Synthesis of a hairpin pyrrole-imidazole polyamide conjugate containing a quinone methide precursor and vinyl linking group

Article abstract of DOI:10.1016/j.tetlet.2004.02.070

Standard procedures for elaborating a quinone methide precursor for conjugation to a DNA ligand was not compatible with the presence of a vinyl group. Instead, an acrylate linker was attached by Heck coupling subsequent to o-substitution of the phenolic p

Full text of DOI:10.1016/j.tetlet.2004.02.070

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2887–2889
Synthesis of a hairpin pyrrole–imidazole polyamide conjugate
containing a quinone methide precursor and vinyl linking group
Dalip Kumar and Steven E. Rokita*
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
Received 22 January 2004; accepted 13 February 2004
Abstract—Standard procedures for elaborating a quinone methide precursor for conjugation to a DNA ligand was not compatible
with the presence of a vinyl group. Instead, an acrylate linker was attached by Heck coupling subsequent to o-substitution of the
phenolic precursor. This transformation required protection of the phenolic group and use of ethyl acrylate rather than acrylic acid.
The presence of the vinyl group also rendered the quinone methide precursor more labile to alkaline conditions than its equivalent
saturated derivative and required mild conditions for coupling to the pyrrole–imidazole polyamide.
ꢀ 2004 Elsevier Ltd. All rights reserved.
An important class of antitumor agents acts by cross-
-
OTBS
O
L
linking DNA. Unfortunately, the specificity of these
agents is not yet sufficient to avoid many deleterious
complications. Considerable effort has therefore been
directed toward design and discovery of new compounds
that exhibit greater chemical and biological specificity.1
A variety of natural and synthetic compounds including
mitomycin, bizelesin, and merchlorethamine exhibit an
intrinsic preference for cross-linking at particular
nucleotide sequences.2 Further selectivity is often gained
by conjugating a DNA binding ligand to a reactive
appendage, essentially creating an affinity reagent.3
Pyrrole–imidazole polyamides are especially attract for
this purpose due to their predictable association with
base pairs through the minor groove of DNA and their
compatibility with cellular conditions.4 Such ligands
have been conjugated with alkylating agents related to
the cyclopropylpyrroloindole group of CC-1065 and
Duocarmycin and to the cross-linking N-mustard
chorambucil.5 A range of linkers have been examined in
these conjugates and a relatively rigid trans acrylate
moiety appears highly effective.6
AcO
OAc
AcO
OAc
-
F
L
OH
L
O
DNA
DNA
AcO
L
Scheme 1.
favors reaction with the 2-amino group of guanine in the
minor groove,8 a conjugate directing reaction to this site
has only now been prepared as reported here. A previ-
ous strategy7c for adapting a nascent quinone methide
for coupling to a sequence-directing ligand and cross-
linking to DNA was not appropriate for the acrylate
derivative (9, Scheme 2). Attempts to hydroxymethylate
ortho to the phenolic position of 4-hydroxycinnamic
acid with formaldehyde under alkaline conditions gen-
erated only polymer. Accordingly, substitution at the
ortho positions was performed first using a Mannich
reaction to generate bis(morpholinomethyl)phenol 1 as
described by Crisp and Turner.9 Next, the linker arm
was attached and activated for conjugation to the
polyamide (Scheme 2).
Our laboratory has focused on quinone methides as
DNA alkylating and cross-linking agents (Scheme 1).7
Although an innate selectivity based on product stability
Keywords: Quinone methide; Pyrrole–imidazole polyamide; DNA
alkylation; Minor groove ligand.
* Corresponding author. Tel.: +1-301-405-1816; fax: +1-301-405-9376;
e-mail: rokita@umd.edu
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.02.070

