A remarkable series of vinblastine analogues displaying enhanced activity and an unprecedented tubulin binding steric tolerance: C20′ urea derivatives

Article abstract of DOI:10.1021/jm3015684

A systematic series of previously inaccessible key C20′ urea and thiourea derivatives of vinblastine were prepared from 20′- aminovinblastine that was made accessible through a unique Fe(III)/NaBH ₄-mediated alkene functionalization reaction of anhydrovinblastine. Their examination defined key structural features of the urea-based analogues that contribute to their properties and provided derivatives that match or exceed the potency of vinblastine by as much as 10-fold in cell-based functional assays, which is directly related to their relative tubulin binding affinity. In contrast to expectations based on apparent steric constraints of the tubulin binding site surrounding the vinblastine C20′ center depicted in an X-ray cocrystal structure, remarkably large C20′ urea derivatives are accommodated.

Full text of DOI:10.1021/jm3015684

Article
pubs.acs.org/jmc
A Remarkable Series of Vinblastine Analogues Displaying Enhanced
Activity and an Unprecedented Tubulin Binding Steric Tolerance:
C20′ Urea Derivatives
Erick K. Leggans, Katharine K. Duncan, Timothy J. Barker, Kristin D. Schleicher, and Dale L. Boger*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: A systematic series of previously inaccessible key C20′ urea
and thiourea derivatives of vinblastine were prepared from 20′-amino-
vinblastine that was made accessible through a unique Fe(III)/NaBH4-
mediated alkene functionalization reaction of anhydrovinblastine. Their
examination defined key structural features of the urea-based analogues that
contribute to their properties and provided derivatives that match or exceed
the potency of vinblastine by as much as 10-fold in cell-based functional
assays, which is directly related to their relative tubulin binding affinity. In
contrast to expectations based on apparent steric constraints of the tubulin
binding site surrounding the vinblastine C20′ center depicted in an X-ray
cocrystal structure, remarkably large C20′ urea derivatives are accom-
modated.
relatively insensitive to the reaction parameters. The interest in
this Fe(III)/NaBH4-mediated reaction emerged not only from
its use in accessing vinblastine but the opportunity it presented
for the late-stage, divergent12 preparation of otherwise
inaccessible vinblastine analogues incorporating alternative
C20′ functionality. Although this site is known to be critical
to the properties of vinblastine13 and is found deeply embedded
in the tubulin bound complex (Figure 1),14 the prior
exploration of C20′ substituent effects has been limited to
semisynthetic O-acylation of the C20′ alcohol or its elimination
and subsequent alkene reduction or superacid-catalyzed
additions.15 These invariably led to substantial reductions in
biological potency of the resulting derivative, albeit with
examination of only a limited number of key analogues.
Consequently, in the course of the development of the Fe(III)/
NaBH4-mediated alkene functionalization reaction, its use was
extended to the preparation of a series of key vinblastine
analogues bearing alternative C20′ functionality. Those of
initial interest included the C20′ azide 6 and amine 7, both of
which proved to be approximately 100-fold less potent than
vinblastine (1) and 10-fold less potent than 20′-deoxyvinblas-
tine (3). However, acylation of the C20′ amine improved
activity 10-fold and installation of the unsubstituted C20′ urea
11 or thiourea 12 provided compounds that nearly matched the
potency of vinblastine itself (Figure 1). Herein and based on
these observations, we report a systematic exploration of C20′
amine, urea, and thiourea derivatives of vinblastine that have
not only provided C20′ urea-based analogues that substantially
INTRODUCTION
■
Vinblastine (1) and vincristine (2) are the most widely
recognized members of the Vinca alkaloids as a result of their
clinical use as antitumor drugs, and their discovery represents
one of the earliest important contributions that plant-derived
natural products have made to cancer chemotherapy (Figure
1).1 Originally isolated from the leaves of Catharanthus roseus
(L) G. Don in trace quantities,2,3 vinblastine and vincristine
were among the first small molecules shown to bind tubulin
and to inhibit microtubule formation and mitosis.1,4 Because of
their clinical importance, low natural abundance, and structural
complexity, they have been the subject of extensive and
continuing biological and synthetic investigations.4−6
We previously reported the total synthesis of vinblastine6 and
its extension to the total synthesis of related natural products
including vincristine and key analogues that utilizes a one-pot,
two-step, biomimetic Fe(III)-promoted single electron oxida-
tive coupling of catharanthine and vindoline and a subsequent
Fe(III)/NaBH4-mediated in situ alkene oxidation to generate
vinblastine directly.7−9 Recently, we detailed the results of
investigation of the Fe(III)/NaBH4-mediated free radical
oxidation of the anhydrovinblastine trisubstituted alkene used
to introduce the vinblastine C20′ tertiary alcohol,6b extending
the reaction to provide a simple method for direct
functionalization of unactivated alkenes.10,11 In these studies,
the broad alkene substrate scope was defined, the exclusive
Markovnikov addition regioselectivity was established, the
excellent functional group tolerance was revealed, alternative
free radical traps were introduced, the Fe(III) salt and initiating
hydride source were examined, and remarkably mild reaction
conditions (0−25 °C, 5−30 min) were introduced that are
Received: October 24, 2012
© XXXX American Chemical Society
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Scheme 1
Scheme 2
Figure 1. Top: Natural products and initial C20′ vinblastine
analogues. Bottom: X-ray structure of vinblastine bound to tubulin
highlighting the region surrounding vinblastine C20′ site (PDB ID:
1Z2B).14
exceed the potency of vinblastine but that also exhibit good
activity against a Pgp (P-glycoprotein)-overexpressing, vinblas-
tine-resistant tumor cell line. Just as remarkably and in contrast
to expectations based on the steric constraints of the tubulin
binding site surrounding the vinblastine C20′ center as
depicted in the X-ray cocrystal structure of a tubulin bound
complex,14 large C20′ urea derivatives are accommodated,
exhibiting potent functional activity in cell-based proliferation
assays and effectively binding tubulin.
analogues. Alternatively, treatment of 20′-isothiocyanovinblas-
tine (5),10 made from the reaction of 20′-aminovinblastine (7)
and carbon disulfide (82%,) with available amines (method 4)
also provided a set of C20′ thiourea vinblastine analogues.
A series of N,N-disubstituted C20′ urea and thiourea
vinblastine analogues were also prepared using methods 2
and 4 and commercially available N,N-disubstituted amines.
Additionally, the carbamate analogue 45 was synthesized for
direct comparison with what proved to be the potent C20′ urea
and thiourea derivatives. Thus, vinblastine was treated with the
N,N-dimethylcarbamyl chloride to provide the C20′ carbamate
45 (eq 1).
Biological Activity. The C20′ substituted vinblastine
analogues were examined for cell growth inhibitory activity
against the HCT116 (human colon cancer) and HCT116/
VM46 (resistant human colon cancer) tumor cell lines, the
latter of which exhibits resistance (100-fold) to vinblastine
through overexpression of Pgp.16,17 As we reported, the
unsubstituted urea 11 on which the studies are based
approached but did not match the potency of vinblastine.
RESULTS AND DISCUSSION
■
Chemistry. The targeted vinblastine C20′ urea analogues
were prepared by two methods (Scheme 1). The first method
(method 1) involved treating the recently accessible 20′-
aminovinblastine (7)10 with available isocyanates to provide the
corresponding ureas. In the instances when the isocyanates
were not readily available, method 2 was used. This entailed
treating 20′-aminovinblastine (7) with p-nitrophenylchlorofor-
mate to provide the activated carbamate 13, which was then
treated with a series of amines to provide the additional C20′
urea analogues.
The vinblastine C20′ thiourea analogues were prepared also
using two complementary methods (Scheme 2). Thus,
treatment of 20′-aminovinblastine (7) with a small series of
commercially available isothiocyanates (method 3) versus
isocyanates provided the corresponding C20′ thiourea
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modest loss in activity given expectations. Introduction of a
polar group that can serve as either a H-bond donor or acceptor
on the alkyl substituent in 22 maintained the activity observed
with other small alkyl substituents in the HCT116 cell line;
however, it had a deleterious effect in the potency against the
resistant cell line (IC50 >1000 nM, HCT116/VM46), similar to
that seen in the parent urea 11. The cell growth inhibition
activity against the L1210 (mouse leukemia) tumor cell line was
also measured, and the results were qualitatively and
quantitatively (IC50) nearly identical to those observed with
the HCT116 cell line, providing IC50 values for the urea
derivatives that matched or exceeded the activity of vinblastine
itself (see Supporting Information for L1210 cytotoxic activity
data).
The results of the examination of the systematically varied
monosubstituted C20′ urea derivatives are summarized in
Figure 2 alongside those of 1 and 11. Terminal N-alkyl
The examination of the monosubstituted C20′ urea
derivatives bearing N-aryl substituents (23−30) proved even
more unexpected. All exhibited cell growth inhibitory activity at
levels exceeding the parent urea 11, matching or surpassing the
potency of vinblastine itself. Electron-withdrawing or electron-
donating substituents on the parent N-phenyl urea 23 are well
tolerated, and although no strongly polar substituents were
examined, it is notable that the p-methoxy substituent proved
to be among the best of the p-substituents. As a result, the
impact of a m- and o-methoxy phenyl substituent was also
examined. Significantly, 29 and 30 bearing N-(m-methoxy-
phenyl) and N-(o-methoxyphenyl) urea substituents, respec-
tively, exhibited exceptional activity, substantially exceeding the
potency of vinblastine nearly 10-fold (IC50 770 pM vs 6.8 nM,
HCT116) and displaying uniquely potent activity against the
vinblastine-resistant HCT116 cell line (IC50 = 80 and 65 nM,
HCT116/VM46). This activity along with that of 15 and 18
represents a 10-fold improvement over vinblastine and may
represent activity at a level of clinical significance.
Placement of a one or two methylene spacer between the
phenyl ring and urea nitrogen (31 and 32) maintained activity,
with both derivatives matching the activity of both 23 and
vinblastine itself. Replacement of the phenyl ring in 31 with a
heteroaromatic ring (furan or pyridine) provided 33 and 34,
which displayed comparable activity to the parent 31, matching
or slightly exceeding the potency of vinblastine. All of these
observations are unexpected given the apparent steric
constraints of the tubulin binding site surrounding the
vinblastine C20′ center observed in the X-ray crystal structure
of a tubulin bound complex.14 To further probe just how much
space may be available to a C20′ derivative, the rigid N-
biphenyl urea 35 was prepared and examined. Remarkably, it
displayed cell growth inhibitory activity at a level indistinguish-
able from vinblastine (Figure 3). This result indicates that
sterically demanding modifications to such C20′ urea
derivatives are possible, even beyond what we have probed
herein. As a result and in addition to improvements in potency,
this may be a superb site for modulating the physical and
chemical properties of the drug that impact additional features
including Pgp efflux,18 in vivo drug distribution, selective
cellular uptake, and metabolism.
Figure 2. Activity of vinblastine C20′ urea analogues.
substituents were not only well tolerated, but provided
significant enhancements in activity, improving on the potency
of 11 and providing derivatives that substantially surpass that of
vinblastine itself. Most notable of these are the urea derivatives
14−18, bearing small N-alkyl substituents, some of which
exhibited IC50s of 700−800 pM against HCT116, improving
activity against HCT116 10-fold relative to 11 and substantially
surpassing the potency of vinblastine itself (ca. 10-fold). Even
the larger N-alkyl derivatives 19 and 21 matched or slightly
surpassed the activity of vinblastine, and only 20, bearing the
large t-butyl substituent, experienced a small, but surprisingly
A series of thiourea derivatives were also examined (Figure
4). Although the unsubstituted thiourea derivative 12
approached the activity of the corresponding urea 11 in our
original studies (IC50 = 7.7 vs 7.5 nM, HCT116), the
monosubstituted N-alkyl or N-aryl derivatives 36 and 37
proved to be 3- to 4-fold less active than the corresponding
ureas 21 and 23 (HCT116). However, it is notable that the
activity difference between sensitive and vinblastine-resistant
C
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Figure 3. Unprecedented C20′ steric tolerance.
Figure 5. Activity of vinblastine C20′ disubstituted urea and thiourea
analogues.
importance of the C20′ amine versus C20′ alcohol function-
alization, suggesting the H-donor capabilities of the former may
be important.
