Synthesis of mutual azo prodrugs of anti-inflammatory agents and peptides facilitated by α-aminoisobutyric acid

Article abstract of DOI:10.1021/jo201358e

Reported is the synthesis of azo mutual prodrugs of the nonsteroidal anti-inflammatory agents (NSAIDs) 4-aminophenylacetic acid (4-APAA) or 5-aminosalicylic acid (5-ASA) with peptides, including an antibiotic peptide temporin analogue modified at the amin

Full text of DOI:10.1021/jo201358e

Article
pubs.acs.org/joc
Synthesis of Mutual Azo Prodrugs of Anti-inflammatory Agents and
Peptides Facilitated by α-Aminoisobutyric Acid
David A. Kennedy,*,† Nagarajan Vembu,‡ Frank R. Fronczek,§ and Marc Devocelle†
†Centre for Synthesis and Chemical Biology, Department of Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons in
Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland
‡Post Graduate and Research Department of Chemistry, Urumu Dhanalakshmi College, Tiruchirappalli - 620 019, India
§Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, United States
S
* Supporting Information
ABSTRACT: Reported is the synthesis of azo mutual prodrugs of the
nonsteroidal anti-inflammatory agents (NSAIDs) 4-aminophenylacetic acid (4-
APAA) or 5-aminosalicylic acid (5-ASA) with peptides, including an antibiotic
peptide temporin analogue modified at the amino terminal by an α-
aminoisobutyric acid (Aib) residue. These prodrugs are designed for colonic
delivery of two agents to treat infection and inflammation by the bacterial
pathogen Clostridium difficile.
originally developed as the azo prodrug sulfasalazine.11,12 Azo
prodrugs release therapeutically active amine drugs upon
INTRODUCTION
■
The bacterium Clostridium difficile can colonize the human
colon where it causes diseases such as diarrhea and
pseudomembranous colitis, typically associated with disruption
reduction site-specifically by bacterial extracellular azoreductase
enzymes and the redox potential in the human colon.13,14 The
azo prodrug APAZA releases the nonsteroidal anti-inflamma-
of the normal commensal gut microflora by a course of
antibiotic treatment.1 There have been several outbreaks2 of
tory agents 5-aminosalicylic acid and 4-aminophenylacetic acid
(4-APAA) 1a, protecting against damage to the colonic
strains of C. difficile that are resistant to many antibiotics of
epithelium that is caused by toxin of C. difficile.15
different classes.3 Recommended routine treatment against
infection by C. difficile is mainly limited clinically to either
metronidazole or the cyclic glycopeptide vancomycin.4
However, a course of therapy with these antibiotics tends to
disturb the gastrointestinal microflora, allowing recolonization
by C. difficile causing multiple recurrences of infection after
treatment.4 Due to the possibility of emergence of resistance
against existing antibiotics, there is a need for the development
of new antibiotics for treatment of this infection.
Cationic antimicrobial peptides offer potential to be
developed as new antibiotics.5 Antibiotic peptides are expressed
as components of innate immunity in diverse living organisms,
including humans,5 and in the intestines, where they have a role
in maintaining the normal gut symbiotic microflora.6 As a class,
antibiotic peptides exhibit potent and broad-spectrum direct
microbistatic or microbicidal activity against pathogenic Gram-
positive and -negative bacteria; fungi, including yeasts;
protozoa; and viruses.7 Some cationic antimicrobial peptides
have selective antitumor and anticancer activity.8 The antibiotic
peptide temporin analogues originally isolated from the skin
secretions of the frog Rana temporaria are selectively potent
against Gram-positive bacteria including clostridia.9
Our proposed approach to generate new agents targeting
infection and disease caused by C. difficile is to generate mutual
azo prodrugs for concerted site-specific delivery of an
antimicrobial peptide and an NSAID (Scheme 1). Protection
of the ammonium terminal, which contributes toward the
overall positive charge that is important for the activity of an
antimicrobial peptide displaying a low net charge, by an azo
bond with an anilinic anti-inflammatory agent is employed with
the aim of maintaining both components in an inactive state
before reaching the colon, thereby avoiding ulceration side
effects from the NSAID or disruption of commensal microflora
by the antibiotic in the upper gastrointestinal tract.16−18
The structural design of the azo mutual prodrugs is shown in
Scheme 2. The introduction of an α,α-dialkyl α-amino acid as
the N-terminal residue was chosen to avoid an α-proton that
could be abstracted allowing tautomerization of the azo to a
hydrazone, which could be hydrolyzed in vivo and release a
toxic hydrazino metabolite. The synthesis results in linkage of
the anti-inflammatory agent via an azo bond to an α-
methylalanine or α-aminoisobutyric acid (Aib) residue. α-
Aminoisobutyric acid residues are important constituents
toward the activity of peptaibiotics and peptaibols, which are
classes of antibiotic peptides of fungal origin.19,20
C. difficile secretes toxins that cause disease by inflammation
of the colonic epithelium.10 A classical treatment for
inflammatory bowel diseases, which include Crohn’s disease
and ulcerative colitis, is the nonsteroidal anti-inflammatory drug
(NSAID) 5-aminosalicylic acid (5-ASA) 1b which was
Received: June 29, 2011
Published: October 25, 2011
© 2011 American Chemical Society
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Scheme 1. Activation of Peptide and Anti-inflammatory Agent Mutual Prodrug
Scheme 2. Azo Prodrug Structural Design
In the next step, acid-labile protecting groups, benzhydryl
and tert-butyl esters, were chosen for the carboxyl groups, to
allow their deprotection concomitantly to protecting groups of
side-chains of amino acid residues. tert-Butyl ester as a
protecting group was chosen also for its bulkiness, to favor
selective deprotection of the methyl ester by alkaline hydrolysis
(vide infra). Benzhydryl esterification was performed with
diphenyldiazomethane,26 with reaction rate enhanced by a
protic mixture of water in 1,4-dioxane solvent, or alternatively,
as in the case of preparation of benzhydryl ester 7 (Scheme 5),
sterically hindered tert-butyl alcohol in dimethylformamide
solvent. tert-Butyl ester 4a was prepared by reaction with di-tert-
butyldicarbonate catalyzed by 4-(dimethylamino)pyridine.27
Esterification by tert-butyl alcohol catalyzed by sulfuric acid
with magnesium sulfate desiccant in dichloromethane28 gave
the product 4b satisfactorily, although the phenolic position is
not tert-butylated by this method (Table 1).
