Optimisation of tetrahydroisoquinoline-based chimeric microtubule disruptors

Article abstract of DOI:10.1002/cmdc.201402025

Tetrahydroisoquinoline (THIQ)-based chimeric microtubule disruptors were optimised through modification of the N-benzyl motif, in concert with changes at C3 and C7, resulting in the identification of compounds with improved in vitro antiproliferative activities (e.g. 15: GI₅₀ 20 nM in DU-145). The broad anticancer activity of these novel structures was confirmed in the NCI 60-cell line assay, with 12 e,f displaying MGM values in the 40 nM region. In addition, their profiles as inhibitors of tubulin polymerisation and colchicine binding to tubulin were confirmed. Compound 15, for example, inhibited tubulin polymerisation with an IC₅₀ of 1.8 μM, close to that of the clinical drug combretastatin A-4, and also proved effective at blocking colchicine binding. Additionally, compound 20 b was identified as the only phenol in the series to date showing both better in vitro antiproliferative properties than its corresponding sulfamate and excellent antitubulin data (IC₅₀=1.6 μM). Compound 12 f was selected for in vivo evaluation at the NCI in the hollow fibre assay and showed very good activity and wide tissue distribution, illustrating the value of this template for further development.

Full text of DOI:10.1002/cmdc.201402025

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DOI: 10.1002/cmdc.201402025
Optimisation of Tetrahydroisoquinoline-Based Chimeric
Microtubule Disruptors
Wolfgang Dohle,[a] Mathew P. Leese,[a] Fabrice L. Jourdan,[a] Christopher J. Chapman,[a]
Ernest Hamel,[b] Eric Ferrandis,[c] and Barry V. L. Potter*[a]
Tetrahydroisoquinoline (THIQ)-based “chimeric” microtubule
disruptors were optimised through modification of the
N-benzyl motif, in concert with changes at C3 and C7, resulting
in the identification of compounds with improved in vitro anti-
proliferative activities (e.g. 15: GI50 20 nm in DU-145). The
broad anticancer activity of these novel structures was con-
firmed in the NCI 60-cell line assay, with 12e,f displaying MGM
values in the 40 nm region. In addition, their profiles as inhibi-
tors of tubulin polymerisation and colchicine binding to tubu-
lin were confirmed. Compound 15, for example, inhibited tu-
bulin polymerisation with an IC50 of 1.8 mm, close to that of the
clinical drug combretastatin A-4, and also proved effective at
blocking colchicine binding. Additionally, compound 20b was
identified as the only phenol in the series to date showing
both better in vitro antiproliferative properties than its corre-
sponding sulfamate and excellent antitubulin data (IC50 =
1.6 mm). Compound 12 f was selected for in vivo evaluation at
the NCI in the hollow fibre assay and showed very good activi-
ty and wide tissue distribution, illustrating the value of this
template for further development.
Introduction
Chimeras in mythology are creatures composed from the ele-
ments of multiple animals or animals and man. Small or large
molecules that combine the essential features of two or more
separate entities are also termed chimeras. The chimeric fusion
protein bcr-abl,[1] derived from the naturally occurring onco-
genic gene fusion that drives the growth of chronic myeloge-
nous leukemia,[2] serves as an example of the latter. A long-
standing interest in the development of microtubule disruptors
as anticancer agents led us to explore whether, through com-
bining the key pharmacophore elements of two series of col-
chicine site binders, we might generate “chimeric” small mole-
cules with similar activity. In a preliminary report[3] we outlined
how, by introducing the A,B-ring elements of the pharmaco-
phore from a series of steroidal microtubule disruptors 1[4–9]
(e.g., 2-methoxyestradiol-3,17-O,O-bis-sulfamate, 2-MeOE2bis-
MATE, STX140, 1a) into a tetrahydroisoquinoline (THIQ) motif
and connecting this to a trimethoxyaryl motif commonly
found in a range of colchicine site binding natural products
(e.g., colchicine 2), we could generate novel “chimeric” micro-
tubule disruptors 3,4 (Figure 1).
Figure 1. Generation of small-molecule chimeric microtubule disruptors
(Y=H-bond acceptor).
These chimeras, and their sulfamates 4a,b in particular, are
notable for their excellent physicochemical properties, their in
vitro and in vivo activity against various cancer cell lines, in-
cluding drug-resistant types, and also their structural simplicity
when compared to existing clinical agents. One notable ad-
vantage is the synthetic ease with which these compounds
can be constructed. In parallel work, we also applied the same
THIQ core to generate steroidomimetic microtubule disruptors
that exhibit a distinct structure–activity relationship (SAR) to
the chimeras, yet share their favourable physicochemical prop-
erties.[10] In the present study, we explore optimisation of our
chimeric system through modification of the trimethoxyaryl
component, knowing that in some colchicine site binding mi-
crotubule disruptor series replacement of this motif with alter-
nate trisubstituted systems can have a dramatic effect on po-
[a] Dr. W. Dohle, Dr. M. P. Leese, Dr. F. L. Jourdan, Dr. C. J. Chapman,
Prof. B. V. L. Potter
Medicinal Chemistry, Department of Pharmacy and Pharmacology
University of Bath
Bath, BA2 7AY (UK)
E-mail: B.V.L.Potter@bath.ac.uk
[b] Dr. E. Hamel
Sceening Technologies Branch, National Cancer Institute
Frederick, MD 21702 (USA)
[c] Dr. E. Ferrandis
Institut de Recherche Henri Beaufour
91966 Les Ulis Cedex (France)
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tency.[11] We therefore synthesised candidate chimeric microtu-
bule disruptors in which one or more of the methoxy groups
are exchanged for an alternate functionality.
Results and Discussion
Scheme 2. Synthesis of 7-ethyl-THIQs. Reagents and conditions: a) 3-Bromo-
4,5-dimethoxybenzyl bromide, DIPEA, DMF, 808C, 20 h, 40%; b) H2NSO2Cl,
DMA, 258C, 20 h, 47%.
Chemistry
Having previously established synthetic approaches to protect-
ed 6-hydroxy-7-methoxy and 6-hydroxy-7-ethyl THIQs in pre-
liminary studies, the logic was already in hand.[3] The major
modification over foregoing work was introduction of an ap-
propriately substituted benzyl motif at N2, followed by se-
quential deprotection and sulfamoylation of the 6-hydroxy
group. This was achieved by transforming THIQs 5[3] and
6a,b[12,13] into the corresponding functionalised N-benzylated
compounds 8a–d[12] and 10a–f using various direct N-benzyla-
tion methods or N’-(3-dimethylaminopropyl)-N-ethylcarbodi-
imide (EDCI) coupling with the corresponding benzoic acids
and subsequent reduction of product with lithium aluminium
hydride (LiAlH4). The protected phenols were either
treated with hydrogen and palladium on carbon (Pd/
C; for 8a–d) or tetra-n-butylammonium fluoride
(TBAF; for 10a–f), furnishing phenols 11 a–j in good
yields. Subsequent treatment of 11 a–j with sulfamoyl
chloride in N,N-dimethylacetamide (DMA)[14] gave the
corresponding sulfamates 12a–j (Scheme 1).
We also modified the potential hydrogen bonding effects
around C6, while retaining the C7-methoxy and the N-2-
(3’,4’,5’-trimethoxybenzyl) groups unchanged, to establish if in
vitro activity could be further improved. Compound 17a was
synthesised from the commercially available 6,7-dimethoxy-
THIQ salt 16, 3,4,5-trimethoxybenzyl chloride and DIPEA in
DMF. Compounds 17b,c were accessed from phenol 3a by
treatment with acetic anhydride or methanesulfonyl chloride,
respectively (Scheme 3).
Compound 15 was elaborated in two synthetic
steps by direct N-benzylation of the unprotected
phenol 13[3] with 3-bromo-4,5-dimethoxybenzyl bro-
mide[15] and diisopropylethylamine (DIPEA) in N,N-di-
methylformamide (DMF) and subsequent treatment
Scheme 3. Synthesis of C6-modified THIQs. Reagents and conditions: a) 3,4,5-Trimethoxy-
benzyl chloride, Et3N, EtOH, 1308C, MW, 1 h, 62%; b) Ac2O, Et3N, CHCl3, 258C, 24 h, 79%;
c) CH3SO2Cl, pyridine, 258C, 73%.
of 14 with sulfamoyl chloride in DMA to give the cor-
responding sulfamate 15 in moderate overall yield
(Scheme 2).
Another objective was to
study the effect of deletion of
the group at C7 since this had
not previously been explored. A
range of functionalised benzyl
groups was then introduced at
N2, as described above, starting
from compounds 18a–c.[16,17]
Direct benzylation methods, or
by coupling with the corre-
sponding benzoic acids or ben-
zoyl chlorides and successive re-
duction of product with LiAlH4,
afforded compounds 20a–i, usu-
ally in moderate yield. An exten-
sion of the linker between the
THIQ core and the aryl motif
connected to it was also ach-
ieved by the same strategy and
Scheme 1. Synthesis of 7-methoxy-THIQs. Reagents and conditions: a) ArCH2Cl, Et3N, EtOH, 1308C, MW; b) ArCH2Br,
DIPEA, DMF, 808C; c) ArCO2H, EDCI, CH2Cl2/THF, 258C; d) LiAlH4, THF, reflux; e) H2, Pd/C, THF/MeOH, 258C; f) TBAF,
THF, 258C; g) H2NSO2Cl, DMA, 258C.
gave 20j in good overall yield
(Scheme 4).
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Table 1. Antiproliferative [mm] activity of THIQs against DU-145 human
prostate and MDA MB-231 human breast cancer cells in vitro.[a]
Compd R1
R2
R3
R4
R5
R6
DU-145
0.004
MDA MB-231
Taxol
1a
N/A
N/A
N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A
H
H
0.002
0.28
0.62
0.329
>100
11.9
4.17
0.491
>100
>100
>100
3.15
0.4
0.34
0.65
0.297
3 a
H
OMe OMe OMe
OMe OMe OMe
H
H
H
H
H
H
4a
SO2NH2
H
Scheme 4. Synthesis of C7-hydrogen-substituted THIQs. Reagents and condi-
tions: a) ArCH2Cl, Et3N, EtOH, 1308C, MW; b) ArCH2Br, DIPEA, DMF, 808C;
c) ArCO2H, EDCI, Et3N, CH2Cl2/THF, 258C; d) LiAlH4, THF, 258C; e) ArCOCl,
Et3N, CH2Cl2, 258C; f) H2, Pd/C, THF/MeOH, 258C, 1.5 h, 74%; g) H2NSO2Cl,
DMA, 258C, 24 h, 88%.