Although less pronounced, but as detailed in our initial
work,10 the amide 9 and methyl carbamate 10 were found to be
more than 10-fold less active than the urea 11, further
highlighting a unique role the urea terminal nitrogen plays in
potentiating the activity (Figure 6). As a result and given the
size of substituents tolerated, even the intermediate p-
nitrophenylcarbamate 13 used to prepare the ureas herein
was tested and proved to be a surprisingly effective agent (IC50
Figure 4. Activity of vinblastine C20′ thiourea analogues.
cell lines diminishes in this thiourea series (15- to 25-fold vs
100-fold), suggesting they may be transported by Pgp
somewhat less effectively.18
A small, but important series of N,N-disubstituted ureas and
thioureas were also examined in order to establish whether the
terminal urea nitrogen could be fully substituted or whether the
derivatives require or benefit from the presence of an N−H H-
bond donor (Figure 5). Remarkably, all the N,N-disubstituted
ureas exhibited potent cell growth inhibitory activity matching
or surpassing the activity of vinblastine and indicating that a
terminal H-bond donor site is not important to their functional
activity. However, both 39 and 40 were less active than the
corresponding monosubstituted urea derivatives 14 and 15.
Ureas with cyclic substituents on the terminal nitrogen, 41 and
42, exhibited a similar potency to the acyclic derivatives 39 and
40. An analogous observation was made with the N,N-dimethyl
thiourea derivative 43, which approached the potency of
vinblastine but exhibited activity slightly lower than the
corresponding N,N-dimethyl urea 38. Interestingly, and like
the thiourea derivatives 36 and 37, 43 and especially the urea
derivatives 39 and 42 exhibited a diminished activity difference
between sensitive and vinblastine-resistant HCT116 cell lines
(10-fold vs 100-fold). Finally, comparison of the N,N-dimethyl
urea or thiourea 39 and 43 with the N,N-dimethylcarbamate 44
(>1000-fold less active) clearly illustrates the distinction and
Figure 6. Importance of the C20′ amine versus alcohol functionaliza-
tion and distinctions between urea/thiourea versus carbamate/amide
derivatives.
D
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= 55 nM, HCT116), matching the activity of the methyl
carbamate 10.
In recent work, the incorporation of a fluorine atom at the
10′ position provided a potent molecule (48) with an 8-fold
improvement in activity over vinblastine itself.9a We were
interested in determining whether the incorporation of both
the 10′-F substituent and a C20′ urea would have an additive
effect in enhancing the potency of vinblastine. An analogue 49
with both functionalities was prepared and evaluated (Figure
9). Only a modest improvement in potency was observed in 49
Finally, two additional C20′ amines (45 and 46) were
prepared by reductive amination (H2CO (5 equiv), NaBH3CN
(20 equiv), THF, 4 h, 32% (45) NHMe, 33% (46) NMe2) of
20′-aminovinblastine (7) in order to establish the generality of
the observations made with the unsubstituted primary amine 7
itself (Figure 7). Although the C20′ methylamine proved more
Figure 7. Activity of C20′ amines.
Figure 9. Urea derivative of 10′-fluorovinblastine.
potent than 7, it was still 10-fold less active than vinblastine and
the C20′ dimethylamine derivative 46 was the least active of the
C20′ amines, suggesting the importance of a H-bond donor
with regard to biological activity.
Finally, the 20′-(methylamino)vinblastine (45) was enlisted
to establish whether the active urea derivatives require or
benefit from the H-bond donor site of the derivatized C20′
amine. Thus, treatment of 45 with ethyl isocyanate provided
the N-methyl urea 47 for comparison with 15 (Figure 8). There
relative to 15 and 48, suggesting that these modifications are
not fully additive. Nonetheless, the modified vinblastine 49 is at
least 10-fold more potent than vinblastine, exhibiting a
subnanomolar IC50 for cell growth inhibition (620 pM,
HCT116) and a nearly 10-fold improved activity against a
vinblastine-resistant cell line (IC50 = 70 nM, HCT116/VM46).
Binding to Tubulin. Given the apparent steric constraints
of the tubulin binding site surrounding the vinblastine C20′
center (Figure 1) and the size of the C20′ urea substituents that
support and improve on the functional potency of vinblastine
itself in the cytotoxic cell growth assays, it was not clear
whether these effects could be related to their target (tubulin)
binding affinity or derived from their impact on other
properties of the molecules (e.g., cell permeability, metabolism,
solubility). As a result, two representative C20′ urea derivatives
35 and 39 were examined in a well-established tubulin binding
assay conducted by measuring the competitive displacement of
3H-vinblastine from porcine tubulin (Figure 10).19 Notably, 35
contains the large biphenyl urea substituent yet matches the
functional activity of vinblastine, whereas 39 bears the much
smaller N,N-dimethylurea whose functional activity slightly
exceeds that of vinblastine (≤3-fold). Importantly, these
binding studies confirmed that 39 binds tubulin with a slightly
better affinity than vinblastine and further established that even
35, bearing the large biphenyl substituent, remarkably binds
with an affinity matching or even slightly exceeding that of
vinblastine. Thus, the effects of the urea 39 as well as 35
observed in the functional assays correlate directly with their
target tubulin binding affinities. These unanticipated observa-
tions with 35 highlight that the vinblastine interaction with
tubulin surrounding the C20′ center is flexible and capable of
reorganization to accommodate even a very large substituent. It
is notable that this site is adjacent to the nucleotide binding site
involving the T5 loop in the N-terminal β1 tubulin nucleotide
binding domain and adjacent to the H6 helix and H6−H7 loop
Figure 8. Impact of C20′ nitrogen H-bond donor.
was a 700-fold decrease in activity between 15 and 47
(HCT116), clearly illustrating the importance of a H-bond
donor site on the C20′ position. This observation clearly
suggests that the incorporation of a urea functionality maintains
a key H-bond site directly attached to the C20′ position and
that it best approximates the acidity of the vinblastine C20′
alcohol while allowing for further functionalization on the urea
terminal nitrogen that maintains or in many cases improves the
activity of the compound.
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(250 and 500 μm) F254 glass plates. Optical rotations were determined
on a Rudolph Research Analytical Autopol III automatic polarimeter
using the sodium D line (λ = 589 nm) at room temperature (23 °C)
and are reported as follows: [α]D23, concentration (c = g/100 mL), and
solvent. FT-IR spectroscopy was recorded on a Nicolet 380 FT-IR
instrument. 1H NMR was recorded on a Bruker 600 MHz
spectrometer. Chemical shifts are reported in ppm from an internal
1
standard of residual CHCl3 (δ 7.26 for H). Proton chemical data are
reported as follows: chemical shift (δ), multiplicity (ovlp =
overlapping, br = broad, s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet), coupling constant, and integration. High
resolution mass spectra were obtained on an Agilent ESI-TOF/MS
using Agilent ESI-L low concentration tuning mix as internal high
resolution calibration standards. The purity of each tested compound
(>95%) was determined on an Agilent 1100 LC/MS instrument using
a ZORBAX SB-C18 column (3.5 mm, 4.6 mm ×50 mm, with a flow
rate of 0.75 mL/min and detection at 220 and 254 nm) with a 10−
98% acetonitrile/water/0.1% formic acid gradient (two different
gradients).
General Methods for the Synthesis of Ureas. Method 1. A
solution of 20′-aminovinblastine (7, 3.5 mg, 0.004 mmol) in THF (3
mL) was treated with an isocyanate (0.008 mmol). The reaction
mixture was stirred for 2 h at 25 °C and then was quenched with the
addition of distilled H2O (3 mL). The mixture was extracted with 10%
MeOH in CH2Cl2, and the combined organic extracts were washed
with saturated aqueous NaCl (3 mL). The organic layer was dried over
Na2SO4 and concentrated under reduced pressure. PTLC (SiO2,
EtOAc:MeOH:Et3N = 97:3:3) provided the ureas (15, 19−21, 23−
32, and 34−35); yields 35−98%. Isocyanates used include: ethyl
isocyanate, n-butyl isocyanate, t-butyl isocyanate, cyclohexyl isocya-
nate, phenyl isocyanate, benzyl isocyanate, phenethyl isocyanate, o-
methoxyphenyl isocyanate, m-methoxyphenyl isocyanate, p-methox-
yphenyl isocyanate, p-fluorophenyl isocyanate, p-chlorophenyl iso-
cyanate, p-tolyl isocyanate, p-trifluoromethylphenyl isocyanate, furfuryl
isocyanate, and p-biphenyl isocyanate.
Figure 10. Tubulin binding assay.
that links the nucleotide binding domain to the intermediate
domain. It is likely this region is capable of significant
reorganization to accommodate the binding of 35 or that the
urea substituent may extend into the nucleotide binding site
and displace a bound nucleotide.
Method 2. A solution of 20′-aminovinblastine (7, 5.7 mg, 0.007
mmol) in THF (3 mL) was treated with 4-nitrophenyl chloroformate
(2.1 mg, 0.011 mmol, 1.5 equiv). The reaction mixture was stirred for
4 h at 25 °C and then was concentrated under reduced pressure.
PTLC (SiO2, EtOAc:MeOH = 95:5) provided 13 (4.6 mg, 67%, off-
white solid). A solution of 13 (4.0 mg, 0.004 mmol) in THF (3 mL)
was treated with an amine (0.008 mmol). The reaction mixture was
stirred for 1 h at 25 °C and then was quenched with the addition of
distilled H2O (3 mL). The mixture was extracted with 10% MeOH in
CH2Cl2, and the combined organic extracts were washed with
saturated aqueous NaCl (3 mL). The organic layer was dried over
Na2SO4 and concentrated under reduced pressure. PTLC (SiO2,
EtOAc:MeOH:Et3N = 97:3:3) provided the ureas (14, 16−18, 22, 33,
and 39−42); yields 40−99%. The amines used include: methylamine,
propylamine, isopropylamine, cyclopropylamine, dimethylamine, di-
ethylamine, 2-(aminomethyl)pyridine, morpholine, piperidine, and
ethanolamine.
General Methods for the Synthesis of Thioureas. Method 3.
A solution of 20′-aminovinblastine (7, 8.0 mg, 0.010 mmol) in THF (4
mL) was treated with an isothiocyanate (0.031 mmol). The reaction
mixture was stirred for 2 h at 25 °C and then was quenched with the
addition of distilled H2O (3 mL), the mixture was extracted with 10%
MeOH in CH2Cl2, and the combined organic extracts were washed
with saturated aqueous NaCl (3 mL). The organic layer was dried over
Na2SO4 and concentrated under reduced pressure. PTLC (SiO2,
EtOAc:MeOH:Et3N = 97:3:3) provided the thiourea (37); yields 70%.
The isothiocyanate used: phenylisothiocyanate.
CONCLUSIONS
■
A remarkable series of previously inaccessible C20′ urea
derivatives of vinblastine were prepared and found to match
or substantially exceed the potency of vinblastine in functional
cell-based growth inhibition assays. In addition to defining
structural features of the urea required for or potentiating their
activity that are directly related to their relative tubulin binding
affinity, the studies established an unprecedented steric
tolerance for the size of a C20′ substituent. A H-bond donor
on the C20′ position was unequivocally shown to be an
important feature of the potent vinblastine analogues. Although
this site is known to be critical to the properties of vinblastine
and is located deeply embedded in the tubulin bound complex
where such substituents would be apparently sterically
constrained, the studies revealed that sterically demanding
ureas are not only tolerated but that functionalization of this
site offers a superb opportunity for enhancing potency as much
as 10-fold. In addition to improvements in potency, this may
also be a superb site for modulating the physical and chemical
properties of the drug that impact additional features including
Pgp efflux, in vivo drug distribution, selective cellular uptake,
and metabolism.