RESULTS AND DISCUSSION
■
The complete synthesis of azo mutual prodrugs, as achieved for
the peptide temporin with an anti-inflammatory agent, is
depicted in Schemes 3 and 7. The azo bond was prepared by
Scheme 3. Preparation of Azo and Protection of
a
Carboxylate
Attempted deprotection of the methyl ester of 4c by base
hydrolysis using sodium hydroxide was not selective over the
benzhydryl ester. Lithium hydroxide in a mixture of water and
tetrahydrofuran deprotected the methyl ester of 4a selectively
in the presence of the tert-butyl ester. However, the resulting α-
azo carboxylate 5 decomposes by decarboxylation, expected to
be facilitated by the basicity and mesomeric electronic
withdrawal of the azo and the Thorpe−Ingold geminal dialkyl
effect (Scheme 4).
a
Reagents and conditions: i. 1. HCl, NaNO2. 2. HBF4. ii.
Scheme 4. Decarboxylation
Methyltrimethylsilyl dimethylketene acetal, THF, CH3CN. iii.
Boc2O, t-BuOH, DMAP (cat.). iv. t-BuOH, H2SO4, MgSO4,
CH2Cl2. v. Diphenyldiazomethane, 1,4-dioxane, H2O.
aliphatic diazonium coupling of a silyl ketene acetal to a
diazonium fluoborate salt prepared by diazotization of the
anilinic21,22 anti-inflammatory agent starting material. The
particular case of diazonium fluoborate 2 was obtainable from
the reaction solution of the diazonium chloride by precipitation
using fluoboric acid23 but not sodium fluoborate,24 despite
sodium fluoborate rather than fluoboric acid being recom-
mended in general for obtaining increased yields of diazonium
precipitate.25 The yield for the preparation of the 5-azosalicylic
acid 3b was lower than for the 4-azophenylacetic acid 3a
(Scheme 3).
Nevertheless, α-azo carboxylic acids have been reported.29
The similar α-dimethyl carboxylate 8 (Scheme 5) that has an
amide instead of α-azo, synthesized in parallel for the
Table 1. Esterification of 3
3
4
a
entry
compound
R1
R2
reagents
R1
R2
yield (%)
I
a
b
b
c
CH2CO2H
OH
H
iii
iii
iv
v
CH2CO2t-Bu
OH
H
97
5
II
CO2H
CO2H
H
CO2t-Bu
CO2t-Bu
H
III
IV
OH
OH
52
44
CH2CO2H
CH2CO2Bzh
a
Reagents and conditions: iii. Boc2O, t-BuOH, DMAP (10 mol %). iv. t-BuOH, H2SO4, MgSO4, CH2Cl2. v. diphenyldiazomethane, 1,4-dioxane,
H2O.
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using vacuum apparatus for deoxygenated conditions was not
feasible for the reaction mixture containing solvent quantities of
volatile hydrazine.) In any case, redox potential of the azo bond
from electronic donation by the phenolic substituent tends to
affect the ease of reduction or the propensity for reversion of
the hydrazo product to the azo such as upon exposure to
oxygen. For example, electron-donating substituents decrease
the ease of reduction of aryl azo compounds by clostridial
azoreductase.31 Upon exposure to ordinary atmosphere, the α-
hydrazo carboxylate product 11 is susceptible to facile oxidative
reversion to azo 5 followed by decarboxylative degradation,
thus necessitating deoxygenated reaction and extraction
conditions.
Scheme 5. Amide Isostere
preparation of a negative control, also decarboxylated (Scheme
6) as a side reaction.
The α-hydrazo carboxylic acid 11 gave very poor yields when
coupled to an amino acid by amide bond-forming reagents,
including HATU, DMTMM,32 PyBroP,33 cyanuric fluoride,34
and TFFH (fluoro-N,N,N′,N′-tetramethylformamidinium hexa-
fluorophosphate),35 typically used for difficult couplings in
peptide synthesis. Although hindrance by the α-methyl groups
is an effect widely acknowledged in the difficult coupling of the
amino acid α-methylalanine, other considerations here include
deactivation of the carbonyl by the α-nucleophile effect and
electron donation from the α-hydrazo group, or potential
reaction of the activated carboxyl group with the hydrazo
group. Evidence for the latter contributions was the need to
oxidize the α-hydrazo acid fluoride 12 by exposure to
atmosphere to convert to the α-azo acid fluoride 13 before
Scheme 6. Decarboxylation
Decarboxylation was circumvented by functional group
interconversion, by reduction of the azo 4a to hydrazo 10a
by hydrazine hydrate30 (Scheme 7). Yields for reduction of the
4-azophenylacetate 4a were quantitative. In contrast, reduction
of the 5-azosalicylate 4b by hydrazine could not in this case be
used to continue the synthesis as no necessary solid as an
isolable precipitate was given from the reaction. (Extraction
Scheme 7. Completion of the Synthesis of Mutual Prodrug
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cooled to 0 °C, and a cooled solution of NaNO2 (3.5 g, 50 mmol, 1
equiv) in deionized H2O (17.5 mL) was added in small portions
during 20 min such that the reaction temperature did not exceed 5 °C.