11 a
12a
11 b
12b
11 c
12c
11 d
12d
11 e
12e
11 f
12 f
11 g
12g
11 h
12h
11 i
OMe OMe OMe
H
H
>100
17
7.8
0.66
SO2NH2 OMe OMe OMe
H
OMe
SO2NH2 OMe
OMe
SO2NH2 OMe
H
H
H
H
OMe OMe
OMe OMe
OMe
OMe
H
H
H
H
H
OMe >100
OMe >100
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe OMe
OMe OMe
H
H
H
H
H
H
H
H
H
H
H
H
>100
8.54
Biology
SO2NH2
H
SO2NH2
H
SO2NH2
H
SO2NH2
H
OMe OMe Cl
OMe OMe Cl
OMe OMe Br
OMe OMe Br
OMe OEt OMe
OMe OEt OMe
OEt OEt OEt
OEt OEt OEt
0.9
0.04
0.3
0.03
0.3
0.1
Compounds were evaluated for their ability to inhibit DU-145
(prostate cancer) and MDA MB-231 (breast cancer) cell prolifer-
ation and compared to the first generation chimeras 3a and
4a and to 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeO-
E2bisMATE; 1a) and paclitaxel (Taxol) as benchmark drugs
(Table 1). The results obtained across the two cell lines are in
close agreement and therefore only the DU-145 data are used
for SAR discussion herein. As reported previously, in both the
trimethoxybenzyl phenol 3a and 11a-c and sulfamate series
4a and 12a–c, the 3’,4’,5’-trimethoxy system proves optimal
(4a GI50 =297 nm), although the 2’,4’,5’-trimethoxy sulfamate
12b also displays good activity (GI50 =660 nm).[12] Deletion of
the 5’-methoxy group of 4a to give the 3’,4’-dimethoxy com-
pound 12d results in a near 30-fold reduction in activity, thus
illustrating the highly preferred status of trisubstitution in this
series of chimeras.[12] Replacement of the 5’-methoxy with
either chlorine in 12e, or bromine in 12 f, however, delivers
a dramatic 8- to 10-fold increase in antiproliferative activity,
with the corresponding phenols 11 e,f also exhibiting signifi-
cant activity. The 3’,4’-dimethoxy-5’-bromo derivatives 11 f and
12 f are exceptionally active. Substitution of the 4’-methoxy of
4a with an ethoxy group also delivers improved activity for
phenol 11 g and sulfamate 12g, revealing a degree of steric
flexibility at this position in the chimeric series in strong con-
trast to the steroidomimetic series.[12,13] Replacement of the
3’,4’,5’-trimethoxybenzyl group with a 3’,4’,5’-triethoxybenzyl
group results in a >20-fold reduction in activity. Similarly, the
3’,4’,5’-triethyl compounds 11 i and 12i are significantly less
active than 3a and 4a, respectively.
0.04
0.2
0.03
0.3
0.07
>100
4.4
>100
8.5
63
1.8
SO2NH2
H
Et
Et
Et
Et
Et
Et
26
5.7
12i
SO2NH2
[a] Results are GI50 values in mm and are the mean of three determina-
tions. Data for 1a, 3a, 4a, 11 a–d and 12a–d for comparison are taken
from the literature [3,8,12]. N/A: not applicable.
Table 2. Antiproliferative activity [mm] of variously modified 3’,4’-dime-
thoxybenzyl-substituted THIQs against DU-145 human prostate and MDA
MB-231 human breast cancer cells in vitro.[a]
Compd
R1
X
R2
R5
DU-145
0.4
MDA MB-231
11 j[b]
12j[b]
14
H
Me
Me
H
OMe
OMe
Et
Br
Br
Br
0.3
0.04
0.4
SO2NH2
H
0.05
1.2
15
SO2NH2
Me
Ac
H
H
H
H
Et
Br
0.02
>100
0.03
77.6
0.34
0.188
17a
17b
17c
OMe
OMe
OMe
OMe
OMe
OMe
0.407
0.22
SO2Me
[a] Results are GI50 values in mm and are the mean of three determina-
tions. [b] Compounds 11 j and 12j are racemic mixtures.
Having established that the 3’,4’-dimethoxy-5’-bromobenzyl
derivative is the most active of the various trisubstituted
benzyl derivatives, we combined this motif with THIQ core
modifications at C3 and C7 that had delivered enhanced activi-
ty in preceding studies. Introduction of a methyl group at C3
had delivered a modest enhancement in antiproliferative activ-
ity for the 3’,4’,5’-trimethoxybenzyl THIQs,[13] while replacement
of the C7-methoxy group with an ethyl group had afforded
a near 10-fold improvement in activity.[3] As can be seen in
Table 2, when such modifications were made to the THIQ core
bearing the 3’,4’-dimethoxy-5’-bromobenzyl group at N2, only
relatively modest activity changes resulted with a slight im-
provement and a slight reduction in activity resulting from C7
and C3 modification, respectively. Note that the potential
effect of individual enantiomers on the biological activity of
11 j and 12j has not been pursued. Nonetheless, the C7 ethyl
derivative 15 (GI50 =20 nm) is in vitro the most active com-
pound discovered in this series of chimeras to date. We then
focused our attention on the C6 position. Changing the nature
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of the motif at C6 has a clear effect on in vitro antiproliferative
activity (Table 2). Compounds bearing unhindered hydrogen-
bond acceptors, such as in 3a (see Table 1), prove much more
active than hindered ones; for example, 17a is 120-fold less
active (GI50 =77.6 mm in MDA MB-231) than 3a. However, pro-
jection of a group bearing an unhindered hydrogen-bond ac-
ceptor at C6 proves positive with, for example, the sulfamoyl
group of 4a and the carbonyl and sulfonyl groups of 17b,c de-
livering a ca. two- to three-fold better antiproliferative activity
than that shown by phenol 3a.
Next, we examined the effect of deletion of the C7 substitu-
ent that in the steroidomimetic series had proven essential for
activity. In conjunction, we varied the C6 substituent and the
linker at N2 and, in so doing, discovered an SAR strongly con-
trasting to that previously elucidated (Table 3). Here, the com-
Table 4. Antiproliferative activity [mm] of selected compounds against
various cancer cell lines from the NCI-60 cell line panel.[a]
Compd Lung
Colon
CNS
Melanoma Ovarian
Renal MGM
HOP-62 HCT-116 SF-
539
UACC-62 OVCAR-3 SN12-C
1a
0.051
0.869
0.056
0.353
0.08
0.045
0.474
0.039
0.143
0.038
1.05
0.036 <0.01
<0.01
0.187 0.91
0.02 0.066
0.074 0.768
0.018 0.055
0.209 5.29
0.019 0.049
0.126
0.087
0.501
0.039
0.38
0.044
1.17
11 e
12e
11 f
12 f
12i
20b
0.365
0.019
0.177
0.022
0.916
0.036
0.35
0.025
0.305
0.028
0.592
0.049
2.45
0.062
0.035
0.045
[a] Results are GI50 values in mm and are the mean of three determina-
tions. The MGM represents the mean concentration that caused 50%
growth inhibition in all 60 cell lines. Data for 1a are taken from the litera-
ture [9].
Table 3. Antiproliferative activity [mm] of C7 hydrogen-substituted THIQs
against DU-145 human prostate and MDA MB-231 human breast cancer
cells in vitro.[a]
allows activity across a wide range of cancer types to be as-
sessed. Data from six cell lines are presented along with the
mean activity across the whole panel (MGM value). The data
obtained in the assay are consistent with those obtained in
the antiproliferative screens discussed above and confirm the
potential of these compounds against a broad range of cancer
phenotypes with, in particular, 12e,f and 20b proving highly
active.
Compd R1
X
R3
R4
R5
DU-145
MDA MB-231
20a
20b
20c
20d
20e
20 f
20g
20h
20i
Bn
H
Me
CH2
CH2
CH2
OMe OMe OMe >100
>100
0.227
4.89
>100
74
OMe OMe OMe
OMe OMe OMe
0.151
31
We also wished to establish the microtubule disruptor activi-
ty in particular of 12e,f, 12j, 15 and 20b alongside the estab-
lished potent disruptor combretastatin A-4 (CA-4) and the
3’,4’,5’-trimethoxybenzyl THIQ derivatives 3a and 4a,b
(Table 5). The 3’,4’-dimethoxy-5’-halobenzyl THIQs are superior
to the first generation chimeras 3a and 4a as inhibitors of tu-
bulin assembly and approach the activity of CA-4, with 15 dis-
rupting the polymerisation of tubulin with an IC50 value of
1.8ꢀ0.04 mm. In tubulin-based assays, the concentration
needed far exceeds the antiproliferative dose. It presumably
suffices to disrupt microtubule dynamics to arrest the cell
cycle. Additionally, of course, the nominal compound concen-
tration recorded is that of agent added to the culture medium,
and is not the concentration within cells. We also determined
SO2NH2 CH2
OMe OMe OMe >100
H
H
H
H
H
H
CH2
CH2
CH2
CH2
CO
OMe OMe Cl
OMe OMe Br
86
35
25
OMe OEt OMe >100
OEt OEt OEt >100
OMe OMe OMe >100
>100
>100
>100
>100
20j
CH2CH2 OMe OMe OMe >100
[a] Results are GI50 values in mm and are the mean of three determina-
tions.
pound with a hydroxy group at C6, phenol 20b, is by far the
most active (GI50 =151 nm in DU-145 and GI50 =227 nm in
MDA MB-231). Larger C6 substituents are universally less
active, for example 20c (about 20- to 200-fold) or inactive
(e.g., 20a and sulfamate 20d) in the concentration range
tested. Modifying the 3’,4’,5’-trimethoxybenzyl motif at N2 also
proves to have a dramatic effect on antiproliferative activity.
The only compounds to show modest activity are the 3’,4’-di-
methoxy-5’-halobenzyl THIQs 20e,f, albeit they are between
100- to 600-fold less active than 20b. Increasing the size of
one or more groups in this motif similarly delivers inactive
compounds such as 20g,h. Finally, exchanging the methylene
linker between the 3’,4’,5’-trimethoxy aryl motif and N2 for
a carbonyl group or extending it to ethylene proves fruitless
(see compounds 20i,j). Although not shown here, it should be
mentioned that compounds wherein the 3’,4’,5’-trimethoxy-
benzyl motif at N2 in 20b and 20d is replaced by alternate
mono-, di- and other trimethoxybenzyl motifs, and the sulfa-
moylated derivatives of 20e–j show at best only modest activi-
ty (high micromolar GI50s).
Table 5. Activity of selected THIQs as inhibitors of tubulin polymerisation
and [3H]colchicine binding to tubulin.
Compd
Tubulin assembly
IC50 [mm][a]
Colchicine binding
Inhibition at 5 mm inhibitor [%][a]
CA-4
3a
4a
1.2ꢀ0.1
>20 (no activity)[b]
>20 (partial activity)[b]
2.5ꢀ0.3
98ꢀ0.7
4.1ꢀ2
10ꢀ0.9
49ꢀ0.5
32ꢀ3
4b
12e
12 f
12j[c]
15
5.6ꢀ0.7
2.4ꢀ0.4
45ꢀ0.6
50ꢀ4
2.4ꢀ0.2
1.8ꢀ0.04
78ꢀ2
20b
1.6ꢀ0.1
73ꢀ3
[a] Data are the mean ꢀSD of at least two determinations. [b] Compound
4a inhibits tubulin assembly at 20 mm while 3a is inactive at this concen-
tration. [c] Compound 12j is a racemic mixture. Data for CA-4, 3a and
4a,b are taken from the literature [12].
A selection of compounds was also tested at the US National
Cancer Institute (NCI) in the full 60-cell-line assay (Table 4) that
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that sulfamates 12e,f, 12j and 15 inhibit colchicine binding to
tubulin, with 15 being the best, showing 78% inhibition at
5 mm, approaching again the activity of CA-4 (98%). Interest-
ingly, deletion of the methoxy group at C7 in 3a leads to
phenol 20b that shows slightly improved antiproliferative ac-
tivity but also, somewhat surprisingly in comparison, excellent
antitubulin data compared to both 3a and its sulfamate 4a.