EXPERIMENTAL SECTION
Method 4. A solution of 20′-aminovinblastine (7, 8.2 mg, 0.010
mmol) in THF (3 mL) was treated with carbon disulfide (915 μL, 15
mmol). The reaction mixture was stirred for 13 h and then was
quenched with the addition of distilled H2O (5 mL), and the mixture
was extracted with 10% MeOH in CH2Cl2 and washed with saturated
aqueous NaCl (2 mL). The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. PTLC (SiO2, EtOAc:MeO-
H:Et3N = 97:3:3) provided 5 (7.0 mg, 82%, white solid).10 A solution
■
General Procedures. All commercial reagents were used without
further purification unless otherwise noted. THF was distilled prior to
use. All reactions were performed in oven-dried (200 °C) glassware
and under an inert atmosphere of anhydrous Ar unless otherwise
noted. Column chromatography was performed with silica gel 60. TLC
was performed on Whatman silica gel (250 μm) F254 glass plates and
spots visualized by UV. PTLC was performed on Whatman silica gel
F
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of 20′-isothiocyanovinblastine (5, 6.0 mg, 0.007 mmol) in THF (3
mL) was treated with an amine (0.011 mmol). The reaction mixture
was stirred for 1 h at 25 °C and then was quenched with the addition
of distilled H2O (3 mL). The mixture was extracted with 10% MeOH
in CH2Cl2, and the combined extracts were washed with saturated
aqueous NaCl (3 mL). The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. PTLC (SiO2, EtOAc:MeO-
H:Et3N = 97:3:3) provided the thiourea (36, 38, and 43); yields 26−
92%. The amines used include: cyclohexylamine, 4-fluorophenethyl-
amine, and dimethylamine.
10.1, 3.7 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J = 9.3 Hz, 1H), 4.29 (br s,
1H), 4.14 (br s, 1H), 3.96−3.91 (m, 1H), 3.81−3.77 (m, 1H), 3.80 (s,
3H), 3.79 (s, 3H), 3.74 (s, 1H), 3.73−3.71 (m, 1H), 3.60 (s, 3H), 3.37
(d, J = 14.8 Hz, 2H), 3.34−3.26 (m, 2H), 3.23 (t, J = 11.9 Hz, 1H),
3.18−3.11 (m, 2H), 2.83 (d, J = 16.0 Hz, 1H), 2.71 (s, 3H), 2.67 (s,
1H), 2.56 (d, J = 13.6 Hz, 1H), 2.47−2.43 (m, 1H), 2.36 (d, J = 14.4
Hz, 1H), 2.20−2.15 (m, 2H), 2.11 (s, 3H), 1.86−1.73 (m, 4H), 1.47−
1.42 (m, 1H), 1.37−1.29 (m, 3H), 1.20 (dd, J = 6.4, 2.5 Hz, 6H), 0.82
(t, J = 7.4 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H). IR (film) νmax 3471, 3323,
2923, 1741, 1459, 1239, 1041 cm−1. HRESI-TOF m/z 917.4761
(C50H66N6O9 + Na+, required 917.4783); [α]D23 +6.1 (c 0.1, CHCl3).
(R = 20′-NHCONHCH(CH2)2) 18. Yield: 70%, method 2. 1H NMR
(600 MHz, CDCl3) δ 9.86 (br s, 1H), 8.02 (br s, 1H), 7.50 (d, J = 7.8
Hz, 1H), 7.18−7.13 (m, 1H), 7.13−7.07 (m, 2H), 6.62 (s, 1H), 6.10
(s, 1H), 5.85 (dd, J = 10.2, 4.4 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J = 6.6
Hz, 1H), 5.17 (s, 1H), 4.66 (s, 1H), 3.91 (t, J = 13.8 Hz, 1H), 3.80 (s,
6H), 3.74 (s, 1H), 3.56 (s, 3H), 3.41−3.13 (m, 6H), 3.08−3.00 (m,
2H), 2.83 (d, J = 16.2 Hz, 1H), 2.77 (br s, 1H), 2.71 (s, 3H), 2.70−
2.68 (m, 1H), 2.67 (s, 1H), 2.63 (d, J = 13.5 Hz, 1H), 2.47−2.43 (m,
1H), 2.38 (d, J = 12.1 Hz, 1H), 2.24 (d, J = 14.2 Hz, 1H), 2.19 (dd, J =
12.9, 7.7 Hz, 1H), 2.15 (s, 1H), 2.11 (s, 3H), 1.86−1.70 (m, 4H),
1.52−1.49 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H), 0.76 (t, J = 7.3 Hz, 3H),
0.70−0.69 (m, 2H), 0.50−0.48 (m, 2H). IR (film) νmax 3629, 2952,
1740, 1506, 1458, 1245, 998, 748 cm−1. HRESI-TOF m/z 893.4792
1
(R = 20′-NHCO2C6H4NO2) 13. Yield: 67%, method 2. H NMR
(600 MHz, CDCl3) δ 9.87 (br s, 1H), 8.11 (d, J = 9.2 Hz, 2H), 8.04
(br s, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.18−7.14 (m, 1H), 7.14−7.08
(m, 2H), 6.76 (d, J = 9.1 Hz, 2H), 6.63 (s, 1H), 6.11 (s, 1H), 5.85 (dd,
J = 10.0, 3.9 Hz, 1H), 5.48 (s, 1H), 5.30 (d, J = 9.8 Hz, 1H), 4.00 (t, J
= 13.2 Hz 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.72 (s, 1H), 3.63 (s, 3H),
3.60−3.55 (m, 4H), 3.43 (d, J = 13.6 Hz, 1H), 3.37 (dd, J = 15.9, 4.5
Hz, 1H), 3.35−3.26 (m, 2H), 3.20−3.13 (m, 2H), 2.98 (d, J = 13.8
Hz, 1H), 2.85−2.77 (m, 2H), 2.70 (s, 3H), 2.46−2.42 (m, 1H), 2.35
(d, J = 11.9 Hz, 1H), 2.24 (d, J = 13.0 Hz, 1H), 2.20−2.13 (m, 1H),
2.11 (s, 3H), 1.85−1.76 (m, 2H), 1.64 (d, J = 14.8 Hz, 1H), 1.47−1.40
(m, 2H), 1.37−1.29 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H), 0.83 (t, J = 7.4
Hz, 3H). IR (film) νmax 3467, 2924, 1736, 1218 cm−1. HRESI-TOF m/
23
z 975.4492 (C53H62N6O12 + H+, required 975.4498); [α]D −13 (c
23
0.1, CHCl3).
(C50H64N6O9 + H+, required 893.4808); [α]D +19 (c 0.09, CHCl3).
(R = 20′-NHCONHCH3) 14. Yield: 85%, method 2. 1H NMR (600
MHz, CDCl3) δ 9.83 (br s, 1H), 7.97 (br s, 1H), 7.51 (d, J = 7.8 Hz,
1H), 7.19−7.13 (m, 1H), 7.13−7.07 (m, 2H), 6.64 (s, 1H), 6.09 (s,
1H), 5.86 (dd, J = 10.0, 4.4 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J = 10.7 Hz,
2H), 4.54 (br s, 1H), 4.33 (br s, 1H), 3.84 (t, J = 13.2 Hz 1H), 3.80 (s,
6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.39−3.36 (m, 2H), 3.32−3.21 (m,
3H), 3.20−3.10 (m, 2H), 2.86 (d, J = 4.8 Hz, 3H), 2.83 (d, J = 16.6
Hz, 1H), 2.71 (s, 3H), 2.68 (s, 1H), 2.58 (d, J = 13.8 Hz, 1H), 2.45
(dd, J = 16.9, 10.5 Hz, 1H), 2.38 (d, J = 12.8 Hz, 1H), 2.20−2.15 (m,
2H), 2.11 (s, 3H), 1.85−1.74 (m, 4H), 1.69 (d, J = 13.7 Hz, 2H),
1.44−1.30 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H).
IR (film) νmax 3539, 2870, 1721, 1554, 1461, 1223, 1039, 712 cm−1.
HRESI-TOF m/z 867.4631 (C53H62N6O12 + H+, required 867.4651);
(R = 20′-NHCONHCH2CH2CH2CH3) 19. Yield: 45%, method 1. 1H
NMR (600 MHz, CDCl3) δ 9.85 (br s, 1H), 7.98 (br s, 1H), 7.50 (d, J
= 7.8 Hz, 1H), 7.18−7.13 (m, 1H), 7.13−7.07 (m, 2H), 6.64 (s, 1H),
6.09 (s, 1H), 5.86 (dd, J = 10.2, 4.4 Hz, 1H), 5.46 (s, 1H), 5.31 (d, J =
11.1 Hz, 1H), 4.56 (br s, 1H), 4.27 (br s, 1H), 3.80 (s, J = 1.6 Hz, 6H),
3.74 (s, 1H), 3.60 (s, 3H), 3.37 (dd, J = 15.9, 4.7 Hz, 2H), 3.31−3.20
(m, 4H), 3.15 (br s, 2H), 3.09 (dd, J = 14.5, 7.2 Hz, 4H), 2.83 (d, J =
16.2 Hz, 1H), 2.71 (s, 3H), 2.68 (s, 1H), 2.55 (d, J = 13.8 Hz, 1H),
2.47−2.43 (m, 1H), 2.38 (d, J = 12.1 Hz, 1H), 2.20−2.15 (m, 2H),
2.11 (s, 3H), 2.07 (s, 1H), 1.86−1.72 (m, 4H), 1.56−1.49 (m, 2H),
1.38−1.33 (m, 4H), 0.93 (t, J = 7.3 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H),
0.77 (t, J = 7.3 Hz, 3H). IR (film) νmax 3544, 2952, 1736, 1501, 1458,
1240, 1030, 761 cm−1. HRESI-TOF m/z 909.5106 (C51H68N6O9 +
23
23
[α]D −1.5 (c 0.05, CHCl3).
H+, required 909.5121); [α]D +14 (c 0.06, CHCl3).
1
1
(R = 20′-NHCONHCH2CH3) 15. Yield: 95%, method 1. H NMR
(R = 20′-NHCONHC(CH3)3) 20. Yield: 45%, method 1. H NMR
(600 MHz, CDCl3) δ 9.84 (br s, 1H), 7.98 (br s, 1H), 7.51 (d, J = 7.8
Hz, 1H), 7.19−7.14 (m, 1H), 7.14−7.07 (m, 2H), 6.64 (s, 1H), 6.09
(s, 1H), 5.86 (dd, J = 9.9, 4.0 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J = 9.8
Hz, 1H), 4.59 (br s, 1H), 4.29 (br s, 1H), 3.83 (t, J = 12.0 Hz, 1H),
3.80 (s, 6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.37 (d, J = 15.0 Hz, 2H),
3.34−3.19 (m, 4H), 3.18−3.13 (m, 2H), 2.83 (d, J = 16.0 Hz, 1H),
2.71 (s, 3H), 2.68 (s, 1H), 2.57 (d, J = 13.1 Hz, 1H), 2.47−2.43 (m,
1H), 2.38 (d, J = 13.4 Hz, 1H), 2.20−2.15 (m, 2H), 2.11 (s, 3H),
1.86−1.73 (m, 4H), 1.68 (d, J = 14.3 Hz, 2H), 1.41−1.38 (m, 4H),
1.19 (t, J = 7.2 Hz, 3H), 0.82 (t, J = 7.4 Hz, 3H), 0.77 (t, J = 7.4 Hz,
3H). IR (film) νmax 3463, 2921, 1739, 1500, 1459, 1228, 1039, 739
cm−1. HRESI-TOF m/z 881.4797 (C49H64N6O9 + H+, required
(600 MHz, CDCl3) δ 9.83 (br s, 1H), 7.99 (br s, 1H), 7.53 (d, J = 8.0
Hz, 1H), 7.19−7.14 (m, 1H), 7.13−7.07 (m, 2H), 6.65 (s, 1H), 6.09
(s, 1H), 5.86 (dd, J = 10.1, 3.9 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J = 10.2
Hz, 1H), 4.54 (br s, 1H), 4.10 (br s, 1H), 3.83 (t, J = 14.1 Hz, 1H),
3.80 (s, 3H), 3.78 (s, 3H), 3.74 (s, 1H), 3.60 (s, 3H), 3.40−3.22 (m,
5H), 3.17−3.12 (m, 2H), 2.71 (s, 3H), 2.68 (s, 1H), 2.54 (d, J = 13.6
Hz, 1H), 2.47−2.42 (m, 1H), 2.35 (d, J = 13.4 Hz, 1H), 2.21−2.14
(m, 2H), 2.11 (s, 3H), 1.87−1.75 (m, 2H), 1.73−1.62 (m, 4H), 1.49−
1.41 (m, 2H), 1.38 (s, 9H), 1.37−1.31 (m, 2H), 0.82 (t, J = 7.4 Hz,
3H), 0.77 (t, J = 7.4 Hz, 3H). IR (film) νmax 3457, 2941, 1741, 1556,
1458, 1244, 1036, 741 cm−1. HRESI-TOF m/z 909.5128
23
(C51H68N6O9 + H+, required 909.5121); [α]D +3.0 (c 0.1, CHCl3).