2888
D. Kumar, S. E. Rokita / Tetrahedron Letters 45 (2004) 2887–2889
OH
OH
OAc
I
N
N
N
N
AcO
OAc
(i)
(ii)
O
O
O
O
I
3
2
1
OAc
OTBS
OH
AcO
OAc
TBSO
OTBS
HO
OH
(iv)
(v)
(iii)
(vi)
5
6
4
CO2CH3
OTBS
CO2H
CO2H
OTBS
AcO
OAc
AcO
OAc
(vii)
(ix)
(viii)
O
8
7
CO2H
O
O N
O
N
O
O
N
OTBS
H
N
H
N
N
N
N
H
N
N
AcO
OAc
H
N
O
O
O
NH
9
O
O
O
O
N
N
H
O
H
N
N
N
H
N
H
N
N
H
N
H
N
O
N
H
N
N
H
N
N
O
O
Scheme 2. Reagents and conditions: (i) NaI, chloramine-T, 50 ꢁC; (ii) Ac2O, AcOH; (iii) Pd(OAc)2, (o-CH3C6H4)3P, methyl acrylate, Et3N, toluene,
20 h; (iv) 1.25 M H2SO4, THF, 80 ꢁC, 3.5 h; (v) LiOH, H2O, rt, 2 h; (vi) TBS-Cl, imidazole, DMF, 48 h; (vii) FeCl3, Ac2O, 0 ꢁC; (viii) N-hydroxy-
succinimide, EDC, CH2Cl2, rt; (ix) aqueous DMF/CH3CN pH 7.5 pyrrole–imidazole polyamide.
Treatment of 1 with NaI/chloramine-T in DMSO yiel-
ded the p-iodo derivative 2 that was ready for coupling
to the linker arm. A palladium-catalyzed Heck reaction
proceeded in good yield (4, 84%) but only after pro-
tecting the phenolic group with acetate (3) and using
methyl acrylate with Pd(OAc)2, (o-CH3C6H4)3P, and
Et3N (2 equiv) in dry toluene (100 ꢁC).10 No coupling
was detected after 2 was combined with the palladium
catalyst and either acrylic acid or its methyl ester under
a range of temperatures (55–110 ꢁC) and solvents
(DMF, CH3CN, and H2O). A similar lack of reaction
was observed when acrylic acid was treated with the
protected phenol 3 under the anhydrous conditions
above. The sensitivity of this reaction is likely based on
the inhibitory effects of electron donating groups
attached to the aromatic system and the limited quantity
of base that could be added in the presence of this
quinone methide precursor. Deprotection of 4 by
sequential treatment with 1.25 M H2SO4 in 75% aqueous
THF (80 ꢁC) and aqueous LiOH at room temperature
produced 3-[30,50-bis(hydroxymethyl)-40-hydroxyphen-
yl]-2-propenoic acid 5 in 71% yield. Alternative reflux of
the H2SO4 solution in attempt to hydrolyze the three
esters concurrently led to polymerization of the organic
material. The desired trisilyloxy derivative 6 was gen-
erated under the standard conditions of excess TBS-Cl
and imidazole in DMF at room temperature.7c Selective
substitution forming the benzylic acetates was achieved
by treatment with FeCl3 and acetic anhydride under
conditions described by Ganem and Small.11
The quinone methide precursor 7 exhibited unusual
sensitivity to many of the typical procedures used for its
final coupling to DNA ligands. Even stepwise reaction
through an N-hydroxysuccinimide ester was initially
discouraging after a complex mixture of products was
detected under routine addition of 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide (EDC) in DMF.7c
However, the activated ester 8 was successfully isolated
in 64% yield when CH2Cl2 was substituted for DMF.
The presence of the vinyl group also destabilized
attachment of the TBS group as evident by its loss after
exposure to diisopropylethylamine in either CH3CN,
DMF, H2O, or their combination under conditions used