Moreover, 20b is markedly better as an antiproliferative agent
than its respective sulfamate derivative 20d. The origin of
these very considerable improvements remains to be deter-
mined, but might be due to steric effects leading to better ac-
cessibility of the unsubstituted phenolic hydroxy group and/or
an alternate and stronger binding conformation at the tubulin
binding site.
noted to adversely affect their biological properties and
indeed, until recently, it was thought that the trimethoxyaryl
group is critical for efficient binding. A range of combretasta-
tins was synthesised with the trimethoxy motif substituted by
other functionalities. This study demonstrated interestingly
that modifying this motif could significantly reduce cytotoxicity
and enhance antitubulin properties.[19] Our initial series of chi-
meric ligands[12] demonstrated that excellent antiproliferative
in vitro and in vivo activities of this new class of compounds
could be obtained with a chimera possessing a trimethoxyaryl
motif, although the properties of such compounds with re-
spect to inhibition of tubulin polymerisation and colchicine
binding are somewhat less impressive. While it is as yet unclear
if members of our prototype chimeric microtubule disruptors
possessing the trimethoxyaryl motif (e.g., 3–4) bind with this
motif in a position comparable to that of colchicine, the pres-
ent studies do generally support the previous observations
that the trimethoxy motif is not critical[19] and demonstrate
that molecules of a more optimised series possessing function-
alities other than trimethoxy can interact potently with tubulin.
Thus, in direct comparison, for example, the ca. 10-fold im-
provement in DU-145 antiproliferative activity of bromodime-
thoxyaryl-substituted 12 f versus the parent trimethoxyaryl 4a,
coupled with improvement in inhibition of tubulin assembly
and colchicine binding, provide further evidence that excessive
reverence for the trimethoxyaryl motif in optimisation of anti-
mitotic properties should perhaps not remain. With antiproli-
ferative activity maintained and enhanced in our more opti-
mised series, together with the much enhanced antitubulin ac-
tivity, members of the new series reported herein should yield
even more impressive in vivo data in xenograft models of
cancer.
It thus appears reasonable to suggest that the interaction of
the novel THIQ derivatives can at least partially be ascribed to
their ability to disrupt the normal dynamic polymerisation of
tubulin by interaction at, or around, the colchicine binding
site. On the strength of their in vitro antiproliferative activity,
11 f and 12 f were selected for in vivo evaluation at the NCI in
the hollow fibre assay that involves assessment of activity
against the proliferation of various cancer lines in sealed poly-
vinylidine fluoride fibres implanted i.p. or s.c. in mice.[18] A 50%
net cell growth inhibition is awarded a score of 2 and over 48
fibres (12 cell lines ꢁ2 sites ꢁ2 dose levels) a maximum score
of 96 is possible. The results obtained for these compounds
showed the strong difference in activity between the phenol
11 f and its corresponding sulfamate 12 f. Dosing of 11 f i.p. at
150 mgkgꢁ1 resulted in 50% inhibition of cell growth in only
one fibre and thus a score of only 2. In contrast, sulfamate 12 f
when dosed by the same route at 75 mgkgꢁ1 delivered a score
of 34 (16 for i.p. fibres and 18 for s.c. fibres). This demonstrates
both good activity and tissue distribution for the sulfamoylat-
ed THIQs and augers well for further development of this class
of compounds as in vivo agents, with the anticipated better in
vivo performance of the sulfamoyl ester versus the phenol. Al-
though the activity surpasses normal criteria (a score >20) for
further investigations at the NCI, compound 12 f was not se-
lected for additional study. Despite the excellent in vitro and
antitubulin data, and with in vivo data for the related phenol
11 f in hand, phenol 20b, despite its clearly attractive antitubu-
lin activities, is not very likely to show the same excellent in
vivo and bioavailability properties as the sulfamoylated com-
pound 12 f. Therefore, no further studies were carried out so
far with this compound class.
Conclusions
A second generation class of tetrahydroisoquinoline (THIQ)-
based chimeric microtubule disruptors with improved and ex-
cellent activity in vitro and in vivo, combined with a desirable
drug-like profile, was identified. The best compounds possess
antiproliferative activities in the 20–40 nm range, inhibit tubu-
lin assembly, interfere with the colchicine binding site and pos-
sess a pendant N-aryl group without a trimethoxy motif. The
sole sulfamoylated compound to be evaluated in vivo shows
highly promising preliminary activity, validating the chimeric
design and optimisation strategy for this new class of anticanc-
er agents.
The antimitotic natural product agents colchicine, combre-
tastatin A-4, podophyllotoxin and steganacin interact with the
colchicine site on tubulin and all possess a trimethoxyaryl unit.
This unit in colchicine is thought to be derived via the shiki-
mate biosynthetic pathway and that for combretastatin A-4,
currently in clinical trials, is probably derived similarly. This A-
ring unit in colchicine has long been thought to make an addi-
tive contribution to the strength of binding to tubulin, possibly
through hydrogen bonding, and to serve also as an anchor to
maintain the whole molecule in the proper orientation within
the binding site. Alteration of the oxygenation pattern from
a trimethoxy motif on the A-ring of the combretastatins was
Experimental Section
Biology: In vitro studies
Cell Lines: DU-145 (brain metastasis carcinoma of the prostate) and
MDA-MB-231 (metastatic pleural effusion of breast adenocarcino-
ma) established human cell lines were obtained from ATCC Global
Bioresource Center. Cells were maintained in a 5% CO2 humidified
atmosphere at 378C in RPMI-1640 medium, supplemented with
10% fetal bovine serum, penicillin (100 UmLꢁ1), and streptomycin
(0.1 mgmLꢁ1).
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Antiproliferative assays: DU-145 and MDA-MB-231 cells were
seeded into 96-well microtiter plates (5000 cells/well) and treated
with 10ꢁ9–10ꢁ4 m of compounds or with vehicle control. At 96 h
post-treatment, live cell counts were determined by the WST-1 cell
proliferation assay (Roche, Penzberg, Germany), as per the manu-
facturer’s instructions. Viability results were expressed as a percent-
age of mean control values resulting in the calculation of the 50%
growth inhibition (GI50). All experiments were performed in tripli-
cate.
the nearest 0.1 Hz. Mass spectra were recorded at the Mass Spec-
trometry Service Centre, University of Bath, UK. FAB-MS was carried
out using m-nitrobenzyl alcohol (NBA) as the matrix. Melting
points were determined using a Stuart SMP3 or a Stanford research
systems Optimelt MPA100 melting point apparatus (Stanford Re-
search Systems, Sunnyvale, CA, USA) and are uncorrected. All com-
pounds were ꢂ98% pure by reverse phase HPLC run with CH3CN/
H2O or MeOH/H2O (Sunfire C18 reverse phase column, 4.6ꢁ
150 mm, 3.5 mm pore size).
Tubulin assays: Bovine brain tubulin, prepared as described previ-
ously,[20] was used in the studies presented here. Assembly IC50
values were determined as described in detail elsewhere.[21] Briefly,
1.0 mgmLꢁ1 (10 mm) tubulin was preincubated without GTP with
varying compound concentrations for 15 min at 308C. The reaction
mixtures were placed on ice, and GTP (final concentration, 0.4 mm)
was added. The reaction mixtures were transferred to cuvettes,
held at 08C in a recording spectrophotometer. Baselines were es-
tablished at 08C, and increase in turbidity was followed for 20 min
following a rapid (<30 s) jump to 308C. Compound concentrations
required to reduce the turbidity increase by 50% were determined.
The method for measuring inhibition of the binding of
[3H]colchicine to tubulin was described in detail previously.[22] Reac-
tion mixtures contained 0.1 mgmLꢁ1 (1.0 mm) tubulin, 5.0 mm
[3H]colchicine, and potential inhibitor at 5.0 mm. Compounds were
compared to CA-4, a particularly potent inhibitor of the binding of
colchicine to tubulin.[23] Reaction mixtures were incubated 10 min
at 378C, a time point at which the binding of colchicine in control
reaction mixtures is generally 40–60% complete. A minimum of
two experiments was performed for each compound.
2-(3-Chloro-4,5-dimethoxybenzoyl)-7-methoxy-6-(triisopropylsi-
lyloxy)-1,2,3,4-tetrahydroisoquinoline (9a): Compound 6a
(504 mg, 1.5 mmol) and 3-chloro-4,5-dimethoxybenzoic acid
(487 mg, 2.25 mmol) were placed in an oven-dried tube and dis-
solved in CH2Cl2 (3.0 mL) and THF (3.0 mL). N’-(3-Dimethylamino-
propyl)-N-ethylcarbodiimide (EDCI; 573 mg, 3.0 mmol) was added,
and the reaction mixture was stirred at RT for 18 h. HCl (2m,
30 mL) was then added, and the mixture was extracted with CH2Cl2
(2ꢁ50 mL). The combined organics were dried (MgSO4), filtered
and concentrated in vacuo. Flash column chromatography
(hexane!hexane/EtOAc 4:1!EtOAc) afforded compound 9a as
1
a colourless oil (535 mg, 66%): H NMR (270 MHz, CDCl3): d=1.04
(18H, d, J=6.9 Hz), 1.11–1.30 (3H, m), 2.73 (2H, s, br), 3.59 and
3.69 (2H, m), 3.73 (3H, s, br), 3.84 (3H, s, br), 3.85 (3H, s), 4.49 and
4.73 (2H, s, br), 6.37 and 6.58 (1H, s), 6.61 (1H, s), 6.92 (1H, s),
7.03 ppm (1H, d, J=1.9 Hz); HRMS (ES+): m/z [M+H]+calcd for
C28H41ClNO5Si+: 534.2437, found: 534.2431.
2-(3,5-Dimethoxy-4-ethoxybenzoyl)-7-methoxy-6-(triisopropylsi-
lyloxy)-1,2,3,4-tetrahydroisoquinoline (9b): Method as for 9a
using compound 6a (503 mg, 1.5 mmol), 3,5-dimethoxy-4-ethoxy-
benzoic acid[26] (508 mg, 2.25 mmol) and EDCI (574 mg, 3.0 mmol)
in CH2Cl2 (3.0 mL) and THF (3.0 mL) at RT for 18 h. Flash column
chromatography (hexane!hexane/EtOAc 7:3!EtOAc) afforded
Chemistry
All chemicals were either purchased from Aldrich Chemical Co. (Gil-
lingham, UK) or Alfa Aesar (Heysham, UK). Organic solvents of A.R.
grade were supplied by Fisher Scientific (Loughborough, UK) and
used as supplied. The petroleum ether (PE) used for column chro-
matography was of fractions 40–608C. CH2Cl2, CHCl3, N,N-dimethyl-
formamide (DMF), N,N-dimethylacetamide (DMA) and tetrahydro-
furan (THF) were purchased from Aldrich and stored under a posi-
tive pressure of N2 after use. Sulfamoyl chloride was prepared by
an adaptation of the method of Appel and Berger[24] and was
stored in the refrigerator under a positive pressure of N2 as a solu-
tion in toluene as described by Woo et al.[25] An appropriate
volume of this solution was freshly concentrated in vacuo immedi-
ately before use. Compounds 5, 6a,b, 8a–d, 11 a–d, 12a–d, 13
and 18a–c were prepared according to literature proce-
dures.[3,12,13,16,17] Reactions were carried out at room temperature
(RT) unless stated otherwise. Thin-layer chromatography (TLC) was
performed on precoated aluminium plates (Merck, silica gel 60
F254). Product spots were visualised either by UV irradiation at
254 nm or by staining with either alkaline KMnO4 solution or 5%
dodecamolybdophosphoric acid in EtOH, followed by heating.