23
1
881.4808); [α]D +5.4 (c 0.08, CHCl3).
(R = 20′-NHCONHCH2CH2CH3) 16. Yield: 40%, method 2. H
(R = 20′-NHCONHC6H11) 21. Yield: 51%, method 1. H NMR
1
(600 MHz, CDCl3) δ 9.84 (br s, 1H), 7.99 (br s, 1H), 7.52 (d, J = 7.9
Hz, 1H), 7.20−7.14 (m, 1H), 7.14−7.07 (m, 2H), 6.64 (s, 1H), 6.09
(s, 1H), 5.86 (dd, J = 10.3, 4.1 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J = 9.3
Hz, 1H), 4.53 (br s, 1H), 4.22 (br s, 1H), 4.02 (d, J = 9.0 Hz, 1H),
3.83 (t, J = 12.9 Hz, 1H), 3.79 (s, 3H), 3.79 (s, 3H), 3.74 (s, 1H), 3.60
(s, 3H), 3.50−3.45 (m, 1H), 3.39−3.21 (m, 3H), 3.16−3.12 (m, 2H),
2.83 (d, J = 16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.55 (d, J = 13.5
Hz, 1H), 2.47−2.42 (m, 1H), 2.37 (d, J = 12.7 Hz, 1H), 2.21−2.14
(m, 2H), 2.11 (s, 3H), 2.04−1.90 (m, 2H), 1.86−1.74 (m, 4H), 1.64−
1.59 (m, 2H), 1.37−1.31 (m, 6H), 1.20−1.02 (m, 6H), 0.82 (t, J = 7.4
Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H). IR (film) νmax 3460, 2919, 1738,
1556, 1459, 1242, 1033, 752 cm−1. HRESI-TOF m/z 935.5276
(C53H70N6O9 + H+, required 935.5277); [α]D23 +3.8 (c 0.09, CHCl3).
NMR (600 MHz, CDCl3) δ 9.84 (br s, 1H), 7.98 (br s, 1H), 7.51 (d, J
= 7.8 Hz, 1H), 7.18−7.14 (m, 1H), 7.14−7.08 (m, 2H), 6.64 (s, 1H),
6.09 (s, 1H), 5.86 (dd, J = 10.1, 4.3 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J =
9.8 Hz, 1H), 4.63 (br s, 1H), 4.28 (br s, 1H), 3.83 (t, J = 12.0 Hz, 1H),
3.80 (s, 6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.37 (dd, J = 15.9, 4.7 Hz,
2H), 3.34−3.27 (m, 2H), 3.27−3.11 (m, 4H), 2.83 (d, J = 16.2 Hz,
1H), 2.71 (s, 3H), 2.68 (s, 1H), 2.57 (d, J = 13.8 Hz, 1H), 2.47−2.42
(m, 1H), 2.38 (d, J = 13.1 Hz, 1H), 2.24−2.15 (m, 2H), 2.09 (s, 3H),
1.87−1.75 (m, 4H), 1.60−1.52 (m, 6H), 1.40−1.34 (m, 2H), 0.95 (t, J
= 7.4 Hz, 3H), 0.82 (t, J = 7.4 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H). IR
(film) νmax 3436, 2952, 1739, 1547, 1459, 1243, 1032, 755 cm−1.
HRESI-TOF m/z 895.4953 (C50H66N6O9 + H+, required 895.4964);
23
1
[α]D +4.0 (c 0.05, CHCl3).
(R = 20′-NHCONHCH2CH2OH) 22. Yield: 60%, method 2. H
(R = 20′-NHCONHCH(CH3)2) 17. Yield: 59%, method 2. 1H NMR
(600 MHz, CDCl3) δ 9.84 (br s, 1H), 8.00 (br s, 1H), 7.52 (d, J = 7.9
Hz, 1H), 7.17−7.08 (m, 3H), 6.65 (s, 1H), 6.09 (s, 1H), 5.87 (dd, J =
NMR (600 MHz, CDCl3) δ 9.78 (br s, 1H), 7.99 (br s, 1H), 7.52 (d, J
= 7.8 Hz, 1H), 7.19−7.09 (m, 3H), 6.69 (s, 1H), 6.08 (s, 1H), 5.89−
5.85 (m, 1H), 5.48 (s, 1H), 5.31 (d, J = 11.8 Hz, 1H), 4.59 (br s, 1H),
G
dx.doi.org/10.1021/jm3015684 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3.83−3.72 (m, 4H), 3.80 (s, 3H), 3.79 (s, 3H), 3.76 (s, 1H), 3.57 (s,
3H), 3.57−3.54 (m, 1H), 3.42−3.37 (m, 3H), 3.34−3.29 (m, 1H),
3.26−3.17 (m, 4H), 3.08−3.03 (m, 1H), 2.87 (br s, 1H), 2.82 (d, J =
16.0 Hz, 1H), 2.72 (s, 3H), 2.68 (s, 1H), 2.60−2.42 (m, 1H), 2.33−
2.17 (m, 1H), 2.14−2.09 (m, 1H), 2.11 (s, 3H), 1.97−1.77 (m, 2H),
1.75−1.64 (m, 3H), 1.50−1.45 (m, 1H), 1.38−1.34 (m, 2H), 1.16 (dd,
J = 14.3, 4.6 Hz, 2H), 0.83−0.81 (m, 3H), 0.77 (t, J = 7.1 Hz, 3H). IR
(film) νmax 3399, 2927, 1738, 1502, 1458, 1232, 1040 cm−1. HRESI-
Hz, 1H), 4.99 (br s, 1H), 3.90−3.84 (m, 2H), 3.80 (s, 6H), 3.75 (s,
1H), 3.67−3.63 (m, 1H), 3.61 (s, 3H), 3.40−3.36 (m, 2H), 3.33−3.29
(m, 1H), 3.27−3.20 (m, 1H), 3.13−3.10 (m, 1H), 2.83 (d, J = 15.8
Hz, 1H), 2.72 (s, 3H), 2.68 (d, J = 11.9 Hz, 1H), 2.60 (d, J = 13.8 Hz,
1H), 2.48−2.43 (m, 1H), 2.40 (d, J = 13.3 Hz, 1H), 2.33−2.24 (m,
2H), 2.20−2.15 (m, 1H), 2.10 (s, 3H), 2.01 (s, 1H), 1.90−1.87 (m,
2H), 1.84−1.75 (m, 2H), 1.66 (d, J = 14.9 Hz, 2H), 1.40−1.32 (m,
2H), 0.82 (t, J = 7.2 Hz, 6H). IR (film) νmax 3334, 2931, 1716, 1537,
1472, 1232, 1023, 745 cm−1. HRESI-TOF m/z 997.4685
23
TOF m/z 897.4753 (C49H64N6O10 + H+, required 897.4756); [α]D
23
(C54H63F3N6O9 + H+, required 997.4681); [α]D −18 (c 0.04,
−14 (c 0.2, CHCl3).
(R = 20′-NHCONHC6H5) 23. Yield: 87%, method 1. 1H NMR (600
MHz, CDCl3) δ 9.87 (br s, 1H), 7.98 (br s, 1H), 7.47 (d, J = 8.1 Hz,
1H), 7.45 (d, J = 8.0 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.16−7.14 (m,
1H), 7.13−7.05 (m, 3H), 6.62 (s, 1H), 6.09 (s, 1H), 5.85 (dd, J = 10.3,
4.6 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J = 9.4 Hz, 1H), 4.80 (s, 1H), 3.80
(s, 6H), 3.74 (s, 1H), 3.60 (s, 3H), 3.39−3.35 (m, 2H), 3.32−3.28 (m,
1H), 3.22−3.18 (m, 1H), 3.10 (s, 3H), 2.82 (d, J = 16.3 Hz, 1H), 2.71
(s, 3H), 2.67 (s, 1H), 2.59 (d, J = 13.8 Hz, 1H), 2.47−2.42 (m, 1H),
2.36−2.28 (m, 2H), 2.25−2.13 (m, 2H), 2.11 (s, 3H), 2.04 (s, 1H),
1.85−1.76 (m, 3H), 1.71−1.65 (m, 2H), 1.64−1.54 (m, 2H), 1.24−
1.20 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H), 0.80 (t, J = 7.4 Hz, 3H). IR
(film) νmax 3468, 2940, 1742, 1501, 1460, 1237, 1031, 760 cm−1.
HRESI-TOF m/z 929.4807 (C53H64N6O9 + H+, required 929.4807);
CHCl3).
(R = 20′-NHCONH(4-methoxyphenyl)) 28. Yield: 36%, method
1
1. H NMR (600 MHz, CDCl3) δ 9.80 (br s, 1H), 8.01 (br s, 1H),
7.38 (d, J = 7.9 Hz, 1H), 7.24−7.20 (m, 1H), 7.18−7.09 (m, 2H), 6.93
(d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 6.39 (s, 1H), 6.34 (s,
1H), 6.08 (s, 1H), 5.88 (dd, J = 10.0, 4.7 Hz, 1H), 5.41 (s, 1H), 5.30
(d, J = 10.1 Hz, 1H), 4.60 (d, J = 12.0 Hz, 1H), 4.04 (t, J = 12.0 Hz,
1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.77 (s, 1H), 3.66 (s,
3H), 3.65−3.58 (m, 2H), 3.52−3.48 (m, 1H), 3.38−3.33 (m, 2H),
3.31−3.27 (m, 1H), 3.02−2.93 (m, 2H), 2.86−2.79 (m, 2H), 2.73 (s,
3H), 2.66 (s, 1H), 2.48−2.41 (m, 1H), 2.18−2.14 (m, 2H), 2.11 (s,
3H), 2.04−1.97 (m, 1H), 1.83−1.74 (m, 2H), 1.35−1.28 (m, 2H),
0.87 (t, J = 7.3 Hz, 3H), 0.79 (t, J = 7.3 Hz, 3H). IR (film) νmax 3447,
2899, 1742, 1507, 1459, 1234, 1036, 736 cm−1. HRESI-TOF m/z
23
[α]D +16 (c 0.9, CHCl3).
23
959.4917 (C54H66N6O10 + H+, required 959.4913); [α]D −9.4 (c
(R = 20′-NHCONH(4-fluorophenyl)) 24. Yield: 47%, method 1.
1H NMR (600 MHz, CDCl3) δ 9.81 (br s, 1H), 7.97 (br s, 1H), 7.49
(d, J = 8.1 Hz, 1H), 7.42 (dd, J = 8.8, 4.8 Hz, 2H), 7.18−7.14 (m, 1H),
7.12−7.08 (m, 2H), 7.01 (t, J = 8.6 Hz, 2H), 6.63 (s, 1H), 6.09 (s,
1H), 5.86 (dd, J = 10.2, 4.5 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J = 12.1 Hz,
1H), 4.71 (br s, 1H), 3.80 (s, 6H), 3.75 (s, 1H), 3.61 (s, 3H), 3.40−
3.36 (m, 2H), 3.32−3.28 (m, 1H), 3.21−3.06 (m, 4H), 2.82 (d, J =
16.5 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.59 (d, J = 13.7 Hz, 1H),
2.47−2.42 (m, 1H), 2.34 (d, J = 12.7 Hz, 1H), 2.20−2.15 (m, 2H),
2.11 (s, 3H), 1.85−1.75 (m, 4H), 1.59−1.50 (m, 4H), 1.23−1.21 (m,
2H), 0.82 (t, J = 7.4 Hz, 3H), 0.79 (t, J = 7.4 Hz, 3H). IR (film) νmax
3490, 2921, 1745, 1509, 1455, 1228, 1043, 736 cm−1. HRESI-TOF m/
0.04, CHCl3).