The reaction was stirred for a further 30 min until HBF4 (∼8 M, 17
mL) was added. The resulting precipitate was collected by filtration
and sucked dry to obtain a pale solid with discernible needle crystals
(11.3 g, 45 mmol, 91% (this yield was increased to 97% when the
reaction was allowed for ∼2 h)) which were recrystallized by
dissolving in CH3CN, filtering to remove insoluble solid, precipitation
of crystals with Et2O, and chilling to obtain a pale-pink solid (76%).
(Diazonium salts in general are reactive and tend to be unstable in
solution above about 5 °C. Care should be taken to maintain all
solutions at low temperatures and avoid contact with skin by wearing
thick gloves when handling the salts.) Mp 118 °C (dec.); Crystal
coupling to the amino acid. Thus, probably because of the α-
hydrazo group, one-pot solution-phase coupling of carboxylic
acid 11 to an amino acid via the fluorination reagent TFFH was
not successful, despite TFFH being effective for coupling of the
similarly hindered amino acid α-methylalanine to peptides35,36
and dimethylmalonic acid to 7 (Scheme 5). The properties of
acid fluorides in being more reactive toward nitrogen
nucleophiles than oxygen nucleophiles such as water and stable
enough to isolate34 were ideal in these circumstances in which
it was necessary to expose the acid fluoride to atmosphere in
order to oxidize the hydrazo to azo to allow coupling. The
crude acid fluoride 13, prepared using the fluorinating reagent
diethylaminosulfur trifluoride (DAST),37 was coupled to amino
acids without purification.
After coupling to amino acid methyl ester, the methyl ester
14 was deprotected by base hydrolysis before coupling to a
protected peptide sequence by a solution-phase convergent
condensation using carbodiimide coupling chemistry. The
protected peptide was prepared by solid-phase synthesis and
cleaved from the resin under mild acidic conditions.38 The
crude condensation product 16 was deprotected without
isolation or purification to obtain the final product 17, which
was purified by reverse-phase HPLC.
1
structure; H NMR (CD3CN, 400 MHz, δ): 9.46 (br s, 1H, COOH),
8.46 (d, J = 8.93 Hz, 2H, aryl CH), 7.86 (d, J = 7.14 Hz, 2H, aryl CH),
3.97 (s, 2H, benzylic CH2); 13C NMR (CD3CN, 100.6 MHz, δ): 169.5
(COOH), 150.4 (aryl C), 132.8 (aryl CH), 132.0 (aryl CH), 112.2
(aryl C), 40.0 (benzylic CH2).
3-Carboxy-4-hydroxybenzenediazonium Tetrafluoroborate
(2b). 5-Aminosalicylic acid (5.0 g, 0.03 mol, 1 equiv) was suspended
in deionized H2O (17 mL) and acidified with 37% HCl aqueous
solution (13 mL). The mixture was cooled to 0 °C. A solution of
NaNO2 (2.3 g, 0.03 mol, 1 equiv) in deionized H2O (16 mL) was
added portionwise. The mixture was stirred for 1 h 30 min. HBF4
aqueous solution (8 M, 18 mL) was added and stirred before being
allowed to stand. The precipitate was collected by filtration to obtain
7.0 g of crystalline solid. The crude solid was treated with CH3CN and
filtered. Solvent was evaporated from the filtrate, and the remaining
solid was triturated with Et2O and collected by filtration. Purified
product yield: 4.7 g, 18.6 mmol, 57%, as white solid. 1H NMR
(CD3CN, 400 MHz, δ): 8.75 (d, J = 2.80 Hz, 1H, CH), 8.20 (dd, J1 =
9.20 Hz, J2 = 2.80 Hz, 1H, CH), 7.15 (d, J = 9.20 Hz, 1H, CH); 13C
NMR (CD3CN, 100.6 MHz, δ): 171.6 (quaternary C), 168.3
(quaternary C), 138.1 (CH), 138.0 (CH), 121.7 (CH), 115.6
(quaternary C), 101.8 (quaternary C).
CONCLUSION
■
Although azo prodrugs of anti-inflammatory agents and amino
acids have been previously reported for site-specific delivery to
the colon,39,40 to the best of our knowledge, here is presented
the first preparation of mutual prodrug candidates containing
antimicrobial peptides and nonsteroidal anti-inflammatory
agents for colonic delivery. The synthesis of an antibacterial
peptide temporin A analogue L512TA41 linked to the anti-
inflammatory agent 4-aminophenylacetic acid has been
achieved by a strategy that employs an α-methylalanine linker
connected to the anti-inflammatory moiety via an azo bond
involving the α-amino group of the methylalanine residue.
Crystal structures have been obtained for the first four of the
intermediates in the synthesis, compounds 2a, 3a, 4a, 10a. All
steps, except coupling to the hindered α-dimethyl carboxylic
acid, gave good to excellent yields, and the isolated
intermediates are obtained in good purity, in most cases
accompanied by crystallization directly from the workup or
upon standing after extraction. Only one chromatography step
is necessary before coupling to the peptide. This synthetic
strategy can be applied to the preparation of azo mutual
prodrugs of anti-inflammatory agents and peptides. The azo
conjugate of 5-aminosalicylic acid and α-methylalanine methyl
ester has, for example, been successfully prepared by this
methodology for N-terminal modification of antimicrobial
peptide candidates.
