysins from the producing
myxobacteria by fermentation1 has led to recent disclo-
sures for their total synthesis4 and the synthesis of novel
analogs.5 Tubulysins are mixed nonribosomal-polyketide
tetrapeptides that, in addition to isoleucine (Ile), contain
three unique amino acids: N-methylpipecolinic acid
(Mep), tubuvaline (Tuv), and tubuphenylalanine (Tup).
We recently reported the synthesis and initial biological
activity of simplified tubulysin analogs that lack the
N,O-acetal, and have the acetate group of tubuvaline re-
placed with a ketone.5d Systematic modification of the
N-methyl group, stereochemistry, and ring size of Mep re-
vealed preliminary structural requirements. Here we ex-
tend these structure–activity studies by reporting the
Keywords: Tubulysin; Tubulin; Anticancer agents; Cytotoxicity; Nat-
ural products.
Synthesis of the key tripeptide intermediates commenced
with opening of lactams 45d and 55d and esterification to
give benzyl esters 66 and 7 (Scheme 1). Amine deprotec-
*
Corresponding author. Tel.: +1 612 626 6523; fax: +1 612 624
0139; e-mail: fecik001@umn.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.046

R. Balasubramanian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2996–2999
2997
Scheme 1. Synthesis of tripeptide intermediates 11 and 12.
tion and coupling with tubuvaline fragment 85d afforded
dipeptides 9 and 10. Further deprotection and coupling
with N-Boc-L-Ile produced tripeptides 11 and 12.
Desmethyl tripeptide 11 was deprotected at the N-termi-
nus, coupled with N-methyl amino acids 13a–d, and
deprotected to afford tubulysin analogs 14a–d (Scheme
2). Amino group deprotection and coupling of tripeptide
11 with N-Boc protected amino acids 13e–h gave pro-
tected tetrapeptides 14e–h. Removal of the Boc groups
and benzyl esters then gave N-desmethyl tubulysin ana-
logs 15a–d.
Scheme 2. Synthesis of desmethyl tubulysin analogs 14a–d and 15a–d.
At this stage, antiproliferative and tubulin inhibition
screening established that tubulysin analogs lacking
the N-methyl group of Mep, or that have the unnatural
L-configuration at this residue were dramatically less po-
tent.5d Therefore, the full complement of Mep analogs in
the dimethyl tubuphenylalanine series was not synthe-
sized. Deprotection of tripeptide 12, followed by cou-
pling with amino acids 13a and 13b and removal of
the benzyl esters afforded tubulysin analogs 16a and
16b (Scheme 3). Saponification of the benzyl esters re-
quired heating at reflux due to the increased substitution
at the a-position.
Scheme 3. Synthesis of dimethyl tubulysin analogs 16a and 16b.
various Mep analogs are required for activity. The activ-
ity of analog 14a demonstrates that the ring size of Mep
can be changed to N-Me-D-Pro, although this is accom-
panied with a slight loss of activity. Since these data are
consistent with other tubulysin analogs,5d we did not
synthesize the complete complementary series of di-
methyl analogs. Comparison of the antiproliferative
activity of tubulysin analogs 16a and 16b again shows
that while N-Me-D-Pro can be substituted for Mep,
there is a loss of potency. New SAR trends for the
Tup fragment are also revealed here. Modification to
Tup by the incorporation of a geminal dimethyl group
(16a and 16b) results in comparable levels of antiprolif-
erative activity to the corresponding mono-methyl ana-
logs.5d However, unsubstituted Tup variants lacking
All tubulysin analogs were screened for antiproliferative
activity and inhibition of tubulin polymerization using
combretastatin A4 and the hemiasterlin analog HTI-
286/SPA110 as positive controls (Table 1). Their anti-
proliferative activity in 1A9 ovarian cancer cells and
the tubulin polymerization assay was conducted under
standard conditions as previously described.5d,7
The antiproliferative data closely adhere to the struc-
ture–activity relationship (SAR) trends found in our
previous series of tubulysin analogs.5d Within the desm-
ethyl series (14a–d and 15a–d), it is clear that the
N-methyl group and the natural D-configuration of the