(R = 20′-NHCONH(3-methoxyphenyl)) 29. Yield: 50%, method
1
1. H NMR (600 MHz, CDCl3) δ 9.82 (br s, 1H), 7.96 (br s, 1H),
7.47 (d, J = 7.9 Hz, 1H), 7.19 (t, J = 8.1 Hz, 1H), 7.16−7.13 (m, 1H),
7.12−7.06 (m, 3H), 6.99 (d, J = 8.6 Hz, 1H), 6.62 (s, 1H), 6.61 (d, J =
8.1 Hz, 1H), 6.08 (s, 1H), 5.85 (dd, J = 10.2, 4.6 Hz, 1H), 5.46 (s,
1H), 5.30 (d, J = 9.9 Hz, 1H), 4.82 (s, 1H), 3.79 (s, 6H), 3.78 (s, 3H),
3.74 (s, 1H), 3.66−3.62 (m, 3H), 3.59 (s, 3H), 3.38−3.35 (m, 2H),
3.31−3.27 (m, 1H), 3.22−3.18 (m, 1H), 3.15−3.10 (m, 1H), 3.03 (d, J
= 13.3 Hz, 2H), 2.82 (d, J = 16.1 Hz, 1H), 2.71 (s, 3H), 2.66 (s, 1H),
2.58 (d, J = 13.8 Hz, 1H), 2.47−2.40 (m, 1H), 2.34 (d, J = 13.0 Hz,
1H), 2.22−2.14 (m, 2H), 2.10 (s, 3H), 1.84−1.76 (m, 3H), 1.75−1.69
(m, 2H), 1.38−1.31 (m, 2H), 1.23−1.21 (m, 1H), 0.81 (ovlp t, J = 7.4
Hz, 3H), 0.80 (ovlp t, J = 7.4 Hz, 3H). IR (film) νmax 3483, 2985,
1745, 1501, 1454, 1228, 1033, 760 cm−1. HRESI-TOF m/z 959.4905
(C54H66N6O10 + H+, required 959.4913); [α]D23 −19 (c 0.05, CHCl3).
(R = 20′-NHCONH(2-methoxyphenyl)) 30. Yield: 35%, method
1. 1H NMR (600 MHz, CDCl3) δ 9.85 (br s, 1H), 8.07 (d, J = 9.1 Hz,
1H), 8.01 (br s, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.17−7.12 (m, 1H),
7.13−7.07 (m, 2H), 7.05−6.95 (m, 2H), 6.88−6.86 (m, 1H), 6.64 (s,
1H), 6.10 (s, 1H), 5.85 (dd, J = 10.0, 4.7 Hz, 1H), 5.47 (s, 1H), 5.30
(d, J = 10.1 Hz, 1H), 5.13−5.10 (m, 1H), 4.76 (br s, 1H), 4.57 (br s,
1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.80 (s, 3H), 3.74 (s, 1H), 3.56 (s,
3H), 3.40−3.36 (m, 2H), 3.34−3.20 (m, 2H), 3.14−3.07 (m, 2H),
2.71 (s, 3H), 2.67 (s, 1H), 2.60 (d, J = 13.7 Hz, 1H), 2.50−2.42 (m,
1H), 2.39 (d, J = 12.9 Hz, 1H), 2.27 (d, J = 13.7 Hz, 1H), 2.20−2.15
(m, 2H), 2.11 (s, 3H), 2.09−1.99 (m, 2H), 1.84−1.76 (m, 4H), 1.50−
1.39 (m, 2H), 1.38−1.28 (m, 2H), 0.81 (ovlp t, J = 7.4 Hz, 3H), 0.80
(ovlp t, J = 7.4 Hz, 3H). IR (film) νmax 3451, 2919, 1730, 1531, 1461,
1257, 952, 721 cm−1. HRESI-TOF m/z 959.4909 (C54H66N6O10 + H+,
23
z 947.4726 (C53H63FN6O9 + H+, required 947.4713); [α]D −4.4 (c
0.08, CHCl3).
(R = 20′-NHCONH(4-chlorophenyl)) 25. Yield: 63%, method 1.
1H NMR (600 MHz, CDCl3) δ 9.80 (br s, 1H), 7.95 (br s, 1H), 7.40
(d, J = 7.9 Hz, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.19−7.07 (m, 5H), 6.65
(s, 1H), 6.08 (s, 1H), 5.86 (dd, J = 10.2, 4.5 Hz, 1H), 5.47 (s, 1H),
5.31 (d, J = 12.1 Hz, 1H), 4.81 (br s, 1H), 3.79 (s, 6H), 3.75 (s, 1H),
3.60 (s, 3H), 3.36 (d, J = 15.7 Hz, 2H), 3.31−3.26 (m, 2H), 3.22−3.15
(m, 2H), 3.01−2.91 (m, 1H), 2.82 (d, J = 15.7 Hz, 1H), 2.72 (s, 3H),
2.67 (s, 1H), 2.61−2.55 (m, 2H), 2.49−2.43 (m, 2H), 2.21−2.14 (m,
2H), 2.10 (s, 3H), 1.83−1.74 (s, 4H) 1.36−1.27 (m, 4H), 1.16−1.04
(m, 2H), 0.82 (t, J = 7.4 Hz, 3H), 0.79 (t, J = 7.4 Hz, 3H). IR (film)
νmax 3376, 2923, 1736, 1493, 1455, 1240, 1090, 765 cm−1. HRESI-
23
TOF m/z 963.4407 (C53H63ClN6O9 + H+, required 963.4418); [α]D
+21 (c 0.07, CHCl3).
(R = 20′-NHCONH(4-methylphenyl)) 26. Yield: 40%, method 1.
1H NMR (600 MHz, CDCl3) δ 9.86 (br s, 1H), 7.99 (br s, 1H), 7.47
(d, J = 7.9 Hz, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.18−7.07 (m, 3H), 6.61
(s, 1H), 6.08 (s, 1H), 5.85 (d, J = 5.9 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J
= 10.3 Hz, 1H), 4.74 (br s, 1H), 4.30 (br s, 1H), 3.80 (s, 3H), 3.79 (s,
3H), 3.74 (s, 1H), 3.67−3.62 (m, 3H), 3.61 (s, 3H), 3.40−3.34 (m,
2H), 3.31−3.27 (m, 1H), 3.20−3.16 (m, 1H), 2.81 (d, J = 15.7 Hz,
1H), 2.71 (s, 3H), 2.66 (s, 1H), 2.58 (d, J = 13.3 Hz, 1H), 2.46−2.41
(m, 1H), 2.32 (s, 3H), 2.24−2.14 (m, 2H), 2.11 (s, 3H), 2.03 (s, 1H),
1.87 (d, J = 13.8 Hz, 2H), 1.84−1.76 (m, 2H), 1.63−1.53 (m, 4H),
1.38−1.34 (m, 2H), 1.21 (dd, J = 14.5, 5.7 Hz, 2H), 0.81 (t, J = 7.6 Hz,
3H), 0.78 (t, J = 7.6 Hz, 3H). IR (film) νmax 3369, 2912, 1722, 1507,
1445, 1230, 1017, 729 cm−1. HRESI-TOF m/z 943.4949
23
required 959.4913); [α]D +24 (c 0.03, CHCl3).
(R = 20′-NHCONHCH2C6H5) 31. Yield: 96%, method 1. 1H NMR
(600 MHz, CDCl3) δ 9.84 (br s, 1H), 7.98 (br s, 1H), 7.47 (d, J = 8.1
Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.17−7.08
(m, 4H), 6.62 (s, 1H), 6.09 (s, 1H), 5.85 (dd, J = 10.3, 4.6 Hz, 1H),
5.47 (s, 1H), 5.30 (d, J = 9.4 Hz, 1H), 4.68 (s, 1H), 4.48−4.31 (m,
2H), 3.80 (s, 3H), 3.79 (s, 3H), 3.74 (s, 1H), 3.60 (s, 3H), 3.38−3.33
(m, 2H), 3.31−3.26 (m, 1H), 3.22−3.18 (m, 1H), 3.10 (s, 3H), 2.83
(d, J = 16.3 Hz, 1H), 2.70 (s, 3H), 2.67 (s, 1H), 2.59 (d, J = 13.8 Hz,
1H), 2.47−2.42 (m, 1H), 2.36−2.28 (m, 2H), 2.25−2.13 (m, 2H),
2.11 (s, 3H), 2.04 (s, 1H), 1.83−1.71 (m, 3H), 1.71−1.65 (m, 2H),
1.64−1.54 (m, 2H), 1.24−1.20 (m, 2H), 0.83 (t, J = 7.4 Hz, 3H), 0.80
(t, J = 7.4 Hz, 3H). IR (film) νmax 3454, 2992, 1737, 1501, 1459, 1231,
1033, 729 cm−1. HRESI-TOF m/z 943.4950 (C54H66N6O9 + H+,
23
(C54H66N6O9 + H+, required 943.4964); [α]D +36 (c 0.1, CHCl3).
(R = 20′-NHCONH(4-trifluoromethylphenyl)) 27. Yield: 60%,
method 1. 1H NMR (600 MHz, CDCl3) δ 9.82 (br s, 1H), 7.94 (br s,
1H), 7.58 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 7.9
Hz, 1H), 7.18−7.14 (m, 1H), 7.13−7.07 (m, 2H), 6.65 (s, 1H), 6.09
(s, 1H), 5.87 (dd, J = 10.0, 4.2 Hz, 1H), 5.46 (s, 1H), 5.31 (d, J = 10.3
23
required 943.4964); [α]D +29 (c 0.3, CHCl3).
H
dx.doi.org/10.1021/jm3015684 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
(R = 20′-NHCONHCH2CH2C6H5) 32. Yield: 98%, method 1. H
NMR (600 MHz, CDCl3) δ 9.83 (br s, 1H), 7.97 (br s, 1H), 7.40 (d, J
= 8.1 Hz, 1H), 7.31 (t, J = 7.8 Hz, 2H), 7.17−7.08 (m, 6H), 6.39 (s,
1H), 6.07 (s, 1H), 5.89 (dd, J = 10.3, 4.6 Hz, 1H), 5.39 (s, 1H), 5.32
(d, J = 9.4 Hz, 1H), 4.52 (s, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.74 (s,
1H), 3.65 (s, 3H), 3.64−3.63 (m, 2H), 3.49−3.43 (m, 1H), 3.37−3.32
(m, 1H), 3.31−3.19 (m, 1H), 3.10 (s, 3H), 2.99 (d, J = 15 Hz, 1H),
2.95−2.89 (m, 2H), 2.72 (s, 3H), 2.67 (s, 1H), 2.58−2.50 (m, 1H),
2.45−2.37 (m, 1H), 2.40−2.30 (m, 2H), 2.25−2.13 (m, 2H), 2.11 (s,
3H), 2.00−1.94 (m, 1H), 1.84−1.70 (m, 4H), 1.71−1.65 (m, 2H),
1.64−1.54 (m, 2H), 1.34−1.28 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H), 0.83
(t, J = 7.4 Hz, 3H). IR (film) νmax 3448, 2927, 1742, 1555, 1461, 1236,
1038, 749 cm−1. HRESI-TOF m/z 957.5106 (C55H68N6O9 + H+,
cm−1. HRESI-TOF m/z 951.5051 (C53H70N6O8S + H+, required
951.5048); [α]D +9.2 (c 0.2, CHCl3).
23
(R = 20′-NHCSNHC6H5) 37. Yield: 70%, method 3. 1H NMR (500
MHz, CDCl3) δ 9.82 (br s, 1H), 8.09 (br s, 1H), 7.54 (m, 4H), 7.49
(d, J = 8.0 Hz, 1H), 7.40 (t, J = 7.2 Hz, 1H), 7.24−7.19 (m, 1H),
7.19−7.14 (m, 2H), 6.60 (s, 1H), 6.25 (s, 1H), 6.18 (s, 1H), 5.92 (dd,
J = 10.0, 4.3 Hz, 1H), 5.56 (s, 1H), 5.37 (d, J = 10.0 Hz, 1H), 3.89 (s,
3H), 3.88 (s, 3H), 3.80 (s, 1H), 3.70 (s, 3H), 3.49−3.32 (m, 4H),
3.17−3.07 (m, 2H), 2.94−2.80 (m, 3H), 2.78 (s, 3H), 2.65−3.62 (s,
1H), 2.52−2.43 (m, 1H), 2.33−2.22 (m, 4H), 2.19 (s, 3H), 1.94−1.80
(m, 2H), 1.44−1.37 (m, 3H), 1.14 (d, J = 9.2 Hz, 1H), 0.88 (t, J = 6.3
Hz, 6H). IR (film) νmax 3449, 2930, 1737, 1498, 1458, 1231, 1039, 751
cm−1. HRESI-TOF m/z 945.4573 (C53H64N6O8S + H+, required
23
23
945.4579); [α]D +8.1 (c 0.2, CHCl3).
required 957.5121); [α]D −17 (c 0.06, CHCl3).