(4-{[2-(Methoxycarbonyl)propan-2-yl]diazenyl}phenyl)acetic
Acid (3a). To a solution of diazonium tetrafluoborate 2a (5.0 g, 20
mmol, 1.0 equiv) in anhydrous THF (23.3 mL) cooled to −5 °C
under N2 gas in an overdried flask was added methyltrimethylsilyl
dimethylketene acetal (5.8 mL, 28.6 mmol, 1.43 equiv) slowly in 1 mL
portions via syringe. Anhydrous CH3CN (23.3 mL) was added and the
mixture was stirred for two hours. The mixture was concentrated by
rotary evaporation without heat and with the flask covered by
aluminum foil. The residue was treated with H2O and extracted with
CHCl3. The combined CHCl3 extracts were washed with H2O, dried
over anhydrous MgSO4, and filtered, and solvent was evaporated to
obtain an oil that crystallized upon standing (5.6 g, 21.4 mmol, yield:
quantitative). Crystal structure. IR (KBr disk) 3449, 2992, 2954, 2731,
2650, 2556, 2361, 2341, 1738, 1713, 1608, 1523, 1495, 1463, 1433,
1408, 1363, 1336, 1287, 1238, 1200, 1155, 1105, 1014, 994, 934, 846,
1
819, 797, 758, 732, 680, 508 cm−1; H NMR (CDCl3, 400 MHz, δ):
8.80 (br s, 1H, COOH), 7.59 (d, J = 8.40 Hz, 2H, CH), 7.29 (d, J =
8.40 Hz, 2H, CH), 3.67 (s, 3H, CO2CH3), 3.61 (s, 2H, CH2), 1.51 (s,
6H, CH3); 13C NMR (CDCl3, 100.6 MHz, δ): 176.5 (CO2Me), 174.1
(CO2H), 150.8 (aromatic C), 136.4 (aromatic C), 130.1 (CH), 122.7
(CH), 75.6 (CNN), 52.3 (CH3), 40.8 (CH2), 23.2 (CH3); λmax
(CH3CN) ε296.6 nm: 2887 L mol−1 cm−1, ε402.2 nm: 274 L mol−1 cm−1;
Anal. Calcd for C13H16N2O4: C, 59.08; H, 6.10; N, 10.60. Found: C,
59.22; H, 6.15; N, 10.38.
2-Hydroxy-5-{[2-(methoxycarbonyl)propan-2-yl]diazenyl}-
benzoic Acid (3b). Diazonium tetrafluoborate 2b (3.0 g, 12 mmol, 1
equiv) was dissolved in anhydrous THF (40 mL) and anhydrous
CH3CN (60 mL) under N2 gas. Methyl trimethylsilyl dimethylketene
acetal (5 mL, 25 mmol, 2 equiv) was delivered via syringe. The
reaction was performed at −5 °C for 3 h 20 min. Solvent was
evaporated. The residual solid was treated with H2O and extracted
with CHCl3. The chlorinated phase was washed with H2O, and the
aqueous washings were acidified with concentrated 37% HCl solution
(20 mL) and extracted with CHCl3. The combined chlorinated
EXPERIMENTAL SECTION
■
General. Reverse-phase HPLC chromatography employed Phe-
nomenex Gemini and Varian 5 μm, C-18, 110 Å, 4.6 mm × 250 mm
analytical columns; Phenomenex Jupiter 5 μm, C-5, 110 Å, 4.6 mm ×
250 mm analytical column; Phenomenex Gemini 5 μm, C-18, 110 Å,
250 mm × 10 mm and Phenomenex Jupiter 15 μm, C-5, 300 Å, 250
mm × 10 mm semi-preparative columns. Crystal structures were
determined using data collected with Mo Kα radiation at T = 90 K on
a Nonius KappaCCD diffractometer. NMR chemical shifts are not
corrected. NMR peaks assignments were assisted by CH COSY.
4-(Carboxymethyl)benzenediazonium Tetrafluoroborate
(2a). 4-aminophenylacetic acid 1a (7.5 g, 50 mmol, 1 equiv) was
suspended in concentrated aqueous HCl (20 mL). The mixture was
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extracts were washed with H2O, and solvent was evaporated to obtain
a brown solid (2.6 g, 9.8 mmol, 82%). IR (KBr disk) 2992, 2592, 1737,
1671, 1610, 1585, 1519, 1509, 1483, 1467, 1437, 1378, 1360, 1330,
1277, 1250, 1190, 1143, 1073, 1004, 974, 899, 844, 831, 802, 791, 773,
was stirred vigorously at ambient temperature for 1 day. Solvent was
removed by rotary evaporation. The resulting oily residue was purified
by chromatography on silica gel using petroleum ether with increasing
proportion of CH2Cl2 as mobile phase. After evaporation of solvent,
product was obtained as a partially solidified oil (0.4 g, 0.94 mmol,
44%). 1H NMR (CDCl3, 400 MHz, δ): 7.56 (d, 2H, CH), 7.3 (d, 2H,
CH), 7.3−7.1 (overlapping ms, 10H, CH), 6.79 (s, 1H, benzhydrylic
CH), 3.68 (s, 2H, CH2), 3.66 (s, 3H, CO2CH3), 1.51 (s, 6H, CH3);
13C NMR (CDCl3, 100.6 MHz, δ): 174.0 (CO2Me), 170.0 (CO2Bzh),
150.8 (aromatic C), 140.0 (benzhydryl C), 136.7 (aromatic C), 130.1
(CH), 128.6 (benzhydryl CH), 128.0 (benzhydryl CH), 127.0
(benzhydryl CH), 122.7 (CH), 77.4 (benzhydrylic CH), 75.6
(CNN), 52.3 (CH3), 41.4 (CH2), 23.2 (CH3); ES+-MS 431 [M +
1]+, 453 [M + Na]+; HRMS ES+ TOF Calcd for C26H26N2O4Na:
453.1790. Found: 453.1808.
2-({4-[(tert-Butoxycarbonyl)methyl]phenyl}diazenyl)-2-
methylpropanoic Acid (5). Methyl ester 4a (121 mg, 0.38 mmol, 1
equiv) was stirred with 1 M LiOH aqueous solution (0.76 mL, 0.76
mmol, 2 equiv) in THF (5.8 mL) and deionized H2O (2.42 mL) for 1
day. The mixture was treated with 1 M HCl aqueous solution (24 mL)
to bring the pH to neutral or slightly acidic. The mixture was extracted
with EtOAc (20 mL), and solvent was removed by rotary evaporation.
1H NMR (CDCl3, 400 MHz, δ): 9.35 (br s, 1H), 7.62 (d, J = 8.40 Hz,
1
717, 703, 679, 638, 592, 571, 553, 512 cm−1; H NMR (CDCl3, 400
MHz, δ): 10.78 (s, 1H, OH), 8.26 (d, J = 2.48 Hz, 1H, CH), 7.85 (dd,
J1 = 8.93 Hz, J2 = 2.48 Hz, 1H, CH), 6.99 (d, J = 8.93 Hz, 1H, CH),
3.70 (s, 3H, CO2CH3), 1.52 (s, 6H, CH3); 13C NMR (CDCl3, 100.6
MHz, δ): 174.6 (COOH), 173.5 (CO2Me), 164.2 (aryl COH), 144.2
(aryl C), 129.6 (aryl CH), 127.0 (aryl CH), 118.5 (aryl CH), 111.3
(aryl C), 75.3 (CNN), 52.5 (CO2CH3), 23.3 (CH3); λ max
(CH3CN) ε315.2 nm: 2743 L mol−1 cm−1, ε395.6 nm: 193 L mol−1
cm−1; ES−-MS 265 [M − 1]+; HRMS ES+ TOF Calcd for
C12H14N2O5Na: 289.0800. Found: 289.0789.
Methyl 2-({4-[(tert-Butoxycarbonyl)methyl]phenyl}-
diazenyl)-2-methylpropanoate (4a). Carboxylic acid 3a (2.2 g,
8.3 mmol, 1 equiv) was dissolved in t-BuOH (25 mL) at 26 °C,
followed by di-tert-butyl dicarbonate (2.5 g, 11.6 mmol, 1.4 equiv) and
4-(dimethylamino)pyridine (0.1 g, 0.87 mmol, 0.1 equiv). The mixture
was stirred for 2 h and 21 min. The mixture was concentrated by
rotary evaporation, extracted with CH2Cl2, and washed with aqueous
HCl solution and H2O. The chlorinated extract was dried over
anhydrous MgSO4 powder and filtered, and solvent was removed by
rotary evaporation. The product crystallized upon standing after
evaporation of solvent. Product is a yellow solid (2.6 g, 8 mmol, 97%).
Crystal structure; mp 60 °C; IR (KBr disk) 3447, 2983, 2934, 1740,
1608, 1518, 1457, 1435, 1420, 1394, 1371, 1341, 1278, 1232, 1188,
2H, aryl CH), 7.32 (d, J = 8.40 Hz, 2H, aryl CH), 3.52 (s, 2H, benzylic
CH2), 1.51 (s, 6H, CH3), 1.37 (s, 9H, (CH3)3).
tert-Butyl {4-[2-(Propan-2-ylidene)hydrazinyl]phenyl}-
acetate (6). By decarboxylation of α-azo carboxylic acid 5. 1H
NMR (CDCl3, 400 MHz, δ): 7.05 (d, J = 8.5, 2H), 6.90 (d, J = 8.5 Hz,
2H), 3.34 (s, 2H), 1.95 (s, 3H), 1.78 (s, 3H), 1.34 (s, 9H); 13C NMR
(CDCl3, 100.6 MHz, δ): 170.5, 143.8, 143.1, 128.9, 124, 111.9, 79.4,
41.0, 27.0, 24.2, 14.6; HRMS ES− TOF Calcd for C15H21N2O2:
261.1603. Found: 261.1596.
1
1147, 1011, 986, 942, 882, 848, 799, 757, 694, 578, 511 cm−1; H
NMR (CDCl3, 400 MHz, δ): 7.58 (d, J = 8.40 Hz, 2H, CH), 7.29 (d, J
= 8.40 Hz, 2H, CH), 3.68 (s, 3H, CO2CH3), 3.50 (s, 2H, benzylic
CH2), 1.52 (s, 6H, CH3), 1.36 (s, 9H, C(CH3)3); 13C NMR (CDCl3,
100.6 MHz, δ): 174.0 (CO2Me), 170.4 (CO2t-Bu), 150.6 (aromatic
C), 137.7 (aromatic C), 129.8 (aromatic CH), 122.5 (aromatic CH),
81.1 (quaternary C(CH3)3), 75.5 (quaternary CNN), 52.2 (CH3),
42.56 (CH2), 28.0 (CH3), 23.2 (CH3); λmax (CH3CN) ε290.6 nm: 3801
L mol−1 cm−1, ε401.0 nm: 118 L mol−1 cm−1; Anal. Calcd for
C17H24N2O4: C, 63.73; H, 7.55; N, 8.74. Found: C, 63.77; H, 7.64; N,
8.42.