Flash column chromatography was performed using gradient elu-
tion (solvents indicated in text) on either prepacked columns (Iso-
lute) on a Flashmaster II system (Biotage, Uppsala, Sweden) or on
a CombiFlash Rf Automated Flash Chromatography System (Tele-
dyne Isco, Lincoln, NE, USA) with RediSep Rf disposable flash col-
umns. 1H NMR and 13C NMR spectra were recorded with either
a Delta JMN-GX 270 (Jeol, Peabody, MA, USA) at 270 and 67.5 MHz,
respectively, or a Mercury VX 400 NMR spectrometer (Varian, Paolo
Alto, CA, USA) at 400 and 100 MHz, respectively. Chemical shifts
are reported in parts per million (ppm) relative to tetramethylsilane
(TMS) as internal standard. Coupling constants J are recorded to
1
compound 9b as a colourless oil (449 mg, 55%): H NMR (270 MHz,
CDCl3): d=1.04 (18H, d, J=6.9 Hz), 1.11–1.28 (3H, m), 1.32 (3H, t,
J=7.0 Hz), 2.73 and 2.80 (2H, s, br), 3.60 and 3.69 (2H, m), 3.73
(3H, s, br), 3.80 (6H, s), 4.03 (2H, q, J=7.1 Hz), 4.48 and 4.74 (2H, s,
br), 6.35 and 6.60 (1H, s), 6.60 (1H, s), 6.64 ppm (2H, s); HRMS
(ES+): m/z [M+H]+ calcd for C30H46NO6Si+: 544.3089, found:
544.3093.
7-Methoxy-2-(3,4,5-triethoxybenzoyl)-6-(triisopropylsilyloxy)-
1,2,3,4-tetrahydroisoquinoline (9c): Method as for 9a using com-
pound 6a (503 mg, 1.5 mmol), 3,4,5-triethoxybenzoic acid (571 mg,
2.25 mmol) and EDCI (573 mg, 3.0 mmol) in CH2Cl2 (3.0 mL) and
THF (3.0 mL) at RT for 18 h. Flash column chromatography
(hexane!hexane/EtOAc 7:3!EtOAc) afforded compound 9c as
1
a colourless oil (550 mg, 64%): H NMR (270 MHz, CDCl3): d=1.06
(18H, d, J=6.9 Hz), 1.14–1.29 (3H, m), 1.33 (3H, t, J=7.2 Hz), 1.39
(6H, t, J=6.9 Hz), 2.72 and 2.80 (2H, s, br), 3.60 and 3.69 (2H, m),
3.75 (3H, s, br), 4.03 (2H, q, J=7.2 Hz), 4.06 (4H, q, J=7.4 Hz), 4.48
and 4.74 (2H, s, br), 6.35 and 6.62 (1H, s), 6.62 ppm (3H, s); HRMS
(ES+): m/z [M+H]+ calcd for C32H50NO6Si+: 572.3402, found:
572.3405.
7-Methoxy-2-(3,4,5-triethylbenzoyl)-6-(triisopropylsilyloxy)-
1,2,3,4-tetrahydroisoquinoline (9d): Method as for 9a using com-
pound 6a (503 mg, 1.5 mmol) and 3,4,5-triethylbenzoic acid[27,28]
(483 mg, 2.25 mmol) and EDCI (572 mg, 3.0 mmol) in CH2Cl2
(3.0 mL) and THF (3.0 mL) at RT for 18 h. Flash column chromatog-
raphy using (hexane!hexane/EtOAc 9:1!EtOAc) afforded com-
pound 9d as a colourless oil (342 mg, 43%): 1H NMR (270 MHz,
CDCl3): d=1.07 (18H, d, J=6.9 Hz), 1.10–1.31 (3H, m), 1.12 (3H, t,
J=7.7 Hz), 1.21 (6H, t, J=7.7 Hz), 2.58–2.76 (2H, m), 2.66 (4H, q,
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J=7.7 Hz), 2.68 (2H, q, J=7.7 Hz), 3.61 and 3.68 (2H, m), 3.76 and
3.93 (3H, s, br), 4.51 and 4.77 (2H, s, br), 6.35 and 6.62 (1H, s), 6.62
(1H, s), 7.10 ppm (2H, s); HRMS (ES+): m/z [M+H]+ calcd for
C32H50NO3Si+: 524.3555, found: 524.3562.
7-Methoxy-2-(3,4,5-triethylbenzyl)-6-(triisopropylsilyloxy)-
1,2,3,4-tetrahydroisoquinoline (10e): Method as for 10a using
compound 9d (340 mg, 0.65 mmol) and LiAlH4 (74 mg, 1.95 mmol)
in THF (4.0 mL) at RT for 2 h. Flash column chromatography
(hexane/EtOAc 19:1!19:1 and 2% Et3N) afforded compound 10e
as a viscous yellow oil (289 mg, 87%): 1H NMR (270 MHz, CDCl3):
d=1.07 (18H, d, J=6.9 Hz), 1.13–1.32 (3H, m), 1.15 (3H, t, J=
7.6 Hz), 1.23 (6H, t, J=7.6 Hz), 2.61–2.69 (2H, m) 2.66 (4H, q, J=
7.5 Hz), 2.67 (2H, q, J=7.7 Hz), 2.70–2.78 (2H, m), 3.56 (2H, s), 3.60
(2H, s), 3.72 (3H, s), 6.45 (1H, s), 6.58 (1H, s), 7.04 ppm (2H, s);
HRMS (ES+): m/z [M+H]+ calcd for C32H52NO2Si+: 510.3762, found:
510.3769.
2-(3-Chloro-4,5-dimethoxybenzyl)-7-methoxy-6-(triisopropylsilyl-
oxy)-1,2,3,4-tetrahydroisoquinoline (10a): LiAlH4 (103 mg,
2.7 mmol) was placed in an oven-dried tube and covered with THF
(1.0 mL). 9a (481 mg, 0.9 mmol) was dissolved in THF (4.4 mL) and
added dropwise via syringe. The reaction mixture was stirred at RT
for 2 h. EtOAc (5 mL) was added carefully. The mixture was then di-
luted with EtOAc (100 mL) and left without stirring in a beaker for
0.5 h. The mixture was filtered through Celite that was then
washed with EtOAc (4ꢁ10 mL), and the filtrate was concentrated
in vacuo. Flash column chromatography (hexane/EtOAc 9:1!9:1
and 2% Et3N) afforded compound 10a as a pale yellow oil
(328 mg, 70%): 1H NMR (270 MHz, CDCl3): d=1.07 (18H, d, J=
6.9 Hz), 1.13–1.32 (3H, m), 2.61–2.69 (2H, m), 2.70–2.78 (2H, m),
3.52 (2H, s), 3.54 (2H, s), 3.71 (3H, s), 3.85 (6H, s), 6.44 (1H, s), 6.58
(1H, s), 6.88 (1H, d, J=1.7 Hz), 6.96 ppm (1H, d, J=1.6 Hz); HRMS
(ES+): m/z [M+H]+ calcd for C28H43ClNO4Si+: 520.2644, found:
520.2632.
(ꢀ)-2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-3-methyl-6-
(triisopropylsilyloxy)-1,2,3,4-tetrahydroisoquinoline
(10 f):
Method as for 10b using compound 6b (419 mg, 1.2 mmol), 3-
bromo-4,5-dimethoxybenzyl bromide[15] (449 mg, 1.45 mmol) and
DIPEA (310 mg, 2.4 mmol) in DMF (3.6 mL) at 808C for 18 h. Flash
column chromatography (hexane/EtOAc 9:1!9:1 and 2% Et3N) af-
forded compound 10 f as a yellow oil (598 mg, 86%): 1H NMR
(270 MHz, CDCl3): d=1.07 (18H, d, J=6.6 Hz), 1.09 (3H, d, J=
5.0 Hz), 1.14–1.32 (3H, m), 2.45 (1H, dd, J=16.1, 5.9 Hz), 2.85 (1H,
dd, J=16.0, 4.7 Hz), 3.04 (1H, sext, J=6.1 Hz), 3.40–3.73 (4H, m),
3.71 (3H, s), 3.83 (3H, s), 3.84 (3H, s), 6.41 (1H, s), 6.57 (1H, s), 6.89
(1H, d, J=1.9 Hz), 7.11 ppm (1H, d, J=1.6 Hz); HRMS (ES+): m/z
[M+H]+ calcd for C29H45BrNO4Si+: 578.2296, found: 578.2251.
2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-6-(triisopropylsilyl-
oxy)-1,2,3,4-tetrahydroisoquinoline
(10b):
Compound
6a
(369 mg, 1.1 mmol) was dissolved in DMF (3.3 mL), and diisopropyl-
ethylamine (DIPEA; 287 mg, 2.2 mmol) and 3-bromo-4,5-dimethox-
ybenzyl bromide[15] (335 mg, 1.2 mmol) were added. The mixture
was stirred at 808C for 18 h, cooled to RT, diluted with EtOAc
(100 mL) and washed with H2O (100 mL) and NH4Cl (sat., 5 mL).
The aqueous layer was extracted with EtOAc (100 mL). The com-
bined organics were dried (MgSO4), filtered and concentrated in
vacuo. Flash column chromatography (hexane/EtOAc 19:1!19:1
and 2% Et3N) afforded compound 10b as a yellow oil (272 mg,
2-(3-Chloro-4,5-dimethoxybenzyl)-6-hydroxy-7-methoxy-1,2,3,4-
tetrahydroisoquinoline (11e): Compound 10a (286 mg,
0.55 mmol) was dissolved in THF (2.5 mL). Tetra-n-butylammonium
fluoride (TBAF; 0.66 mL, 1m in THF, 0.66 mmol) was added drop-
wise via syringe, and the reaction mixture was stirred at 08C for
0.5 h. MeOH (5 mL) and CH2Cl2 (30 mL) were added, and the mix-
ture was concentrated in vacuo. Flash column chromatography
(CHCl3/acetone 9:1!9:1 and 2% MeOH) afforded compound 11 e
1
43%): H NMR (270 MHz, CDCl3): d=1.06 (18H, d, J=6.9 Hz), 1.13–
1
as a white solid (168 mg, 84%): mp: 193–1958C; H NMR (270 MHz,
1.32 (3H, m), 2.61–2.69 (2H, m), 2.70–2.78 (2H, m), 3.52 (2H, s),
3.55 (2H, s), 3.71 (3H, s), 3.83 (3H, s), 3.84 (3H, s), 6.44 (1H, s), 6.58
(1H, s), 6.93 (1H, d, J=1.7 Hz), 7.12 ppm (1H, d, J=1.4 Hz); HRMS
(ES+): m/z [M+H]+ calcd for C28H43BrNO4Si+: 564.2139, found:
564.2132.
CDCl3): d=2.61–2.68 (2H, m), 2.69–2.76 (2H, m), 3.46 (2H, s), 3.51
(2H, s), 3.75 (3H, s), 3.80 (6H, s), 6.42 (1H, s), 6.57 (1H, s), 6.88 (1H,
d, J=1.6 Hz), 6.90 ppm (1H, d, J=1.6 Hz); 13C NMR (67.5 MHz,
CDCl3): d=28.0, 50.6, 55.5, 55.8, 56.0, 60.6, 62.0, 108.9, 111.5, 114.3,
122.2, 125.3, 126.4, 127.6, 134.6, 144.1, 145.1, 153.7 ppm; LC-MS
(ES+): m/z 364.2 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for
C19H23ClNO4+: 364.1310, found: 364.1305.