(R = 20′-NHCONHCH2(2-pyridyl)) 33. Yield: 79%, method 2. 1H
NMR (600 MHz, CDCl3) δ 9.84 (br s, 1H), 8.52 (s, 1H), 8.04 (br s,
1H), 7.66 (t, J = 8.1 Hz, 1H), 7.48 (d, J = 7.9 Hz, 2H), 7.39 (d, J = 7.7
Hz, 1H), 7.18−7.08 (m, 3H), 6.67 (s, 1H), 6.09 (s, 1H), 5.87−5.83
(m, 1H), 5.54 (br s, 1H), 5.47 (s, 1H), 5.30 (d, J = 10.4 Hz, 1H),
4.62−4.52 (m, 2H), 3.87−3.83 (m, 1H), 3.80 (s, 6H), 3.74 (s, 1H),
3.57 (s, 3H), 3.39−3.28 (m, 3H), 3.23−3.17 (m, 1H), 3.10−3.02 (m,
1H), 2.83 (d, J = 15.0 Hz, 1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.57 (d, J =
13.8 Hz, 1H), 2.48 (q, J = 9.5 Hz, 1H), 2.35 (d, J = 14.6 Hz, 1H), 2.28
(d, J = 9.8 Hz, 1H), 2.21−2.16 (m, 1H), 2.11 (s, 3H), 1.99 (s, 1H),
1.45−1.32 (m, 1H), 1.86−1.70 (m, 8H), 1.27−1.22 (m, 2H), 0.80 (t, J
= 6.8 Hz, 3H), 0.73 (t, J = 7.1 Hz, 3H). IR (film) νmax 3375, 2925,
1737, 1503, 1459, 1230, 1039 cm−1. HRESI-TOF m/z 944.4913
(R = 20′-NHCSNHCH2CH2(4-fluorophenyl)) 38. Yield: 91%,
method 4. 1H NMR (600 MHz, CDCl3) δ 9.83 (br s, 1H), 8.01 (br s,
1H), 7.51 (d, J = 8.8 Hz, 1H), 7.26−7.24 (m, 2H), 7.17−7.08 (m,
3H), 6.97−6.94 (m, 2H), 6.64 (s, 1H), 6.12 (s, 1H), 5.85 (dd, J = 10.1,
3.4 Hz, 1H), 5.75 (br s, 1H), 5.48 (s, 1H), 5.30 (d, J = 10.2 Hz, 1H),
3.80 (s, 3H), 3.79 (s, 3H), 3.77−3.74 (m, 3H), 3.61 (s, 3H), 3.60 (s,
1H), 3.42−3.35 (m, 2H), 3.32−3.28 (m, 1H), 3.25−3.10 (m, 3H),
3.02−2.97 (m, 1H), 2.96−2.91 (m, 1H), 2.83−2.80 (m, 1H), 2.72 (s,
3H), 2.66 (s, 1H), 2.55 (d, J = 13.7 Hz, 1H), 2.47−2.42 (m, 1H), 2.32
(d, J = 13.8 Hz, 1H), 2.22−2.17 (m, 2H), 2.11 (s, 3H), 1.84−1.65 (m,
4H), 1.54−1.44 (m, 2H), 1.37−1.22 (m, 5H), 0.82 (t, J = 7.2 Hz, 3H),
0.74 (t, J = 7.4 Hz, 3H). IR (film) νmax 3468, 2961, 1737, 1507, 1461,
1226, 1038 cm−1. HRESI-TOF m/z 991.4790 (C55H67N6O8S + H+,
23
required 991.4798); [α]D +6.6 (c 0.2, CHCl3).
23
(C53H65N7O9 + H+, required 944.4916); [α]D +2.7 (c 0.2, CHCl3).
(R = 20′-NHCON(CH3)2) 39. Yield: 99%, method 2. 1H NMR (600
MHz, CDCl3) δ 9.84 (br s, 1H), 7.98 (br s, 1H), 7.48 (d, J = 8.2 Hz,
1H), 7.17−7.13 (m, 1H), 7.12−7.07 (m, 2H), 6.62 (s, 1H), 6.09 (s,
1H), 5.86 (dd, J = 10.0, 5.1 Hz, 1H), 5.46 (s, 1H), 5.30 (d, J = 10.3 Hz,
1H), 4.68 (s, 1H), 3.90 (t, J = 14.7 Hz, 1H), 3.79 (s, 3H), 3.79 (s, 3H),
3.73 (s, 1H), 3.58 (s, 3H), 3.38−3.35 (m, 2H), 3.31−3.27 (m, 2H),
3.23−3.19 (m, 2H), 3.17−3.12 (m, 2H), 3.05 (s, 6H), 2.82 (d, J = 16.0
Hz, 1H), 2.70 (s, 3H), 2.67 (s, 1H), 2.58 (d, J = 13.9 Hz, 1H), 2.47−
2.42 (m, 1H), 2.38 (d, J = 13.1 Hz, 1H), 2.23−2.14 (m, 2H), 2.10 (s,
1H), 1.87−1.74 (m, 4H), 1.69 (d, J = 13.9 Hz, 2H), 1.40−1.33 (m,
4H), 0.81 (t, J = 7.4 Hz, 3H), 0.75 (t, J = 7.4 Hz, 3H). IR (film) νmax
3472, 2955, 1723, 1500, 1459, 1258, 1038, 777 cm−1. HRESI-TOF m/
1
(R = 20′-NHCONHCH2(2-furyl)) 34. Yield: 96%, method 1. H
NMR (600 MHz, CDCl3) δ 9.83 (br s, 1H), 7.98 (br s, 1H), 7.50 (d, J
= 8.2 Hz, 1H), 7.35 (s, 1H), 7.17−7.08 (m, 3H), 6.66 (s, 1H), 6.31 (d,
J = 9.0 Hz, 2H), 6.21 (br s, 1H) 6.09 (s, 1H), 5.86 (dd, J = 9.8, 4.7 Hz,
1H), 5.47 (s, 1H), 5.30 (d, J = 10.0 Hz, 1H), 4.40−4.37 (m, 2H), 3.80
(s, 6H), 3.74 (s, 1H), 3.59 (s, 3H), 3.40−3.28 (m, 2H), 3.26−3.20 (m,
1H), 3.22−3.18 (m, 1H), 3.10−3.00 (m, 3H), 2.83 (d, J = 17.3 Hz,
1H), 2.71 (s, 3H), 2.67 (s, 1H), 2.55 (d, J = 14.2 Hz, 1H), 2.47−2.42
(m, 1H), 2.35 (d, J = 12.8 Hz, 1H), 2.25−2.15 (m, 1H), 2.11 (s, 3H),
2.02 (s, 1H), 1.85−1.76 (m, 3H), 1.71−1.65 (m, 4H), 1.37−1.20 (m,
4H), 0.81 (t, J = 7.4 Hz, 3H), 0.72 (t, J = 7.5 Hz, 3H). IR (film) νmax
3388, 2925, 1739, 1504, 1459, 1230, 1039 cm−1. HRESI-TOF m/z
933.4736 (C52H64N6O10 + H+, required 933.4756); [α]D23 +11 (c 0.1,
CHCl3).
23
z 881.4790 (C49H64N6O9 + H+, required 881.4808); [α]D −50 (c
0.04, CHCl3).
1
(R = 20′-NHCON(CH2CH3)2) 40. Yield: 88%, method 2. H NMR
1
(R = 20′-NHCONH(4-biphenyl)) 35. Yield: 44%, method 1. H
(600 MHz, CDCl3) δ 9.87 (br s, 1H), 8.02 (br s, 1H), 7.51 (d, J = 7.8
Hz, 1H), 7.18−7.13 (m, 1H), 7.13−7.07 (m, 2H), 6.62 (s, 1H), 6.10
(s, 1H), 5.85 (dd, J = 10.2, 4.1 Hz, 1H), 5.47 (s, 1H), 5.30 (d, J = 10.2
Hz, 1H), 4.35 (br s, 1H), 3.85 (t, J = 12.7 Hz, 2H), 3.79 (s, 6H), 3.74
(s, 1H), 3.59 (s, 3H), 3.51−3.45 (m, 1H), 3.40−3.20 (m, 3H), 3.17−
3.12 (m, 1H), 2.83 (d, J = 16.2 Hz, 1H), 2.71 (s, 7H), 2.58 (d, J = 13.7
Hz, 1H), 2.47−2.42 (m, 1H), 2.39 (d, J = 12.7 Hz, 1H), 2.23 (d, J =
14.4 Hz, 1H), 2.20−2.14 (m, 1H), 2.11 (s, 3H), 2.08 (s, 1H), 1.88−
1.67 (m, 4H), 1.37−1.32 (m, 1H), 1.29−1.25 (m, 4H), 1.23 (t, J = 7.1
Hz, 6H), 0.81 (t, J = 7.3 Hz, 3H), 0.75 (t, J = 7.4 Hz, 3H). IR (film)
νmax 3486, 2940, 1741, 1520, 1458, 1233, 1042, 709 cm−1. HRESI-
NMR (600 MHz, CDCl3) δ 9.82 (br s, 1H), 7.98 (br s, 1H), 7.58−
7.51 (m, 6H), 7.45 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 7.6 Hz, 2H), 7.31
(t, J = 7.4 Hz, 1H), 7.17−7.13 (m, 1H), 7.13−7.06 (m, 2H), 6.64 (s,
1H), 6.09 (s, 1H), 5.86 (dd, J = 10.4, 4.8 Hz, 1H), 5.47 (s, 1H), 5.31
(d, J = 10.2 Hz, 1H), 4.82 (br s, 1H), 3.82 (t, J = 13.8 Hz, 1H), 3.80 (s,
3H), 3.79 (s, 3H), 3.75 (s, 1H), 3.61 (s, 3H), 3.39−3.36 (m, 2H),
3.32−3.28 (m, 1H), 3.19 (t, J = 11.4 Hz, 1H), 3.14−3.06 (m, 1H),
3.02 (d, J = 12.5 Hz, 2H), 2.82 (d, J = 15.7 Hz, 1H), 2.71 (s, 3H), 2.67
(s, 1H), 2.60 (d, J = 13.8 Hz, 2H), 2.47−2.42 (m, 1H), 2.35 (d, J =
13.0 Hz, 1H), 2.23 (d, J = 14.2 Hz, 1H), 2.21−2.14 (m, 1H), 2.11 (s,
3H), 1.82−1.77 (m, 4H), 1.73−1.65 (m, 4H), 1.37−1.34 (m, 1H),
0.82 (t, J = 7.4 Hz, 6H). IR (film) νmax 3444, 2967, 1735, 1523, 1459,
1240, 1039, 744 cm−1. HRESI-TOF m/z 1005.5122 (C59H68N6O9 +
23
TOF m/z 909.5130 (C51H68N6O9 + H+, required 909.5121); [α]D
−38 (c 0.08, CHCl3).
1
(R = 20′-NHCO-(morpholine)) 41. Yield: 99%, method 2. H
23
H+, required 1005.5121); [α]D −8.8 (c 0.1, CHCl3).
NMR (600 MHz, CDCl3) δ 9.84 (br s, 1H), 8.06 (s, 1H), 7.99 (br s,
1H), 7.51 (d, J = 7.8 Hz, 1H), 7.19−7.14 (m, 1H), 7.13−7.07 (m,
2H), 6.62 (s, 1H), 6.10 (s, 1H), 5.85 (dd, J = 10.1, 4.1 Hz, 1H), 5.47
(s, 1H), 5.30 (d, J = 10.1 Hz, 1H), 4.48 (s, 1H), 3.85 (d, J = 10.9 Hz,
3H), 3.80 (s, 3H), 3.80 (s, 3H), 3.79−3.77 (m, 1H), 3.77−3.64 (m,
11H), 3.62−3.56 (m, 4H), 3.48−3.44 (m, 1H), 3.38−3.36 (m, 1H),
3.33−3.05 (m, 2H), 2.83 (d, J = 16.1 Hz, 1H), 2.71 (s, 3H), 2.67 (s,
1H), 2.60 (d, J = 13.6 Hz, 1H), 2.47−2.42 (m, 1H), 2.40 (d, J = 12.5
Hz, 1H), 2.29−2.22 (m, 2H), 2.20−2.15 (m, 2H), 2.11 (s, 3H), 1.90−
1.74 (m, 2H), 1.70−1.67 (m, 2H), 1.37−1.32 (m, 2H), 0.81 (t, J = 7.3
Hz, 3H), 0.76 (t, J = 7.5 Hz, 3H). IR (film) νmax 3448, 2919, 1740,
1538, 1449, 1247, 1032, 711 cm−1. HRESI-TOF m/z 923.4897
(C51H66N6O10 + H+, required 923.4913); [α]D23 −8.0 (c 0.1, CHCl3).