D. Kumar, S. E. Rokita / Tetrahedron Letters 45 (2004) 2887–2889
2889
to couple the saturated derivative of 8.7c Coupling only
proceeded as expected when the activated ester 8 and
polyamide were combined under mild conditions of 50%
aqueous CH3CN/DMF (2:1) pH 7.5 at room tempera-
ture. The product 9 was isolated by reverse phase (C-18)
chromatography and confirmed by electrospray mass
spectroscopy.
zen, W. F.; Pande, P.; Rokita, S. E. J. Am. Chem. Soc.
2003, 125, 14005–14013.
8. (a) Veldhuyzen, W.; Lam, Y.-f.; Rokita, S. E. Chem. Res.
Toxicol. 2001, 14, 1345–1351; (b) Veldhuyzen, W. F.;
Shallop, A. J.; Jones, R. A.; Rokita, S. E. J. Am. Chem.
Soc. 2001, 123, 11126–11132.
9. Crisp, G. T.; Turner, P. D. Tetrahedron 2000, 56, 407–415.
1
10. 3: Yield 69%, mp 88–89 ꢁC. H NMR (CDCl3): d 2.06 (s,
6H, COCH3), 2.31 (s, 3H, COCH3), 4.95 (s, 4H, –CH2),
7.72 (s, 2H); 13C NMR (CDCl3): d 20.4, 20.7, 60.3, 90.5,
131.3, 138.9, 148.0, 168.7, 170.4, HRMS calcd for
C14H15IO6 405.9913, found 405.9906. 4: The iodobenzene
3 (1.00 g, 2.46 mmol), methyl acrylate (0.480 g, 5.55 mmol),
distilled triethylamine (0.43 g, 4.9 mmol), tri-o-tolylphos-
phine (0.075 g, 0.25 mmol), and palladium (II) acetate
(0.028 g, 0.13 mmol) in dry toluene (8 mL) were heated at
100 ꢁC under nitrogen for 24 h. Solvent was removed at
reduced pressure and the residue extracted with ether
(3 · 10 mL). After removal of ether, the product was
purified by silica-gel chromatography using ethyl acetate
and hexane (3:7) as solvent. Yield 84%; mp 102 ꢁC; 1H
NMR (CDCl3): d 2.06 (s, 6H, COCH3), 2.33 (s, 3H,
COCH3), 3.79 (s, 3H, CO2CH3), 5.02 (s, 4H, –CH2), 6.41
(d, 1H, J ¼ 16, olefinic), 7.56 (s, 2H); 7.64 (d, 1H, J ¼ 16,
olefinic); 13C NMR (CDCl3): d 20.5, 20.8, 51.9, 61.0, 119.1,
129.8, 130.0, 132.9, 143.1, 148.8, 167.1, 168.1, 170.5;
HRMS calcd for C18H29O8 364.1162, found 364.1158. 5:
1H NMR (MeOH-d4): d 4.70 (s, 4H, CH2), 6.29 (d, 1H,
J ¼ 16, olefinic), 7.39 (s, 2H), 7.58 (d, 1H, J ¼ 16, olefinic);
13C NMR (MeOH-d4): d 61.9, 116.2, 127.3, 128.4, 129.1,
146.9, 157.3, 171.1; HRMS calcd for anion C11H10O5
(M)Hþ) 223.0599, found 223.0606. 6: Yield 39%; mp
198 ꢁC; 1H NMR (CDCl3): d 0.09 (s, 12H, Si(CH3)2), 0.18
(s, 6H, Si(CH3)2), 0.94 (s, 18H, C(CH3)3), 1.00 (s, 9H,
C(CH3)3), 4.68 (s, 4H, CH2), 6.32 (d, 1H, J ¼ 16, olefinic),
7.59 (s, 2H), 7.78 (d, 1H, J ¼ 16, olefinic); 13C NMR
(CDCl3): d 14.1, 18.4, 18.8, 22.6, 25.9, 60.4, 114.8, 126.4,
127.7, 132.5, 147.7, 150.6, 172.4. HRMS calcd for
C29H55O5Si3 567.3357, found 557.3362. 7: Yield 43%; mp
The set of functional groups required in the desired
quinone methide precursor containing an acrylate linker
necessitated a new strategy for its construction. Use of a
Heck coupling to attach the linker arm subsequent to
alkylation of the ortho positions will now provide easy
access to quinone methide precursors containing a
broad array of linkers used to connect with site-directing
ligands. Additionally, strongly basic conditions can be
avoided during coupling of labile appendages to site-
directing ligands without adverse effects.
Acknowledgements
This research was supported in part by the National
Institutes of Health (CA-81571).
References and notes
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1
120–121 ꢁC; H NMR (CDCl3): d 0.23 (s, 6H, Si(CH3)2),
1.03 (s, 9H, C(CH3)3), 2.12 (s, 6H, COCH3), 5.10 (s, 4H,
CH2), 6.34 (d, 1H, J ¼ 16, olefinic), 7.51 (s, 2H), 7.73 (d,
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found 421.1682. 8: N-Hydroxysuccinimide (0.013 g,
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1
64%; H NMR (CDCl3): d 0.22 (s, 6H, Si(CH3)2), 1.02 (s,
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9H, C(CH3)3), 2.12 (s, 6H, COCH3), 2.87 (s, 4H, CH2),
5.10 (s, 4H, CH2), 6.49 (d, 1H, J ¼ 16, olefinic), 7.53 (s, 2H,
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)3.5, 18.7, 21.0, 25.7, 25.8, 61.3, 110.4, 127.4, 128.3, 130.1,
149.0, 154.1 162.1, 169.3, 170.7; HRMS calcd for
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0.001 mmol) and then with aqueous MOPS buffer
(250 mM, pH 7.5, 50 lL). The reaction mixture was stirred
at room temperature for 10 min and the product was
purified by reverse phase (C-18) HPLC. Yield ꢀ20%, ESI-
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1785.5.
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