2-(3,5-Dimethoxy-4-ethoxybenzyl)-7-methoxy-6-(triisopropylsil-
yloxy)-1,2,3,4-tetrahydroisoquinoline (10c): Method as for 10a
using compound 9b (435 mg, 0.8 mmol) and LiAlH4 (92 mg,
2.4 mmol) in THF (4.8 mL) at RT for 2 h. Flash column chromatogra-
phy (hexane/EtOAc 9:1!4:1!4:1 and 2% Et3N) afforded com-
pound 10c as a viscous yellow oil (398 mg, 93%): 1H NMR
(270 MHz, CDCl3): d=1.06 (18H, d, J=6.9 Hz), 1.12–1.29 (3H, m),
1.33 (3H, t, J=7.2 Hz), 2.60–2.68 (2H, m) 2.69–2.77 (2H, m), 3.53
(2H, s), 3.56 (2H, s), 3.69 (3H, s), 3.80 (6H, s), 4.02 (2H, q, J=
7.2 Hz), 6.44 (1H, s), 6.57 (1H, s), 6.59 ppm (2H, s); HRMS (ES+):
m/z [M+H]+ calcd for C30H48NO5Si+: 530.3296, found: 530.3303.
2-(3-Bromo-4,5-dimethoxybenzyl)-6-hydroxy-7-methoxy-1,2,3,4-
tetrahydroisoquinoline (11 f): Method as for 11 e using compound
10b (254 mg, 0.45 mmol) and TBAF (0.54 mL, 1m in THF,
0.54 mmol) in THF (2.7 mL) at 08C for 0.5 h. Flash column chroma-
tography (CHCl3/acetone 9:1!9:1 and 2% MeOH) gave compound
1
11 f as a pale yellow solid (148 mg, 80%): mp: 195–1988C; H NMR
(270 MHz, CDCl3): d=2.59–2.67 (2H, m), 2.68–2.76 (2H, m), 3.45
(2H, s), 3.50 (2H, s), 3.74 (3H, s), 3.78 (3H, s), 3.79 (3H, s), 6.41 (1H,
s), 6.55 (1H, s), 6.91 (1H, d, J=1.6 Hz), 7.05 ppm (1H, d, J=1.6 Hz);
13C NMR (67.5 MHz, CDCl3): d=27.9, 50.6, 55.5, 55.8, 55.9, 60.4,
61.9, 108.9, 112.3, 114.3, 117.0, 125.0, 125.2, 126.4, 135.2, 144.1,
145.2, 153.5 ppm; LC-MS (ES+): m/z 408.2 [M+H]+; HRMS (ES+):
m/z [M+H]+ calcd for C19H23BrNO4+: 408.0805, found: 408.0796.
7-Methoxy-2-(3,4,5-triethoxybenzyl)-6-(triisopropylsilyloxy)-
1,2,3,4-tetrahydroisoquinoline (10d): Method as for 10a using
compound 9c (515 mg, 0.9 mmol) and LiAlH4 (104 mg, 2.7 mmol)
in THF (5.4 mL) at RT for 2 h. Flash column chromatography
(hexane/EtOAc 9:1!4:1!4:1 and 2% Et3N) afforded compound
10d as a viscous yellow oil (379 mg, 75%): 1H NMR (270 MHz,
CDCl3): d=1.07 (18H, d, J=6.9 Hz), 1.14–1.30 (3H, m), 1.34 (3H, t,
J=7.2 Hz), 1.39 (6H, t, J=7.2 Hz), 2.59–2.68 (2H, m) 2.69–2.78 (2H,
m), 3.53 (2H, s), 3.55 (2H, s), 3.71 (3H, s), 4.05 (6H, q, J=7.2 Hz),
6.44 (1H, s), 6.59 ppm (3H, s); HRMS (ES+): m/z [M+H]+ calcd for
C32H52NO5Si+: 558.3609, found: 558.3616.
2-(3,5-Dimethoxy-4-ethoxybenzyl)-6-hydroxy-7-methoxy-1,2,3,4-
tetrahydroisoquinoline (11g): Method as for 11 e using com-
pound 10c (371 mg, 0.7 mmol) and TBAF (0.84 mL, 1m in THF,
0.84 mmol) in THF (4.2 mL) at 08C for 0.5 h. Flash column chroma-
tography (CHCl3/acetone 9:1!4:1!4:1 and 2% MeOH) afforded
compound 11 g as
a
yellow glass (194 mg, 74%): 1H NMR
(270 MHz, CDCl3): d=1.34 (3H, t, J=7.0 Hz), 2.65–2.72 (2H, m),
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2.72–2.79 (2H, m), 3.53 (2H, s), 3.58 (2H, s), 3.78 (3H, s), 3.80 (6H,
s), 4.03 (2H, q, J=7.1 Hz), 5.52 (1H, s, br), 6.45 (1H, s), 6.60 (1H, s),
6.61 ppm (2H, s); 13C NMR (67.5 MHz, CDCl3): d=15.5, 28.2, 50.6,
55.7, 55.9, 56.0, 62.8, 68.8, 105.7, 108.8, 114.3, 125.7, 126.8, 133.7,
135.8, 144.1, 145.0, 153.3 ppm; LC-MS (ES+): m/z 374.2 [M+H]+;
HRMS (ES+): m/z [M+H]+ calcd for C21H28NO5+: 374.1962, found:
374.1953.
(270 MHz, CDCl3): d=2.62–2.70 (2H, m), 2.73–2.81 (2H, m), 3.50
(2H, s), 3.52 (2H, s), 3.76 (3H, s), 3.81 (6H, s), 5.98 (2H, s, br), 6.56
(1H, s), 6.83 (1H, d, J=1.6 Hz), 6.92 (1H, d, J=1.7 Hz), 7.04 ppm
(1H, s); 13C NMR (67.5 MHz, CDCl3): d=28.0, 50.4, 55.5, 56.1, 56.1,
60.6, 61.8, 110.9, 111.2, 121.8, 123.8, 126.9, 127.8, 134.0, 134.8,
137.4, 144.3, 149.6, 153.7 ppm; LC-MS (ES+): m/z 443.2 [M+H]+;
HRMS (ES+): m/z [M+H]+ calcd for C19H24ClN2O6S+: 443.1038,
found: 443.1034.
6-Hydroxy-7-methoxy-2-(3,4,5-triethoxybenzyl)-1,2,3,4-tetra-hy-
droisoquinoline (11h): Method as for 11 e using compound 10d
(336 mg, 0.6 mmol) and TBAF (0.72 mL, 1m in THF, 0.72 mmol) in
THF (3.6 mL) at 08C for 0.5 h. Flash column chromatography
(CHCl3/acetone 9:1!9:1 and 2% MeOH) afforded compound 11 h
as a pale yellow solid (189 mg, 78%): mp: 119–1218C; 1H NMR
(270 MHz, CDCl3): d=1.34 (3H, t, J=6.3 Hz), 1.38 (6H, t, J=6.9 Hz),
2.62–2.70 (2H, m), 2.71–2.79 (2H, m), 3.51 (2H, s), 3.55 (2H, s), 3.79
(3H, s), 4.03 (6H, q, J=7.0 Hz), 5.63 (1H, s, br), 6.45 (1H, s), 6.58
(1H, s), 6.62 ppm (2H, s); 13C NMR (67.5 MHz, CDCl3): d=15.0, 15.6,
28.4, 50.6, 55.8, 56.0, 62.8, 64.6, 68.7, 107.4, 108.8, 114.2, 126.1,
127.0, 133.6, 144.0, 144.9, 152.8 ppm; LC-MS (ES+): m/z 402.3 [M+
H]+; HRMS (ES+): m/z [M+H]+ calcd for C23H32NO5+: 402.2275,
found: 402.2268.
2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-
1,2,3,4-tetrahydroisoquinoline (12 f): Method as for 12e using
compound 11 f (122 mg, 0.3 mmol) and sulfamoyl chloride
(0.9 mmol) in DMA (2.5 mL) at RT for 2 h. The residue was stirred in
CH2Cl2/Et2O/hexane (~1:2:2), filtered and dried to afford compound
1
12 f as a pale yellow solid (54 mg, 37%): mp: 132–1368C; H NMR
(270 MHz, CDCl3): d=2.62–2.70 (2H, m), 2.73–2.81 (2H, m), 3.50
(2H, s), 3.53 (2H, s), 3.76 (3H, s), 3.80 (3H, s), 3.80 (3H, s), 5.99 (2H,
s, br), 6.56 (1H, s), 6.87 (1H, d, J=1.6 Hz), 7.04 (1H, s), 7.07 ppm
(1H, d, J=1.6 Hz); 13C NMR (67.5 MHz, CDCl3): d=28.0, 50.4, 55.5,
56.0, 56.1, 60.4, 61.7, 110.9, 112.0, 117.2, 123.8, 124.6, 126.8, 134.0,
135.4, 137.4, 145.4, 149.6, 153.6 ppm; LC-MS (ES+): m/z 487.2 [M+
H]+; HRMS (ES+): m/z [M+H]+ calcd for C19H24BrN2O6S+: 487.0533,
found: 487.0511.
6-Hydroxy-7-methoxy-2-(3,4,5-triethylbenzyl)-1,2,3,4-tetrahydro-
isoquinoline (11i): Method as for 11 e using compound 10e
(255 mg, 0.5 mmol) and TBAF (0.6 mL, 1m in THF, 0.6 mmol) in THF
(3.0 mL) at 08C for 0.5 h. Flash column chromatography (CHCl3/ace-
tone 9:1!9:1 and 2% MeOH) afforded compound 11 i as a yellow
2-(3,5-Dimethoxy-4-ethoxybenzyl)-7-methoxy-6-sulfamoyloxy-
1,2,3,4-tetrahydroisoquinoline (12g): Method as for 12e using
compound 11 g (169 mg, 0.45 mmol) and sulfamoyl chloride
(1.35 mmol) in DMA (4.0 mL) at RT for 2 h. The residue was stirred
in CH2Cl2/Et2O (~1:4), filtered and dried to afford compound 12g
as a pale yellow solid (125 mg, 61%): mp: 127–1308C; 1H NMR
(270 MHz, CDCl3): d=1.34 (3H, t, J=7.0 Hz), 2.66–2.75 (2H, m),
2.76–2.85 (2H, m), 3.57 (2H, s), 3.59 (2H, s), 3.79 (3H, s), 3.82 (6H,
s), 4.03 (2H, q, J=7.1 Hz), 5.05 (2H, s, br), 6.59 (3H, s), 7.06 ppm
(1H, s); 13C NMR (67.5 MHz, CDCl3): d=15.5, 27.9, 50.2, 55.6, 56.1,
56.2, 62.6, 68.9, 105.8, 111.1, 124.0, 127.3, 133.3, 134.4, 136.0, 137.3,
149.4, 153.4 ppm; LC-MS (ES+): m/z 453.2 [M+H]+; HRMS (ES+):
m/z [M+H]+ calcd for C21H29N2O7S+: 453.1690, found: 453.1688.
1
glass (134 mg, 75%): H NMR (270 MHz, CDCl3): d=1.16 (3H, t, J=
7.6 Hz), 1.24 (6H, t, J=7.4 Hz), 2.67 (4H, q, J=7.4 Hz), 2.67–2.82
(6H, m), 3.57 (2H, s), 3.63 (2H, s), 3.80 (3H, s), 5.49 (1H, s, br), 6.48
(1H, s), 6.61 (1H, s), 7.06 ppm (2H, s); 13C NMR (67.5 MHz, CDCl3):
d=15.3, 15.8, 21.2, 25.7, 28.2, 50.5, 55.7, 55.9, 62.5, 108.9, 114.3,
126.0, 126.9, 127.1, 135.2, 138.2, 141.9, 144.0, 144.9 ppm; LC-MS
(ES+): m/z 354.3 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for
C23H32NO2+: 354.2428, found: 354.2414.