Diphenylmethyl (4-aminophenyl)acetate (7). 4-Aminophenyl-
acetic acid 1a (1.0 g, 6.6 mmol, 1 equiv) was dissolved in DMF (10
mL) and t-BuOH (7 mL). A solution of diphenyldiazomethane (∼15
mmol) in DMF (10 mL) was added, and the mixture was stirred at
ambient temperature for 19 h 5 min. The mixture was washed with
deionized H2O (3 × 40 mL) and extracted with CH2Cl2. The extract
was dried over anhydrous MgSO4 powder, and filtered, and solvent
was evaporated to leave a deep red-pink liquid. The crude mixture was
purified by column chromatography, using petroleum ether and
CH2Cl2 mixture mobile phase. The product was obtained as a pale
solid (0.9 g, 3 mmol, 45%). Rf 0.4−0.3 on silica. 1H NMR (CDCl3, 400
MHz, δ): 7.26−7.16 (overlapping ms, 10H, benzhydryl aryl CH), 6.99
(d, J = 8.36 Hz, 2H, aryl CH), 6.78 (s, 1H, benzhydrylic CH), 6.57 (d,
J = 8.40 Hz, 2H, aryl CH), 3.56 (br s, 2H, NH2), 3.54 (s, 2H, benzylic
CH2); 13C NMR (CDCl3, 100.6 MHz, δ): 171.1 (CO2Bzh), 145.4
(aryl C), 138.4 (benzhydryl C), 130.3 (aryl CH), 128.5 (CH), 127.8
(CH), 127.0 (CH), 123.8 (aryl C), 115 (aryl CH), 77 (benzhydrylic
CH), 40.8 (benzylic CH2); ES+-MS 340 [M + Na]+, 657 [2M + Na]+;
HRMS ES+ TOF Calcd for C21H19NO2Na: 340.1313. Found:
340.1317.
tert-Butyl 2-Hydroxy-5-{[2-(methoxycarbonyl)propan-2-yl]-
diazenyl}benzoate (4b, Table 1, entry III). Anhydrous MgSO4
powder (1.2 g, 10 mmol, 4 equiv) was stirred with concentrated
H2SO4 (0.14 mL, 1.3 mmol, 5 equiv) in CH2Cl2 for 15 min.
Carboxylic acid 3b (0.7 g, 2.6 mmol, 1 equiv) was added, followed by
t-BuOH (1.2 mL, 13 mmol, 5 equiv). A stopper was placed over the
neck of the flask, and the mixture was stirred for 17 h 20 min. The
reaction was quenched by stirring with saturated NaHCO3 aqueous
solution (19.3 mL). The separated aqueous phase was extracted with
CH2Cl2. The chlorinated phase was washed with brine (15 + 20 mL)
and H2O (2 × 20 mL), separated, dried over anhydrous MgSO4
powder, and filtered. The solvent was evaporated from the filtrate to
obtain a brown crystalline solid. Yield: 0.4 g, 1.4 mmol, 52%. Crystal
structure; mp 76 °C; IR (KBr disk) 3449, 3125, 2980, 2926, 2853,
1738, 1672, 1616, 1584, 1519, 1474, 1432, 1374, 1356, 1296, 1250,
1222, 1154, 1079, 1014, 992, 911, 847, 799, 758, 746, 719, 699, 569,
3-({4-[(Diphenylmethoxycarbonyl)methyl]phenyl}amino)-
2,2-dimethyl-3-oxopropanoic acid (8). Dimethylmalonic acid
(274 mg, 2.08 mmol, 2.2 equiv) and TFFH (550 mg, 2.08 mmol,
2.2 equiv) were dissolved in CH2Cl2 (2.9 mL) with N,N-
diisopropylethyl amine (0.7 mL, 4.158 mmol, 4.4 equiv) and added
to a solution of diphenylmethyl (4-aminophenyl)acetate 7 (300 mg,
0.945 mmol, 1.0 equiv) in CH2Cl2 (2 mL). The mixture was stirred for
19 h 20 min at ambient temperature. Solvent was evaporated. The
residue was purified by column chromatography through silica gel in n-
hexane, using n-hexane and dichloromethane mixtures as eluent. The
product has higher Rf on silica with dichloromethane mobile phase
than the starting material. Product was obtained as a yellow solid (57.7
1
544, 518 cm−1. H NMR (CDCl3, 400 MHz, δ): 11.35 (s, 1H, OH),
8.12 (d, J = 2.50 Hz, 1H, aryl CH), 7.74 (dd, J1 = 8.92 Hz, J2 = 2.50
Hz, 1H, aryl CH), 6.93 (d, J = 8.92 Hz, 1H, aryl CH), 3.69 (s, 3H,
CO2CH3), 1.57 (s, 9H, (CH3)3), 1.52 (s, 6H, CH3); 13C NMR
(CDCl3, 100.6 MHz, δ): 174.1 (CO2Me), 169.6 (CO2t-Bu), 164.1
(aryl COH), 144.1 (aryl C), 127.8 (aryl CH), 126.7 (aryl CH), 118.3
(aryl CH), 113.6 (aryl C), 83.7 (quaternary C, C(Me)3); 75.1 (CN
N), 52.3 (CO2CH3), 28.2 (CH3, C(CH3)3); 23.3 (CH3); λ max
(CH3CN) ε297.2 nm: 6266 L mol−1 cm−1, ε393.2 nm: 169 L mol−1
cm−1; HRMS ES− TOF Calcd for C16H21N2O5: 321.1450. Found:
321.1447.