2998
R. Balasubramanian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2996–2999
Table 1. Antiproliferative activity of tubulysin analogs in 1A9 ovarian cancer cells and inhibition of tubulin polymerization
b
c
Compound
R1
R2a
Antiproliferative activity IC50 (lM)
Tubulin Inhibition IC50 (lM)
14a (FT-032)
14b (FT-030)
14c (FT-033)
14d (FT-031)
15a (FT-028)
15b (FT-026)
15c (FT-029)
15d (FT-027)
16a (FT-034)
16b (FT-035)
Combretastatin A4
HTI-286 (SPA110)
H
N-Me-D-Pro
N-Me-D-Pip
N-Me-L-Pro
N-Me-L-Pip
D-Pro
24
2.3
0.6
11
H
4
50
H
H
>50
>50
>50
>50
>50
3.2
13
H
>20
4.4
>20
>20
2.7
1.5
1.1
0.7
H
D-Pip
H
L-Pro
H
L-Pip
Me
Me
N-Me-D-Pro
N-Me-D-Pip
0.3
3.6 nM
0.3 nM
a Pro, proline; Pip, pipecolinic acid; all compounds were isolated as their HCl salts.
b Values are means of two independent IC50 determinations against 1A9 ovarian cancer cells with a maximum drug concentration of 50 lM in DMSO
(See Supplementary Data).
c In vitro inhibition of tubulin polymerization (see Supplementary Data).
an a-methyl group are an order of magnitude less active
(compare 14a and 14b to 16a and 16b).
ogenic a-methyl group of tubuphenylalanine, and the (2)
N,O-acetal and (3) stereogenic acetate groups of tubuv-
aline. Our previously published route to tubulysin ana-
logs enabled their efficient synthesis. Clear SAR trends
emerged with regard to antiproliferative activity and
inhibition of tubulin polymerization. Four of these ana-
logs (14a, 14b, 16a, and 16b) inhibit tubulin polymeriza-
tion comparable to the clinical agents combretastatin A4
and HTI-286. Their inhibition of tubulin polymerization
is also similar to the reported value for tubulysin A
(IC50 = 0.75 lM), although differences in assay condi-
tions preclude direct comparison of these values.3 The
reasons for the decreased antiproliferative activity of
these tubulysin analogs relative to combretastatin A4
and HTI-286 are unknown at this time. Further exami-
nation of the promising activity of these and other
tubulysin analogs will be reported in due course.
Results from the in vitro tubulin polymerization inhibi-
tion assay closely mirror the antiproliferative data
(Table 1). In general, the most active tubulysin analogs
are also the most potent tubulin polymerization inhibi-
tors. Notably, all compounds that retain the natural
D-configuration and the N-methyl group of Mep found
in the natural products (14a, 14b, 16a, and 16b) are
the most potent, with low or sub-micromolar IC50 val-
ues that are comparable to combretastatin A4 and
HTI-286. Comparison of analogs 14a and 14b to 16a
and 16b suggests that the lower antiproliferative activity
of 14a and 16a is due to decreased inhibition of tubulin
polymerization caused by the change from N-Me-D-Pip
to N-Me-D-Pro. Alkylation of the a-position of Tup also
influences the biological activity of tubulysin analogs.
While the tubulin polymerization inhibition IC50s of
the dimethyl substituted analogs 16a and 16b are com-
parable to the unsubstituted 14a and 14b, 16a and 16b
are an order of magnitude more active against 1A9 cells.
This suggests that the increased antiproliferative activity
of 16a and 16b is due to their higher lipophilicity which
enables better cell membrane permeability. The only
compound that does not follow these general trends is
analog 15b. It is unclear why 15b is not antiproliferative
despite its effective inhibition of tubulin polymerization.
One possible explanation is that the unsubstituted D-Pip
residue decreases its lipophilicity and is unable to effec-
tively cross the cell membrane.
Acknowledgments
This work was generously supported by the American
Cancer Society, Grant RSG-05-105-01-CDD (to
R.A.F.), and was supported in part by intramural funds
from the National Institute of Child Health and Human
Development, NIH. Funding for NMR instrumentation
in the Department of Biochemistry, Molecular Biology,
and Biophysics was provided by the University of Min-
nesota Medical School, NSF (BIR-961477), and the
Minnesota Medical Foundation.
In summary, we have synthesized and evaluated a new
series of simplified tubulysin analogs that differ at the
Mep and Tup residues. Many functional groups in the
natural products were found to be unnecessary for anti-
proliferative activity and tubulin inhibition: (1) the stere-
Supplementary data
Experimental procedures, spectral data, and copies of
1H and 13C NMR spectra for compounds 6, 7, 9–12,
14a–h, 15a–d, 16a, and 16b. Supplementary data associ-

R. Balasubramanian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2996–2999
2999
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Eichelberger, U.; Sakamuri, S.; Menon, S.; Chen, Q.-Z.;
Lu, Y.; Wessjohann, L. A. Angew. Chem. Int. Ed. 2007, 46,
2347; (d) Sani, M.; Fossati, G.; Huguenot, F.; Zanda, M.
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ated with this article can be found, in the online version,
at doi:10.1016/j.bmcl.2008.03.046.
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