(ꢀ)-2-(3-Bromo-4,5-dimethoxybenzyl)-6-hydroxy-7-methoxy-3-
methyl-1,2,3,4-tetrahydroisoquinoline (11j): Method as for 11 e
using compound 10 f (520 mg, 0.9 mmol) and TBAF (1m in THF,
1.08 mL, 1.08 mmol) in THF (4.5 mL) at 08C for 0.5 h. Flash column
chromatography (CHCl3/acetone 9:1!9:1 and 2% MeOH) afforded
compound 11 j as a yellow solid (342 mg, 90%): mp: 111–1148C;
1H NMR (270 MHz, CDCl3): d=1.11 (3H, d, J=6.3 Hz), 2.46 (1H, dd,
J=16.2, 6.1 Hz), 2.87 (1H, dd, J=16.1, 4.8 Hz), 3.06 (1H, sext, J=
6.1 Hz), 3.41–3.73 (4H, m), 3.78 (3H, s), 3.81 (3H, s), 3.83 (3H, s),
5.32 (1H, s, br), 6.43 (1H, s), 6.59 (1H, s), 6.92 (1H, d, J=1.9 Hz),
7.10 ppm (1H, d, J=1.7 Hz); 13C NMR (67.5 MHz, CDCl3): d=15.2,
34.4, 51.2, 52.2, 55.8, 56.0, 56.3, 60.5, 108.6, 112.0, 114.5, 117.1,
124.5, 125.0, 126.2, 136.7, 144.0, 144.9, 145.1, 153.6 ppm; LC-MS
(ES+): m/z 422.2 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for
C20H25BrNO4S+: 422.0962, found: 422.0942.
7-Methoxy-6-sulfamoyloxy-2-(3,4,5-triethoxybenzyl)-1,2,3,4-tet-
rahydroisoquinoline (12h): Method as for 12e using compound
11 h (160 mg, 0.4 mmol) and sulfamoyl chloride (1.2 mmol) in DMA
(3.0 mL) at RT for 2 h. The residue was stirred in CH2Cl2/Et2O/
hexane (~1:2:2), filtered and dried to afford compound 12h as
a
pale yellow solid (84 mg, 46%): mp: 133–1348C; 1H NMR
(270 MHz, CDCl3): d=1.33 (3H, t, J=6.9 Hz), 1.39 (6H, t, J=6.9 Hz),
2.64–2.72 (2H, m), 2.75–2.83 (2H, m), 3.54 (2H, s), 3.55 (2H, s), 3.79
(3H, s), 4.03 (2H, q, J=7.0 Hz), 4.04 (4H, q, J=7.0 Hz), 5.07 (2H, s,
br), 6.57 (2H, s), 6.59 (1H, s), 7.05 ppm (1H, s); 13C NMR (67.5 MHz,
CDCl3): d=14.9, 15.6, 28.0, 50.2, 55.6, 56.2, 62.6, 64.6, 68.8, 107.4,
111.1, 124.0, 127.5, 133.1, 134.6, 137.0, 137.3, 149.3, 152.8 ppm; LC-
MS (ES+): m/z 481.3 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for
C23H33N2O7S+: 481.2003, found: 481.1989.
2-(3-Chloro-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-
1,2,3,4-tetrahydroisoquinoline (12e): Compound 11 e (146 mg,
0.4 mmol) was placed in an oven-dried 50 mL round-bottom flask
and dissolved in DMA (2.0 mL). Sulfamoyl chloride (0.57m in tolu-
ene, 2.1 mL, 1.2 mmol) was concentrated in vacuo and re-dissolved
in DMA (1.0 mL). This solution was added dropwise via syringe at
08C. The reaction mixture was stirred at RT for 2 h. EtOAc (100 mL)
was added, and the mixture was washed with NaHCO3 (saturated,
50 mL) and H2O (4ꢁ50 mL). The organic layer was dried (NaCl), fil-
tered and concentrated in vacuo. The residue was stirred in
CH2Cl2/Et2O/hexane (~1:2:2), filtered and dried to afford compound
12e as a white solid (85 mg, 48%): mp: 130–1348C; 1H NMR
7-Methoxy-6-sulfamoyloxy-2-(3,4,5-triethylbenzyl)-1,2,3,4-tetra-
hydroisoquinoline (12i): Method as for 12e using compound 11 i
(106 mg, 0.3 mmol) and sulfamoyl chloride (0.9 mmol) in DMA
(2.5 mL) at RT for 2 h. The residue was stirred in CH2Cl2/hexane
(~1:4), filtered and dried to afford compound 12i as a pale yellow
1
solid (65 mg, 50%): mp: 138–1408C; H NMR (270 MHz, CDCl3): d=
1.14 (3H, t, J=7.4 Hz), 1.22 (6H, t, J=7.6 Hz), 2.61–2.75 (4H, m),
2.65 (4H, q, J=7.6 Hz), 2.77–2.84 (2H, m), 3.59 (2H, s), 3.61 (2H, s),
3.79 (3H, s), 5.07 (2H, s, br), 6.61 (1H, s), 7.02 (2H, s), 7.06 ppm
(1H, s); 13C NMR (67.5 MHz, CDCl3): d=15.5, 27.9, 50.2, 55.6, 56.1,
56.2, 62.6, 68.9, 105.8, 111.1, 123.9, 127.3, 133.3, 134.4, 136.0, 137.3,
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149.3, 153.4 ppm; LC-MS (ES+): m/z 433.3 [M+H]+; HRMS (ES+):
m/z [M+H]+ calcd for C23H33N2O4S+: 433.2156, found: 433.2147.
6-Acetoxy-7-methoxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahy-
droisoquinoline (17b): Compound 3a (108 mg, 0.3 mmol), Et3N
(0.21 mL, 1.6 mmol) and Ac2O (0.16 mL, 1.6 mmol) were stirred in
CHCl3 (10 mL) at RT for 24 h. The reaction mixture was diluted with
CHCl3 (30 mL) and washed with H2O (4ꢁ30 mL) and brine, dried
(MgSO4), filtered and concentrated in vacuo. The residue was
stirred in Et2O, filtered and dried in vacuo to afford compound
17b as a white powder (95 mg, 79%): mp: 143–1448C; 1H NMR
(270 MHz, CDCl3): d=2.28 (3H, s), 2.69–2.83 (4H, m), 3.60 (4H, s),
3.75 (3H, s), 3.84 (3H, s), 3.85 (6H, s), 6.58 (1H, s), 6.62 (2H, s),
6.77 ppm (1H, s); 13C NMR (67.5 MHz, CDCl3): d=20.7, 28.3, 50.5,
55.9, 56.0, 56.2, 60.9, 62.8, 105.7, 110.5, 122.7, 124.1, 126.5, 133.3,
136.9, 138.1, 149.1, 153.3, 169.4 ppm; LC-MS (ES+): m/z 402.24
[M+H]+; Anal. calcd for C22H27NO6: C 65.82, H 6.78, N 3.49, found:
C 65.8, H 6.81, N 3.49.
(ꢀ)-2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-3-methyl-6-sul-
famoyloxy-1,2,3,4-tetrahydroisoquinoline (12j): Method as for
12e using compound 11 j (212 mg, 0.5 mmol) and sulfamoyl chlo-
ride (1.5 mmol) in DMA (3.0 mL) at RT for 2 h. The residue was
stirred in CH2Cl2, filtered and dried to afford compound 12j as
a pale yellow solid (241 mg, 96%): mp: 155–1588C; 1H NMR
(270 MHz, CDCl3): d=0.98 (3H, d, J=6.6 Hz), 2.39 (1H, dd, J=16.6,
5.6 Hz), 2.81 (1H, dd, J=16.2, 4.4 Hz), 2.95 (1H, sext, J=5.6 Hz),
3.30–3.62 (4H, m), 3.66 (3H, d, J=2.2 Hz), 3.69 (3H, d, J=2.2 Hz),
3.71 (3H, d, J=2.2 Hz), 6.45 (2H, s), 6.50 (1H, s), 6.76 (1H, s), 6.94
(1H, s), 6.97 ppm (1H, d, J=1.7 Hz); LC-MS (ES+): m/z 501.2 [M+
H]+; HRMS (ES+): m/z [M+H]+ calcd for C20H26BrN2O6S+: 501.0617,
found: 501.0644;.
7-Methoxy-6-methanesulfonyloxy-2-(3,4,5-trimethoxybenzyl)-
1,2,3,4-tetrahydroisoquinoline (17c): Compound 3a (80 mg,
0.22 mmol) was dissolved in pyridine (1.0 mL) and cooled to 08C.
Methanesulfonyl chloride (20 mL, 0.26 mmol) was added, and the
reaction mixture was stirred at 08C for 2 h and then at RT for 4 h.
The mixture was diluted with EtOAc, washed with H2O and brine,
dried (MgSO4), filtered and concentrated in vacuo. Flash column
chromatography (hexane/EtOAc 1:1) gave a solid that was stirred
in Et2O, filtered and dried to afford compound 17c as a white solid
2-(3-Bromo-4,5-dimethoxybenzyl)-7-ethyl-6-hydroxy-1,2,3,4-tet-
rahydroisoquinoline (14): Method as for 10b using compound
13[3] (266 mg, 1.5 mmol), DIPEA (581 mg, 4.5 mmol) and 3-bromo-
4,5-dimethoxybenzyl bromide[15] (511 mg, 1.65 mmol) in DMF
(5 mL) at 808C for 20 h. Flash column chromatography (CHCl3/ace-
tone 4:1) afforded compound 14 as a tan solid (246 mg, 40%): mp:
156–1608C; 1H NMR (270 MHz, CDCl3): d=1.12 (3H, t, J=7.4 Hz),
2.50 (2H, q, J=7.4 Hz), 2.60 (2H, d, J=5.2 Hz), 2.67 (2H, d, J=
5.2 Hz), 3.51 (2H, s), 3.57 (2H, s), 3.75 (3H, s), 3.84 (3H, s), 6.12 (1H,
s), 6.70 (1H, s), 6.98 (1H, s), 7.12 ppm (1H, s); 13C NMR (67.5 MHz,
CDCl3): d=14.1, 22.7, 28.1, 50.7, 55.4, 55.9, 60.5, 62.1, 112.6, 114.8,
117.1, 125.3, 127.0, 128.5, 132.1, 134.8, 145.5, 145.5, 152.5,
153.6 ppm; LC-MS (ES+): m/z 406.2 [M+H]+; HRMS (ES+): m/z
[M+H]+ calcd for C20H25BrNO3+: 406.1012, found: 406.1005.
1
(70 mg, 73%): mp: 134–1358C; H NMR (270 MHz, CDCl3): d=2.68–
2.84 (4H, m), 3.15 (3H, s), 3.57 (2H, s), 3.59 (2H, s), 3.81 (3H, s),
3.83 (3H, s), 3.85 (6H, s), 6.61 (3H, s), 7.04 ppm (1H, s); 13C NMR
(67.5 MHz, CDCl3): d=28.3, 38.2, 50.4, 55.9, 56.1, 56.2, 61.0, 62.9,
105.6, 110.9, 124.5, 127.4, 134.2, 135.0, 136.7, 137, 149.3,
153.3 ppm; LC-MS (ES+): m/z 438.14 [M+H]+; HRMS (ES+): m/z
[M+H]+ calcd for C21H28NO7S+: 438.1581, found: 438.1569.