Methyl 2-({4-[(Diphenylmethoxycarbonyl)methyl]phenyl}-
diazenyl)-2-methylpropanoate (4c, Table 1, Entry IV). Carbox-
ylic acid 3a (0.6 g, 2.16 mmol, 1 equiv) was placed in a 50-mL round-
bottom flask with deionized H2O (4 mL). Diphenyldiazomethane as a
solution in 1,4-dioxane (4 mL) was added, and the mixture of phases
1
mg, 0.13 mmol, 14%). H NMR (CDCl3, 400 MHz, δ): 7.72 (d, 2H,
aryl CH), 7.27−7.15 (overlapping ms, 12H, aryl CH), 6.79 (s, 1H,
benzhydrylic CH), 3.65 (s, 2H, benzylic CH2), 1.41 (s, 6H, CH3); 13
C
NMR (CDCl3, 100.6 MHz, δ): 207.0 (COOH), 172.8 (CONH),
170.0 (CO2Bzh), 139.9 (benzhydryl C), 132.4 (aryl C), 130.2 (aryl
CH), 128.5 (CH), 128.0 (CH), 127.0 (CH), 119.2 (aryl CH), 77.5
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(benzhydrylic CH), 61.8 (quaternary C), 41.2 (benzylic CH2), 17.8
(CH3) (one aryl C not assigned); ES−-MS 430 [M − 1]−, 863 [2M −
1]−; HRMS ES− TOF Calcd for C26H24NO5: 430.1654. Found:
430.1663.
MHz, δ): 7.49 (d, J = 8.32 Hz, 2H, aryl CH), 7.48 (m, 1H, NH), 7.30
(d, J = 8.32 Hz, 2H, aryl CH), 7.17−7.12 (overlapping ms, 3H, Phe
aryl CH), 7.04−6.99 (m, 2H, Phe aryl CH), 4.95 (dt, J1 = 7.96 Hz, J2 =
5.80 Hz, 1H, CH), 3.67(s, 3H, CO2CH3), 3.53 (s, 2H, benzylic CH2),
3.12 (ms, 2H, Phe benzylic CH2), 1.38 (s, 9H, (CH3)3), 1.34 (s, 3H,
CH3), 1.31 (s, 3H, CH3); 13C NMR (CDCl3, 100.6 MHz, δ): 174.4
(CONH), 171.9 (CO2Me), 170.3 (CO2t-Bu), 150.6 (aryl C), 138.1
(aryl C), 135.8 (aryl C), 130.0 (aryl CH), 129.3 (Phe aryl CH), 128.6
(Phe aryl CH), 127.1 (Phe aryl CH), 122.5 (aryl CH), 81.2 (CMe3),
74.2 (CNN), 52.8 (CH), 52.4 (CO2CH3), 42.5 (benzylic CH2),
37.9 (Phe benzylic CH2), 28.0 ((CH3)3), 23.4 (CH3), 23.1 (CH3);
ES+-MS 468 [M + 1]+, 490 [M + Na]+, 958 [2M + Na]+; HRMS ES+
TOF Calcd for C26H33N3O5Na: 490.2318. Found: 490.2314.
Nα-[α-({4-[(tert-Butoxycarbonyl)methyl]phenyl}diazenyl)-
isobutyryl]-Phe-OH (15). Methyl ester 14 (66 mg, 0.14 mmol, 1
equiv) was stirred with LiOH (6.2 mg, 0.26 mmol, 1.8 equiv) in H2O
(0.23 mL) and THF (0.54 mL) for 2 h. The mixture was acidified with
1 M HCl aqueous solution (1.14 mL) and extracted with CH2Cl2.
Solvent was evaporated under vacuum to obtain a yellow micro-
crystalline solid. Yield: 65 mg, 0.14 mmol, quantitative. Mp 101 °C; 1H
NMR (CDCl3, 400 MHz, δ): 9.04 (br s, 1H, COOH), 7.55 (d, J =
7.68 Hz, 1H, NH), 7.46 (d, J = 8.32 Hz, 2H, aryl CH), 7.29 (d, J =
8.32 Hz, 2H, aryl CH), 7.17−7.12 (overlapping ms, 3H, Phe aryl CH),
7.12−7.05 (m, 2H, Phe aryl CH), 4.95 (m, J1 = 7.60 Hz, J2 = 5.9 Hz,
1H, CH), 3.52 (s, 2H, benzylic CH2), 3.18 (ms, 2H, Phe benzylic
CH2), 1.38 (s, 9H, CO2C(CH3)3), 1.33 (s, 3H, CH3), 1.30 (s, 3H,
CH3); 13C NMR (CDCl3, 100.6 MHz, δ): 174.2 (CO), 173.8 (C
O), 169.4 (CO2t-Bu), 149.6 (aryl C), 137.1 (aryl C), 134.6 (aryl C),
128.9 (aryl CH), 128.4 (Phe aryl CH), 127.6 (Phe aryl CH), 126.2
(Phe aryl CH), 121.5 (aryl CH), 80.3 (CMe3), 73.0 (CNN), 51.9
(CH), 41.5 (benzylic CH2), 36.2 (Phe benzylic CH2), 27.0 ((CH3)3),
22.3 (CH3), 22.0 (CH3); ES−-MS 452 [M − 1]−, 906 [2M − 1]−;
HRMS ES+ TOF Calcd for C25H32N3O5: 454.2342. Found: 454.2352.