1
(R = 20′-NHCSNHC6H11) 36. Yield: 26%, method 4. H NMR
(600 MHz, CDCl3) δ 9.85 (br s, 1H), 8.03 (br s, 1H), 7.51 (d, J = 7.7
Hz, 1H), 7.16−7.14 (m, J = 7.0 Hz, 1H), 7.12−7.06 (m, 2H), 6.62 (s,
1H), 6.10 (s, 1H), 5.84 (dd, J = 10.1, 4.1 Hz, 1H), 5.47 (s, 2H), 5.35−
5.33 (m, 1H), 5.29 (d, J = 9.1 Hz, 1H), 3.79 (s, 3H), 3.78 (s, 3H), 3.73
(s, 1H), 3.67−3.63 (m, 2H), 3.60 (s, 3H), 3.44 (s, 1H), 3.39−3.33 (m,
2H), 3.32−3.26 (m, 2H), 3.24−3.14 (s, 2H), 3.09 (q, J = 7.7 Hz, 1H),
2.81 (d, J = 14.7 Hz, 1H), 2.70 (s, 3H), 2.65 (s, 1H), 2.62−2.60 (m,
1H), 2.47−2.42 (m, 1H), 2.38−2.26 (m, 3H), 2.24−2.14 (m, 3H),
2.10 (s, 3H), 2.08−1.97 (m, 5H), 1.83−1.66 (m, 4H), 1.44−1.36 (m,
4H), 1.20−1.06 (m, 2H), 0.87 (t, J = 6.9 Hz, 3H), 0.81 (t, J = 6.9 Hz,
3H). IR (film) νmax 3467, 2912, 1727, 1506, 1456, 1230, 1087, 774
I
dx.doi.org/10.1021/jm3015684 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(R = 20′-NHCO-(piperidine)) 42. Yield: 34%, method 2. 1H NMR
(600 MHz, CDCl3) δ 9.85 (br s, 1H), 8.02 (br s, 1H), 7.50 (d, J = 7.4
Hz, 1H), 7.17−7.14 (m, 1H), 7.11−7.09 (m, 2H), 6.59 (s, 1H), 6.09
(s, 1H), 5.84 (dd, J = 10.1, 4.1 Hz, 1H), 5.45 (s, 1H), 5.29 (d, J = 10.4
Hz, 1H), 4.45 (s, 1H), 3.82 (t, J = 14.7 Hz, 1H), 3.80 (s, 3H), 3.79 (s,
3H), 3.73 (s, 1H), 3.59 (s, 3H), 3.52−3.48 (m, 2H), 3.43−3.40 (m,
2H), 3.38−3.34 (m, 2H), 3.31−3.26 (m, 4H), 3.18−3.12 (m, 2H),
2.82 (d, J = 16.6 Hz, 1H), 2.70 (s, 3H), 2.66 (s, 1H), 2.47−2.38 (m,
4H), 2.28−2.20 (m, 2H), 2.18−2.14 (m, 2H), 2.10 (s, 3H), 2.07 (s,
1H), 1.90−1.76 (m, 4H), 1.69 (s, 1H), 1.35−1.27 (m, 5H), 0.80 (t, J =
7.3 Hz, 3H), 0.75 (t, J = 7.3 Hz, 3H). IR (film) νmax 3458, 2958, 1791,
1509, 1466, 1251, 1061, 737 cm−1. HRESI-TOF m/z 921.5103
(C52H68N6O9 + H+, required 921.5121); [α]D23 −9.2 (c 0.02, CHCl3).
(R = 20′-NHCSN(CH3)2) 43. Yield: 71%, method 4. 1H NMR (600
MHz, CDCl3) δ 9.83 (br s, 1H), 7.98 (br s, 1H), 7.50 (d, J = 7.8 Hz,
1H), 7.18−7.13 (m, 1H), 7.12−7.08 (m, 2H), 6.64 (d, J = 3.9 Hz,
1H), 6.10 (s, 1H), 5.86 (dd, J = 10.0, 5.1 Hz, 1H), 5.54 (s, 1H), 5.47
(s, 1H), 5.31 (d, J = 10.0 Hz, 1H), 4.70−4.67 (m, 1H), 3.81 (s, 3H),
3.80 (s, 3H), 3.75 (s, 1H), 3.59 (s, 3H), 3.44 (s, 6H), 3.38 (d, J = 12.2
Hz, 2H), 3.31 (d, J = 4.7 Hz, 1H), 3.27−3.15 (m, 3H), 3.14−3.03 (m,
2H), 2.83 (d, J = 15.2 Hz, 1H), 2.72 (s, 3H), 2.70 (s, 1H), 2.67 (s,
1H), 2.54 (d, J = 13.7 Hz, 1H), 2.48−2.43 (m, 1H), 2.38 (d, J = 14.1
Hz, 1H), 2.23−2.16 (m, 2H), 2.11 (s, 3H), 1.96 (d, J = 15.2 Hz, 1H),
1.85−1.74 (m, 2H), 1.38−1.29 (m, 4H), 0.81 (t, J = 7.4 Hz, 3H), 0.75
(t, J = 7.4 Hz, 3H). IR (film) νmax 3458, 2963, 1728, 1536, 1474, 1235,
1061, 737 cm−1. HRESI-TOF m/z 897.4559 (C49H64N6O8S + H+,
3H), 2.67 (s, 3H), 2.46−2.41 (m, 1H), 2.38 (s, 6H), 2.31−2.29 (m,
1H), 2.18−2.14 (m, 1H), 2.11 (s, 3H), 1.87−1.73 (m, 6H), 1.49−1.42
(m, 2H), 1.36−1.31 (m, 2H), 0.83−0.78 (m, 6H). IR (film) νmax 3404,
2951, 1734, 1501, 1459, 1227, 1037, 731 cm−1. HRESI-TOF m/z
838.4752 (C48H63N5O8 + H+, required 838.4749); [α]D23 +16 (c 0.01,
CHCl3).
(R = 20′-NMeCONHCH2CH3) 47. Yield: 95%, method 1. 1H NMR
(600 MHz, CDCl3) δ 9.83 (br s, 1H), 7.95 (br s, 1H), 7.51 (d, J = 8.0
Hz, 1H), 7.16−7.09 (m, 3H), 6.66 (s, 1H), 6.08 (s, 1H), 5.86 (dd, J =
9.9, 3.9 Hz, 1H), 5.48 (s, 1H), 5.31 (d, J = 10.6 Hz, 1H), 4.54 (br s,
1H), 4.29 (br s, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.74 (s, 1H), 3.70−
3.65 (m, 1H), 3.57 (s, 3H), 3.43−3.37 (m, 2H), 3.33−3.18 (m, 5H),),
3.05−3.02 (m, 2H), 2.98 (br s, 3H), 2.82 (d, J = 16.1 Hz, 1H), 2.71 (s,
3H), 2.67 (s, 1H), 2.56 (d, J = 14.2 Hz, 1H), 2.47−2.43 (m, 1H), 2.36
(d, J = 11.6 Hz, 1H), 2.24−2.16 (m, 2H), 2.11 (s, 3H), 1.87−1.75 (m,
3H), 1.38−1.32 (m, 5H), 1.19 (t, J = 7.2 Hz, 3H), 0.83 (t, J = 7.4 Hz,
3H), 0.79 (t, J = 7.3 Hz, 3H). IR (film) νmax 3462, 3400, 2926, 1735,
1504, 1460, 1243, 1039 cm−1. HRESI-TOF m/z 895.4956
23
(C50H66N6O9 + H+, required 895.4964); [α]D +17 (c 0.1, CHCl3).
Compound 49. Iron(III) chloride hexahydrate (55 mg, 0.21
mmol) was added to a solution of (−)-vindoline (19 mg, 0.041 mmol)
and 10′-fluorocatharanthine9a (15 mg, 0.041 mmol) in CF3CH2OH
(0.1 mL), aqueous 0.1 N HCl (1.0 mL), and H2O (1.0 mL) at 23 °C
under Ar. The reaction mixture was stirred for 2 h at 23 °C.
Meanwhile, in a separate flask, a mixture of iron(III) oxalate
hexahydrate (198 mg, 0.41 mmol) in degassed H2O (40 mL) was
cooled to 0 °C and placed under Ar. CsN3 (215 mg, 1.23 mmol) was
added to the mixture at 0 °C, followed by the vindoline coupling
solution and NaBH4 (31 mg, 0.81 mmol) in H2O (1 mL). The
resulting mixture was stirred for 30 min before being quenched by
addition of 28−30% aqueous NH4OH (4 mL). The mixture was
extracted with 10% MeOH in CH2Cl2, and the organic layer was dried
over Na2SO4 and concentrated under reduced pressure. PTLC (SiO2,
EtOAc:MeOH:Et3N = 97:3:3) provided 10′-fluoro-20′-azidovinblas-
tine (50, 2.4 mg, 7%, white solid) and 10′-fluoro-20′-azidoleurosidine
(6.0 mg, 17%, white solid). A solution of compound 50 (2.4 mg,
0.0028 mmol) in THF/H2O (1/1 mL) was treated with CoCl2· 6H2O
(35 mg, 0.15 mmol) followed by NaBH4 (17 mg, 0.45 mmol). The
reaction mixture was stirred for 2 h before being quenched with the
addition of saturated NaHCO3 (1 mL). The mixture was extracted
with 10% MeOH in CH2Cl2 and washed with saturated aqueous NaCl
(1 mL). The organic layer was dried over Na2SO4 and concentrated
under reduced pressure. PTLC (SiO2, EtOAc:MeOH = 90:10)
provided 10′-fluoro-20′-aminovinblastine (51, 1.6 mg, 70%, white
solid). A solution 51 (1.6 mg, 0.0019 mmol) in THF (3 mL) was
treated with ethyl isocyanate (2 μL, 0.025 mmol). The reaction
mixture was stirred for 4 h at 25 °C and then was concentrated under
reduced pressure. PTLC (SiO2, CH2Cl2:MeOH = 92:8) provided 49
23
required 897.4579); [α]D −56 (c 0.07, CHCl3).
(R = 20′-OCON(CH3)2) 44. Yield: 88%. 1H NMR (600 MHz,
CDCl3) δ 9.86 (br s, 1H), 8.02 (br s, 1H), 7.50 (d, J = 7.8 Hz, 1H),
7.17−7.12 (m, 1H), 7.12−7.07 (m, 2H), 6.61 (s, 1H), 6.09 (s, 1H),
5.84 (dd, J = 10.2, 4.1 Hz, 1H), 5.46 (s, 1H), 5.29 (d, J = 10.0 Hz, 1H),
4.34 (s, 1H), 3.84 (t, J = 12.7 Hz, 1H), 3.79 (s, 6H), 3.74 (s, 1H), 3.58
(s, 3H), 3.47 (dd, J = 14.6, 7.2 Hz, 2H), 3.40−3.19 (m, 4H), 3.16−
3.12 (m, 2H), 2.82 (d, J = 16.2 Hz, 1H), 2.70 (s, 9H), 2.66 (s, 1H),
2.57 (d, J = 13.7 Hz, 1H), 2.46−2.41 (m, 1H), 2.38 (d, J = 12.7 Hz,
1H), 2.22 (d, J = 14.4 Hz, 1H), 2.20−2.13 (m, 1H), 2.10 (s, 3H), 2.07
(s, 1H), 1.86−1.76 (m, 2H), 1.72 (d, J = 14.5 Hz, 2H), 1.38−1.30 (m,
2H), 0.80 (t, J = 7.3 Hz, 3H), 0.75 (t, J = 7.4 Hz, 3H). IR (film) νmax
3472, 2969, 1731, 1539, 1459, 1224, 1048, 740 cm−1. HRESI-TOF m/
23
z 882.4642 (C49H63N5O10 + H+, required 882.4648); [α]D −64 (c
0.03, CHCl3).