2-(3-Bromo-4,5-dimethoxybenzyl)-7-ethyl-6-sulfamoyloxy-
1,2,3,4-tetrahydroisoquinoline (15): Method as for 12e using
compound 14 (150 mg, 0.37 mmol) and sulfamoyl chloride
(1.48 mmol) in DMA (1.0 mL) at RT for 20 h. Flash column chroma-
tography (CHCl3/acetone 4:1) afforded compound 15 as a pale
2-(3-Chloro-4,5-dimethoxybenzoyl)-6-hydroxy-1,2,3,4-tetrahy-
droisoquinoline (19a): Method as for 9a using compound 18b
(345 mg, 1.5 mmol), 3-chloro-4,5-dimethoxybenzoic acid (357 mg,
1.65 mmol), EDCI (575 mg, 3.0 mmol) and Et3N (0.25 mL, 1.8 mmol)
in CH2Cl2 (10 mL) at RT for 18 h. The mixture was diluted with
CH2Cl2, washed with H2O (10 mL), citric acid (10%, 10 mL), Na2CO3
(sat.) and brine, dried (MgSO4), filtered and concentrated in vacuo.
Flash column chromatography (hexane!hexane/EtOAc 1:1) afford-
ed compound 19a as an off-white solid (313 mg, 60%): mp: 151–
1
yellow foam (85 mg, 47%): H NMR (270 MHz, CDCl3): d=1.16 (3H,
t, J=7.6), 2.64 (2H, q, J=7.6 Hz), 2.70 (2H, t, J=5.5 Hz), 2.84 (2H, t,
J=5.5 Hz), 3.56–3.58 (4H, m), 3.84 (6H, s), 6.88–6.93 (2H, m), 7.08–
7.13 ppm (2H, m); 13C NMR (67.5 MHz, CDCl3): d=14.3, 22.7, 28.4,
50.1, 55.2, 56.1, 60.6, 61.7, 112.3, 117.3, 121.4, 125.0, 127.9, 133.1,
133.2, 134.4, 135.0, 145.6, 146.7, 153.7 ppm; LC-MS (ES+):
m/z 485.2 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for
C20H26BrN2O5S+: 485.0740, found: 485.0736.
1
1648C; H NMR (270 MHz, CDCl3): d=2.75–2.82 (2H, m), 3.61 (1H,
s, br), 3.81 (3H, s, br), 3.86 (3H, s), 3.85–3.92 (1H, m), 4.49 (1H, s,
br), 4.74 (1H, s, br), 6.56–6.90 (4H, m), 7.02 (1H, s), 7.80 ppm (1H,
s, br); LC-MS (ES+): m/z 348.4 [M+H]+; HRMS (ES+): m/z [M+H]+
calcd for C18H19ClNO4+: 348.0997, found: 348.0987.
6,7-Dimethoxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroiso-
quinoline (17a): Compound 16 (0.23 g, 1.0 mmol), 3,4,5-trime-
thoxybenzyl chloride (0.26 g, 1.2 mmol) and Et3N (0.5 mL,
3.6 mmol) were placed in a 10 mL microwave vessel and dissolved
in EtOH (2.5 mL). The mixture was then irradiated at 1308C for 1 h
in the microwave oven. After cooling to RT, the mixture was
poured into H2O and extracted with EtOAc. The organic layer was
washed with H2O and brine, dried (MgSO4), filtered and concentrat-
ed in vacuo. Flash column chromatography (hexane/EtOAc 6:1!
1:1) gave a white solid that was stirred in Et2O, filtered and dried
to afford compound 17a as a white powder (230 mg, 62%): mp:
118–1198C; 1H NMR (270 MHz, CDCl3): d=2.70 (2H, t, J=5.6 Hz),
2.81 (2H, t, J=5.6 Hz), 3.55 (2H, s), 3.59 (2H, s), 3.81 (3H, s), 3.83
(3H, s), 3.84 (3H, s), 3.85 (6H, s), 6.50 (1H, s), 6.60 (1H, s), 6.62 ppm
(2H, s); 13C NMR (67.5 MHz, CDCl3): d=28.8, 50.7, 55.9, 56.0, 56.2,
61.0, 63.1, 105.6, 109.5, 111.4, 126.3, 126.8, 134.5, 136.9, 147.3,
147.6, 153.2 ppm; LC-MS (ES+): m/z 374.27 [M+H]+; Anal. calcd
for C21H27NO5: C 67.54, H 3.75, N 7.29, found: C 67.5, H 3.76, N
7.10.
2-(3,5-Dimethoxy-4-ethoxybenzoyl)-6-hydroxy-1,2,3,4-tetrahy-
droisoquinoline (19b): Method as for 19a using compound 18b
(345 mg,
1.5 mmol),
4-ethoxy-3,5-dimethoxybenzoic
acid[26]
(373 mg, 1.65 mmol), EDCI (575 mg, 3.0 mmol) and Et3N (0.25 mL,
1.8 mmol) in CH2Cl2 (10 mL) at RT for 18 h. Flash column chroma-
tography (hexane!hexane/EtOAc 1:4) afforded compound 19b as
a white foam (296 mg, 55%): 1H NMR (270 MHz, CDCl3): d=1.34
(3H, t, J=6.9 Hz), 2.75–2.86 (2H, m), 3.60–3.67 (1H, m), 3.81 (6H,
s), 3.81–3.90 (1H, m), 4.06 (2H, q, J=6.9 Hz), 4.51 (1H, s, br), 4.76
(1H, s, br), 6.60–6.97 ppm (5H, m); LC-MS (ES+): m/z 358.1 [M+
H]+; HRMS (ES+): m/z [M+H]+ calcd for C20H24NO5+: 358.1649,
found: 358.1637.
6-Hydroxy-2-(3,4,5-triethoxybenzoyl)-1,2,3,4-tetrahydroisoquino-
line (19c): Method as for 19a using compound 18b (690 mg,
3.0 mmol), 3,4,5-triethoxybenzoic acid (1.14 g, 4.5 mmol), EDCI
(1.15 g, 6.0 mmol) and Et3N (0.5 mL, 3.6 mmol) in CH2Cl2 (10 mL)
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and THF (5 mL) at RT for 18 h. Flash column chromatography
(hexane!hexane/EtOAc 3:2) afforded compound 19c as a white
solid (731 mg, 63%): mp: 157–1588C; 1H NMR (270 MHz, CDCl3):
d=1.32–1.42 (9H, m), 2.80 (2H, s, br), 3.62 and 3.92 (2H, s, br),
3.98–4.11 (6H, m), 4.51 and 4.75 (2H, s, br), 5.97 (1H, s, br), 6.62–
8.2 Hz); 13C NMR (67.5 MHz, [D6]DMSO): d=15.5, 28.5, 50.5, 55.4,
56.0, 63.0, 68.9, 106.3, 113.7, 115.1, 125.4, 127.5, 132.8, 135.0, 135.9,
153.3, 154.9 ppm; LRMS (ES+): m/z 344.4 [M+H]+; HRMS (ES+):
m/z [M+H]+ calcd for C20H26NO4+: 344.1856, found: 344.1842.
6-Sulfamoyloxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroiso-
quinoline (20d): Method as for 12e using compound 20b
(100 mg, 0.3 mmol) and sulfamoyl chloride (0.6 mmol) in DMA
(1.0 mL) at RT for 24 h. Flash column chromatography (hexane/
EtOAc 3:1!EtOAc) afforded compound 20d as a white powder
6.66 (4H, m), 6.67 and 7.00 ppm (1H, s, br); LC-MS (APCI+): m/z
386.5 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for C22H28NO5
:
+
386.1962, found: 386.1960.
6-Hydroxy-2-(3,4,5-trimethoxyphenacetyl)-1,2,3,4-tetrahydroiso-
1
quinoline (19d): Method as for 19a using compound 18b
(345 mg, 1.5 mmol) and 3,4,5-trimethoxyphenylacetic acid (373 mg,
1.65 mmol), EDCI (575 mg, 3.0 mmol) and Et3N (0.25 mL, 1.8 mmol)
in CH2Cl2 (10 mL) at RT for 18 h. Flash column chromatography
(hexane!EtOAc) afforded compound 19d as a gummy foam
(110 mg, 88%): mp: 173–1748C; H NMR (270 MHz, [D6]DMSO): d=
2.65 (2H, t, J=5.5 Hz), 2.84 (2H, t, J=5.5 Hz), 3.56 (2H, s), 3.58 (2H,
s), 3.64 (3H, s), 3.75 (6H, s), 6.65 (2H, s), 6.98–7.02 (2H, m), 7.12
(1H, d, J=8.0 Hz), 7.91 ppm (2H, br); 13C NMR (67.5 MHz,
[D6]DMSO): d=28.8, 49.7, 55.1, 55.8, 60.0, 61.8, 105.4, 119.5, 121.8,
127.7, 133.3, 134.2, 135.8, 136.3, 148.3, 152.8 ppm; HRMS (ES+):
m/z [M+H]+ calcd for C19H25N2O6S+: 409.1428, found: 409.1428;
Anal. calcd for C19H24N2O6S: C 55.87, H 5.92, N 6.86, found: C 55.6,
H 6.02, N 6.51.
1
(470 mg, 88%): H NMR (270 MHz, CDCl3): d=2.66 and 2.78 (2H, t,
J=6.2 Hz), 3.64 and 3.81 (2H, t, J=6.2 Hz), 3.70–3.82 (11H, m), 4.55
and 4.67 (2H, s), 5.50 (1H, s, br), 6.39 and 6.46 (2H, s), 6.56–6.68
(2H, m), 6.83 and 6.97 ppm (1H, d, J=8.1 Hz); LC-MS (APCI): m/z
358.3 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for C20H24NO5
:
+
2-(3-Chloro-4,5-dimethoxybenzyl)-6-hydroxy-1,2,3,4-tetrahydroi-
soquinoline (20e): Method as for 10a using compound 19a
(203 mg, 0.58 mmol) and LiAlH4 (111 mg, 2.9 mmol) in THF (8 mL)
at RT for 18 h. The reaction mixture was cooled to 08C, and H2O
(0.15 mL) was added slowly followed by aq NaOH (15%, 0.15 mL)
and then H2O (0.45 mL). The mixture was stirred at RT for 0.25 h,
then diluted with EtOAc (100 mL) and stirred another 0.25 h, fil-
tered, dried (MgSO4) and concentrated in vacuo. Flash column
chromatography (hexane!hexane/EtOAc 2:3) afforded compound
358.1649, found: 358.1643.
6-Benzyloxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoqui-
noline (20a): Method as for 17a using compound 18c (431 mg,
1.8 mmol), 3,4,5-trimethoxybenzyl chloride (433 mg, 2 mmol) and
Et3N (0.5 mL, 3.6 mmol) in EtOH (2.5 mL) at 1308C for 1.5 h in the
microwave oven. Flash column chromatography (hexane/EtOAc
3:1!1:1) gave a solid that was stirred in Et2O, filtered and dried to
afford compound 20a as a yellow powder (450 mg, 60%): mp:
103–1048C; 1H NMR (270 MHz, CDCl3): d=2.69 (2H, t, J=5.8 Hz),
2.86 (2H, t, J=5.8 Hz), 3.58 (2H, s), 3.60 (2H, s), 3.84 (3H, s), 3.85
(6H, s), 5.03 (2H, s), 6.62 (2H, s), 6.74–6.78 (2H, m), 6.92 (1H, d, J=
8.0 Hz), 7.26–7.43 ppm (5H, s); 13C NMR (67.5 MHz, CDCl3): d=29.4,
50.3, 55.7, 56.1, 60.8, 63.0, 69.9, 105.5, 112.8, 114.3, 127.4, 127.5,
127.9, 128.5, 134.4, 135.6, 136.7, 137.1, 153.1, 157.1 ppm; LC-MS
(ES+): m/z 420.25 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for
C26H30NO4+: 420.2169, found: 420.2158; Anal. calcd for C26H29NO4:
C 74.44, H 6.97, N 3.34, found: C 74.3, H 6.91, N 3.30.
1
20e as a colourless solid (134 mg, 69%): mp: 155–1578C; H NMR
(270 MHz, CDCl3): d=2.69 (4H, s), 3.51 (2H, s), 3.56 (2H, s), 3.73
(3H, s), 3.84 (3H, s), 6.25 (1H, d, J=2.3 Hz), 6.45 (1H, dd, J=8.3,
2.3 Hz), 6.76 (1H, d, J=8.3 Hz), 6.93 ppm (2H, s); 13C NMR
(67.5 MHz, CDCl3): d=28.6, 50.5, 55.4, 56.0, 60.6, 62.2, 111.8, 113.6,
115.1, 122.5, 125.5, 127.6, 127.7, 134.1, 135.1, 144.4, 153.8,
154.6 ppm; LC-MS (ES+): m/z 333.9 [M+H]+; HRMS (ES+): m/z
[M+H]+ calcd for C18H21ClNO3+: 334.1204, found: 334.1197; Anal.
calcd for C18H20ClNO3: C 64.77, H 6.04, N 4.20, found: C 64.7, H
6.03, N 4.19.
6-Hydroxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoqui-
noline (20b): Compound 20a (300 mg, 0.72 mmol) was treated
with Pd/C (10%, 40 mg) in THF (20 mL) and MeOH (20 mL) under
H2 at RT for 1.5 h. The reaction mixture was then filtered through
Celite and washed with MeOH (4ꢁ10 mL). The combined filtrates
were concentrated in vacuo, and the residue was stirred in EtOAc,
filtered and dried to afford compound 20b as a white powder
2-(3-Bromo-4,5-dimethoxybenzyl)-6-hydroxy-1,2,3,4-tetrahydro-
isoquinoline (20 f): Method as for 10b using compound 18b
(200 mg, 0.87 mmol), 3-bromo-4,5-dimethoxybenzyl bromide[15]
(296 mg, 0.96 mmol), DIPEA (790 mg, 6.1 mmol) in DMF (5.0 mL) at
808C for 60 h. Flash column chromatography (hexane!hexane/
EtOAc 2:3) afforded compound 20 f as a white solid (246 mg,
85%): mp: 161–1658C; 1H NMR (270 MHz, CDCl3): d=2.68–2.72
(4H, m), 3.52 (2H, s), 3.56 (2H, s), 3.76 (3H, s), 3.83 (3H, s), 6.33
(1H, d, J=2.2 Hz), 6.49 (1H, dd, J=8.1, 2.2 Hz), 6.78 (1H, d, J=
8.1 Hz), 6.96 (1H, d, J=1.5 Hz), 7.09 ppm (1H, d, J=1.5 Hz);
13C NMR (67.5 MHz, CDCl3): d=28.7, 50.5, 55.4, 56.0, 60.6, 62.1,
112.4, 113.5, 115.0, 117.2, 125.2, 125.8, 127.6, 135.0, 135.2, 145.5,
153.7, 154.5 ppm; LC-MS (ES+): m/z 377.7 [M+H]+; HRMS (ES+):
m/z [M+H]+ calcd for C18H21BrNO3+: 378.0699, found: 378.0685;
Anal. calcd for C18H20BrNO3: C 57.15, H 5.33, N 3.70, found: C 57.0,
H 5.39, N 3.61.
1
(175 mg, 74%): mp: 197–1998C; H NMR (270 MHz, [D6]DMSO): d=
2.60 (2H, t, J=5.4 Hz), 2.72 (2H, t, J=5.4 Hz), 3.45 (2H, s), 3.54 (2H,
s), 3.64 (3H, s), 3.75 (6H, s), 6.48–6.53 (2H, m), 6.65 (2H, s), 6.81
(1H, d, J=8.0 Hz), 9.11 ppm (1H, s); 13C NMR (67.5 MHz, [D6]DMSO):
d=28.8, 50.2, 55.2, 55.8, 60.0, 62.1, 105.5, 113.0, 114.6, 125.2, 127.3,
134.3, 135.1, 136.2, 152.8, 155.4 ppm; LC-MS (ESꢁ): m/z 328.16
ꢁ
+
[MꢁH]
; :
HRMS (ES+): m/z [M+H]+ calcd for C19H24NO4
330.1700, found: 330.1689; Anal. calcd for C19H23NO4: C 69.28, H
7.04, N 4.25, found: C 69.1, H 7.33, N 4.56.
6-Methoxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoqui-
noline (20c): Method as for 17a using compound 18a (300 mg,
1.5 mmol), 3,4,5-trimethoxybenzyl chloride (325 mg, 1.5 mmol) and
Et3N (0.5 mL, 3.6 mmol) in EtOH (5 mL) at 1208C for 1 h in the mi-
crowave oven. Flash column chromatography (hexane!hexane/
EtOAc 1:1) afforded compound 20c as a white solid (81 mg, 16%):
mp: 102–1038C; 1H NMR (270 MHz, CDCl3): d=2.69 (2H, t, J=
7.8 Hz), 2.87 (2H, t, J=7.8 Hz), 3.58 (2H, s), 3.59 (2H, s), 3.77 (3H,
s), 3.84 (3H, s), 3.85 (6H, s), 6.62–6.71 (4H, m), 6.92 ppm (1H, d, J=
2-(3,5-Dimethoxy-4-ethoxybenzyl)-6-hydroxy-1,2,3,4-tetrahydro-
isoquinoline (20g): Method as for 20e using compound 19b
(280 mg, 0.78 mmol) and LiAlH4 (149 mg, 3.9 mmol) in THF (8 mL)
at RT for 18 h. Flash column chromatography (hexane!EtOAc) af-
forded compound 20g as a colourless solid (179 mg, 67%): mp:
154–1578C; 1H NMR (270 MHz, CDCl3): d=1.33 (3H, t, J=7.2 Hz),
2.68 (4H, s), 3.52 (2H, s), 3.59 (2H, s), 3.75 (6H, s), 4.02 (2H, q, J=
7.2 Hz), 6.23 (1H, d, J=1.9 Hz), 6.44 (1H, dd, J=8.3, 1.9 Hz), 6.61
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1783 – 1793 1792

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[2] S. Faderl, M. Talpaz, Z. Estrov, H. M. Kantarjian, Ann. Intern. Med. 1999,
131, 207–219.
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(2H, s), 6.74 ppm (1H, d, J=8.3 Hz); LC-MS (ES+): m/z 344.0 [M+
H]+; HRMS (ES+): m/z calcd for C20H26NO4+: 344.1856, found:
344.1842; Anal. calcd for C20H25NO4: C 69.95, H 7.34, N 4.08, found:
C 69.60, H 7.35, N 4.00.
6-Hydroxy-2-(3,4,5-triethoxybenzyl)-1,2,3,4-tetrahydroisoquino-
line (20h): Method as for 20e using compound 19c (258 mg,
0.67 mmol) and LiAlH4 (127 mg, 3.4 mmol) in THF (8 mL) at RT for
18 h. Flash column chromatography (hexane!hexane/EtOAc 1:4)
afforded compound 20h as a white solid (75 mg, 38%): mp: 140–
1428C; 1H NMR (400 MHz, CDCl3): d=1.34 (3H, t, J=7.2 Hz), 1.35
(6H, t, J=6.8 Hz), 2.68 (4H, s), 3.52 (2H, s), 3.57 (2H, s), 3.97 (4H, q,
J=6.8 Hz), 4.04 (2H, q, J=7.2 Hz), 6.27 (1H, d, J=2.4 Hz), 6.45 (1H,
dd, J=8.4, 2.4 Hz), 6.59 (2H, s), 6.75 ppm (1H, d, J=8.4 Hz);
13C NMR (100 MHz, CDCl3): d=14.9, 15.6, 28.6, 50.5, 55.4, 63.0, 64.5,
68.8, 107.7, 113.6, 115.1, 125.5, 127.5, 132.6, 135.1, 136.8, 152.7,
154.7 ppm; LC-MS (ES+): m/z 372.4 [M+H]+; HRMS (ES+): m/z
[M+H]+ calcd for C22H30NO4+: 372.2169, found: 372.2156; Anal.
calcd. for C22H29NO4: C 71.13, H 7.87, N 3.77, found: C 70.9, H 7.87,
N 3.77.
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6-Hydroxy-2-(3,4,5-trimethoxybenzoyl)-1,2,3,4-tetrahydroisoqui-
noline (20i): Compound 18b (200 mg, 0.87 mmol) and 3,4,5-trime-
thoxybenzoyl chloride (200 mg, 0.87 mmol) were dissolved in
CH2Cl2 (10 mL) and cooled to 08C. Et3N (0.36 mL, 2.6 mmol) was
added. The reaction mixture was stirred at RT for 18 h, then diluted
with CH2Cl2, washed with H2O (10 mL) and brine, dried (MgSO4)
and concentrated in vacuo. Flash column chromatography
(hexane!hexane/EtOAc 1:4) afforded compound 20i as an off-
white solid (200 mg, 67%): mp: 175–1768C; 1H NMR (270 MHz,
CDCl3): d=2.78–2.86 (2H, m), 3.62 (1H, s, br), 3.82–3.88 (10H, m),
4.51 (1H, s, br), 4.76 (1H, s, br), 6.60–6.70 ppm (5H, m); LC-MS
(ES+): m/z 344.0 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for
C19H22NO5+: 344.1492, found: 344.1480.
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6-Hydroxy-2-(3,4,5-trimethoxyphenethyl)-1,2,3,4-tetrahydroiso-
quinoline (20j): Method as for 20e using compound 19d
(200 mg, 0.56 mmol) and LiAlH4 (106 mg, 2.79 mmol) in THF (8 mL)
at RT for 18 h. Flash column chromatography (hexane!EtOAc) af-
forded compound 20j as a colourless solid (120 mg, 63%): mp:
1
180–1868C; H NMR (270 MHz, CDCl3): d=2.72–2.87 (8H, m), 3.64
(2H, s), 3.82 (3H, s), 3.84 (6H, s), 5.02 (1H, s, br), 6.44 (2H, s), 6.54
(1H, d, J=2.5 Hz), 6.59 (1H, dd, J=8.3, 2.5 Hz), 6.89 ppm (1H, d,
J=8.3 Hz); 13C NMR (67.5 MHz, CDCl3): d=28.7, 34.0, 51.0, 55.4,
56.1, 60.2, 60.8, 105.6, 113.8, 115.2, 125.4, 127.6, 135.1, 135.8, 136.3,
153.1, 154.9 ppm; LC-MS (ES+): m/z 344.3 [M+H]+; HRMS (ES+):
m/z [M+H]+ calcd for C20H26NO4+: 344.1856, found: 344.1840;
Anal. calcd for C20H25NO4·0.25H2O: C 69.04, H 7.39, N 4.03, found: C
69.1, H 7.31, N 3.91.
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Acknowledgements
This work was supported by Sterix Ltd., a member of the IPSEN
Group. We thank Ms Alison Smith (University of Bath) for techni-
cal support and the NCI DTP for providing in vitro and in vivo
screening resources.
Keywords: chimeras
· colchicine binding · microtubule
disruptors · tetrahydroisoquinolines · tubulin assembly
[1] a) P. C. Nowell, D. A. Hungerford, Science 1960, 132, 1497; b) P. C.
Nowell, J. Clin. Invest. 2007, 117, 2033–2035; c) R. Kurzrock, H. M. Kant-
arjian, B. J. Druker, M. Talpaz, Ann. Intern. Med. 2003, 138, 819–830.
Received: February 12, 2014
Published online on May 12, 2014
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1783 – 1793 1793