(R = 20′-NHCH3) 45. A solution 20′-aminovinblastine (8.8 mg,
0.011 mmol) in THF (3 mL) was treated with a 37% formaldehyde in
water solution (4 μL, 0.05 mmol). The reaction mixture was stirred for
4 h at 25 °C and then was treated with sodium cyanoborohydride (12
mg, 0.20 mmol). The reaction mixture was stirred for 1 h at 25 °C and
then was quenched with distilled H2O (3 mL). The mixture was
extracted with 10% MeOH in CH2Cl2, and the combined organic
extracts were washed with saturated aqueous NaCl (3 mL). The
organic layer was dried over Na2SO4 and concentrated under reduced
pressure. PTLC (SiO2, EtOAc:MeOH:Et3N = 97:3:3) provided 45
(2.8 mg, 32%, off white solid) and 46 (2.9 mg, 33%, off-white solid).
1
(1.0 mg, 59%, white solid). 50: H NMR (600 MHz, CDCl3) δ 9.76
(br s, 1H), 8.00 (br s, 1H), 7.39 (dd, J = 8.8, 5.1 Hz, 1H), 6.86−6.82
(m, 1H), 6.76 (dd, J = 9.6, 2.4 Hz, 1H), 6.56 (s, 1H), 6.10 (s, 1H),
5.86 (dd, J = 10.3, 4.6 Hz, 1H), 5.48 (s, 1H), 5.29 (d, J = 10.2 Hz, 1H),
3.93 (t, J = 14.1 Hz, 1H), 3.82−3.76 (m, 1H), 3.80 (s, 6H), 3.72 (s,
1H), 3.63 (s, 3H), 3.40−3.33 (m, 2H), 3.30−3.25 (m, 2H), 3.13−3.07
(m, 1H), 2.95 (d, J = 14.4 Hz, 1H), 2.83−2.73 (m, 3H), 2.69 (s, 3H),
2.66 (s, 1H), 2.62 (s, 1H), 2.43−2.40 (m, 2H), 2.25 (d, J = 13.9 Hz,
1H), 2.18−2.14 (m, 1H), 2.10 (s, 3H), 2.07 (s, 1H), 1.85−1.77 (m,
2H), 1.63−1.52 (m, 2H), 1.47−1.41 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H),
0.78 (t, J = 7.4 Hz, 3H). IR (film) νmax 2925, 2109, 1734, 1614, 1460,
1228, 1038 cm−1. HRESI-TOF m/z 854.4218 (C46H516FN7O8 + H+,
1
For 45: H NMR (600 MHz, CDCl3) δ 9.87 (br s, 1H), 8.01 (br s,
1H), 7.51 (d, J = 7.5 Hz, 1H), 7.18−7.14 (m, 1H), 7.11−7.08 (m,
2H), 6.58 (br s, 1H), 6.09 (s, 1H), 5.85 (dd, J = 9.9, 4.0 Hz, 1H), 5.46
(s, 1H), 5.30 (d, J = 9.8 Hz, 1H), 4.07−3.91 (m, 2H), 3.80 (s, 3H),
3.79 (s, 3H), 3.73 (s, 1H), 3.61 (s, 3H), 3.36 (dd, J = 16.3, 4.6 Hz,
1H), 3.31−3.26 (m, 1H), 3.12−3.04 (m, 2H), 2.83 (d, J = 16.1 Hz,
1H), 2.71 (s, 3H), 2.67 (s, 3H), 2.46−2.41 (m, 1H), 2.37−2.31 (m,
1H), 2.31−2.20 (m, 1H), 2.17−2.13 (m, 1H), 2.11 (s, 3H), 1.87−1.73
(m, 2H), 1.18−1.08 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H), 0.76 (t, J = 7.4
Hz, 3H). IR (film) νmax 3487, 2953, 1729, 1505, 1461, 1239, 1037, 735
cm−1. HRESI-TOF m/z 824.4576 (C47H61N5O8 + H+, required
23
required 854.4247); [α]D −6.0 (c 0.08, CHCl3). 51: H NMR (600
MHz, CDCl3) δ 9.81 (br s, 1H), 8.00 (br s, 1H), 7.40−7.37 (m, 1H),
6.89−6.85 (m, 1H), 6.78 (d, J = 9.2 Hz, 1H), 6.51 (s, 1H), 6.10 (s,
1H), 5.89 (dd, J = 10.1, 4.0 Hz, 1H), 5.45 (s, 1H), 5.31 (d, J = 10.1 Hz,
1H), 3.92 (t, J = 14 Hz, 1H), 3.82−3.77 (m, 1H), 3.81 (s, 6H), 3.74 (s,
1H), 3.63 (s, 3H), 3.38 (dd, J = 16.0, 4.7 Hz, 2H), 3.32−3.27 (m, 1H),
3.10−3.05 (m, 2H), 2.84 (d, J = 15.7 Hz, 1H), 2.75−2.68 (m, 3H),
2.71 (s, 3H), 2.64 (s, 1H), 2.50−2.45 (m, 1H), 2.17 (d, J = 3.4 Hz,
1H), 2.11 (s, 3H), 2.08 (s, 1H), 2.05 (s, 1H), 1.87−1.75 (s, 4H),
1.42−1.15 (m, 6H), 0.88 (t, J = 7.4 Hz, 3H), 0.78 (t, J = 7.3 Hz, 3H).
23
824.4593); [α]D −114 (c 0.03, CHCl3).
1
(R = 20′-N(CH3)2) 46. H NMR (600 MHz, CDCl3) δ 9.89 (br s,
1H), 7.98 (br s, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.17−7.11 (m, 3H),
6.57 (br s, 1H), 6.10 (s, 1H), 5.85 (dd, J = 9.5, 4.3 Hz, 1H), 5.46 (s,
1H), 5.30 (d, J = 10.1 Hz, 1H), 3.97−3.92 (m, 1H), 3.80 (s, 3H), 3.79
(s, 3H), 3.73 (s, 1H), 3.61 (s, 3H), 3.38−3.27 (m, 4H), 3.20 (d, J =
13.8 Hz, 1H), 3.17−3.12 (m, 1H), 2.84 (d, J = 16.2 Hz, 1H), 2.71 (s,
J
dx.doi.org/10.1021/jm3015684 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
HRESI-TOF m/z 828.4311 (C46H58FN5O8 + H+, required 828.4311);
[α]D23 −23 (c 0.10, CHCl3). 49: 1H NMR (600 MHz, CDCl3) δ 9.77
(br s, 1H), 7.95 (br s, 1H), 7.39 (dd, J = 8.8, 5.2 Hz, 1H), 6.86 (t, J =
9.4 Hz, 1H), 6.77 (d, J = 9.3 Hz, 1H), 6.58 (s, 1H), 6.09 (s, 1H), 5.88
(dd, J = 9.8, 4.6 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J = 11.4 Hz, 1H), 4.41
(br s, 1H), 4.21 (br s, 1H), 3.84−3.77 (m, 2H), 3.80 (s, 6H), 3.75 (s,
1H), 3.61 (s, 3H), 3.40 (dd, J = 17.0, 5.2 Hz, 2H), 3.35−3.19 (m, 4H),
3.16−3.08 (m, 2H), 2.82 (d, J = 14.9 Hz, 1H), 2.71 (s, 3H), 2.65 (s,
1H), 2.57 (d, J = 14.1 Hz, 1H), 2.47−2.43 (m, 1H), 2.39 (d, J = 12.8
Hz, 1H), 2.22−2.17 (m, 2H), 2.11 (s, 3H), 1.87−1.75 (m, 4H), 1.69
(d, J = 14.6 Hz, 2H), 1.43−1.38 (m, 3H), 1.19 (t, J = 7.1 Hz, 3H), 0.79
(t, J = 7.4 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H). HRESI-TOF m/z
899.4713 (C49H63FN6O9 + H+, required 899.4713).
Cell Growth Inhibition Assay. Compounds were tested for their
cell growth inhibition of L1210 (ATCC no. CCL-219, mouse
lymphocytic leukemia, see Supporting Information) cells, HCT116
(ATCC no. CCL-247, human colorectal carcinoma) cells, and
HCT116/VM46 (a vinblastine-resistant strain of HCT116) cells in
culture. A population of cells (>1 × 106 cells/mL as determined with a
hemocytometer) was diluted with an appropriate amount of
Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing
10% fetal bovine serum (FBS, Gibco) to a final concentration of 30000
cells/mL. To each well of a 96-well plate (Corning Costar), 100 μL of
the cell media solution was added with a multichannel pipet. The
cultures were incubated at 37 °C in an atmosphere of 5% CO2 and
95% humidified air for 24 h. The test compounds were added to the
plate as follows: test substances were diluted in DMSO to a
concentration of 1 mM and 10-fold serial dilutions were performed
on a separate 96-well plate. Fresh culture medium (100 μL) was added
to each well of cells to constitute 200 μL of medium per well followed
by 2 μL of each test agent. Compounds were tested in duplicate (n =
2−8 times) at six concentrations between 0−1000 nM or 0−10000
nM. Following addition, cultures were incubated for an additional 72
h.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support of the National
Institutes of Health (CA042056, CA115526). K.K.D. is a
Skaggs Fellow and T.J.B. is a NIH postdoctoral fellow
(CA165303).
REFERENCES
■
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A phosphatase assay was used to establish the IC50 values as follows:
the media in each cell was removed and 100 μL of phosphatase
solution (100 mg phosphatase substrate in 30 mL of 0.1 M NaOAc,
pH 5.5, 0.1% Triton X-100 buffer) was added to each well. The plates
were incubated at 37 °C for either 5 min (L1210) or 15 min (HCT116
and HCT116/VM46). After the given incubation time, 50 μL of 0.1 N
NaOH was added to each well and the absorption at 405 nm was
determined using a 96 well plate reader. As the absorption is directly
proportional to the number of living cells, the IC50 values were
calculated and the reported values represent of the average of 4−16
determinations (SD 10%).
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of a modification of the Polonovski reaction to the synthesis of
vinblastine-type alkaloids. J. Am. Chem. Soc. 1976, 98, 7017−7024.
(b) Kuehne, M. E.; Matson, P. A.; Bornmann, W. G. Enantioselective
syntheses of vinblastine, leurosidine, vincovaline, and 20′-epi-vincova-
line. J. Org. Chem. 1991, 56, 513−528. (c) Bornmann, W. G.; Kuehne,
M. E. A common intermediate providing syntheses of Ψ-tabersonine,
coronaridine, iboxyphylline, ibophyllidine, vinamidine, and vinblastine.
J. Org. Chem. 1992, 57, 1752−1760. (d) Yokoshima, S.; Ueda, T.;
Kobayashi, S.; Sato, A.; Kuboyama, T.; Tokuyama, H.; Fukuyama, T.
Stereocontrolled total synthesis of (+)-vinblastine. J. Am. Chem. Soc.
2002, 124, 2137−2139. (e) Kuboyama, T.; Yokoshima, S.; Tokuyama,
H.; Fukuyama, T. Stereocontrolled total synthesis of (+)-vincristine.
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(6) (a) Ishikawa, H.; Colby, D. A.; Boger, D. L. Direct coupling of
catharanthine and vindoline to provide vinblastine: total synthesis of
(+)- and ent-(−)-vinblastine. J. Am. Chem. Soc. 2008, 130, 420−421.
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Rayl, T. J.; Hwang, I.; Boger, D. L. Total synthesis of vinblastine,
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Tubulin Binding Competition Assay. The competitive displace-
3
ment of H-vinblastine (obtained from Moravek Biochemicals, Inc.)
from purified porcine tubulin (tubulin and general tubulin buffer
obtained from Cytoskeleton, Inc.) was measured using a procedure
previously described.19 As described, a 100 μL sample of tubulin
solution diluted with 850 μL of buffer was incubated with 25 μL of 7.2
× 10−5 M 3H-vinblastine for 15 min at 37 °C, after which 25 μL of 7.2
× 10−5 M unlabeled alkaloid was added and incubation continued for
60 min. Tubulin-bound 3H-vinblastine was adsorbed onto DEAE filter
paper and counted directly. Millipore Steriflip filter units were used to
wash the filter paper with buffer under mild suction.
ASSOCIATED CONTENT
■
S
* Supporting Information
(7) (a) Ishikawa, H.; Elliott, G. I.; Velcicky, J.; Choi, Y.; Boger, D. L.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 858-784-7522. Fax: 858-784-7550. E-mail: boger@
scripps.edu.
K
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Journal of Medicinal Chemistry
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