General Procedure for Peptide Synthesis. Solid-Phase
Synthesis. Peptides were synthesized using automated peptide
synthesizer using Fmoc Nα-protection strategy on a Rink amide
MBHA resin solid support (typical substitution: 0.7 mmol/g).
Protected peptides for solution phase were synthesized on a Sieber
amide resin.
Cleavage and Deprotection. Deprotection of amino acid residue
side groups and cleavage of peptide from resin was achieved by stirring
with a mixture of TFA (6 mL), deionized H2O (450 μL),
triisopropylsilane (450 μL), and thioanisole (900 μL). If peptide
contained tryptophan, methionine, or cysteine, 1,2-ethanedithiol (450
μL) was also added to the cleavage mixture. Stirring was allowed for 2
h plus an additional 30 min for each arginine residue up to a maximum
of 4 h. The resin was then removed by filtration over sintered glass or
frit. The peptide was precipitated and washed by the addition of Et2O
to the filtrate and collecting as a pellet by centrifugation at 2.8 × 1000
rpm. The pellet was dissolved in H2O, with CH3CN if necessary, and
lyophilized.
Purification. Crude peptides were purified by semipreparative
reverse-phase HPLC. Unless otherwise stated, purification employed a
Phenomenex Gemini 5 μm, C-18, 110 Å, 250 mm × 10 mm and
elution involved a 30 min gradient of H2O (0.1% TFA)/CH3CN
(0.1% TFA) 95:5−35:65, followed by isocratic elution.
Diphenylmethyl {4-[(2-Methylpropanoyl)amino]phenyl}-
acetate (9). Side product by decarboxylation of carboxylic acid 8,
1
isolated by column chromatography. H NMR (CDCl3, 400 MHz, δ):
7.39 (d, J = 8.40 Hz, 2H, aryl CH), 7.3−7.15 (overlapping ms, 10H,
benzhydryl aryl CH), 7.10 (d, J = 8.32 Hz, 2H, aryl CH), 6.77 (s, 1H,
benzhydrylic CH), 3.59 (s, 2H, benzylic CH2), 2.39 (septet, J = 6.80
Hz, 1H, CH), 1.13 (d, J = 6.80 Hz, 6H, CH3); 13C NMR (CDCl3,
100.6 MHz, δ): 175.2 (CONH), 170.5 (CO2Bzh), 140.1 (benzhydryl
C), 137.2 (aryl C), 129.9 (aryl CH), 128.5 (benzhydryl CH), 127.9
(benzhydryl CH), 127.0 (benzhydryl CH), 119.8 (aryl CH), 77
(benzhydrylic CH), 41.0 (benzylic CH2), 36.7 (CH), 19.6 (CH3);
ES−-MS 386 [M − 1]−; HRMS ES+ TOF Calcd for C25H25NO3Na:
410.1732. Found: 410.1738.
Methyl 2-(2-{4-[(tert-Butoxycarbonyl)methyl]phenyl}-
hydrazinyl)-2-methylpropanoate (10a). Azo 4a (1.2 g, 3.7
mmol, 1 equiv) was dissolved in EtOH (100 mL) heated to 60 °C.
NH2NH2 hydrate (20 mL) was added, and the solution was stirred for
2 h. The mixture was poured on ice and concentrated by rotary
evaporation until a white precipitate appeared. The white solid was
collected by filtration and washed with deionized H2O. The analytical
sample was obtained by recrystallization by dissolving in cold EtOH,
adding deionized H2O, and chilling to −5 °C. White flakes. Yield:
quantitative. Crystal structure; mp 79 °C; 1H NMR (CDCl3, 400
MHz, δ): 7.00 (d, J = 8.40 Hz, 2H, aryl CH), 6.78 (d, J = 8.40 Hz, 2H,
aryl CH), 5.45 (br s, 1H, NH), 4.01 (br s, 1H, NH), 3.69 (s, 3H,
CO2CH3), 3.34 (s, 2H, benzylic CH2), 1.36 (s, 9H, C(CH3)3), 1.27 (s,
6H, CH3); 13C NMR (CDCl3, 100.6 MHz, δ): 176.5 (CO2Me), 170.6
(CO2t-Bu), 148.1 (aryl C), 128.6 (aryl CH), 123.6 (aryl C), 111.8
(aryl CH), 79.4 (C(CH3)3), 61.3 (CNHNH), 51.2 (CO2CH3), 40.8
(benzylic CH2), 27.0 (C(CH3)3), 22.4 (CH3); λmax (CH3CN)
ε295.4 nm: 625 L mol−1 cm−1.
2-(2-{4-[(tert-Butoxycarbonyl)methyl]phenyl}hydrazinyl)-2-
methylpropanoic Acid (11). Methyl ester 10a (1 equiv) was stirred
with LiOH (2 equiv) in deoxygenated deionized H2O (20 mL per
gram of ester) and deoxygenated THF (46.7 mL per gram of ester) for
24 h. Reaction was quenched by 1 M HCl aqueous solution (11.7 mL
per gram of carboxylate) and extracted with deoxygenated CH2Cl2.
1
Solvent was evaporated to yield a pale solid. Yield: quantitative. H
NMR (CDCl3, 400 MHz, δ): 7.02 (d, J = 8.36 Hz, 2H), 6.88 (d, J =
8.32 Hz, 2H), 3.36 (s, 2H), 1.38 (s, 6H), 1.36 (s, 9H); 13C NMR
(CDCl3, 100.6 MHz, δ): 181.8, 171.8, 149.0, 129.72, 124.9, 113.0,
80.6, 62.1, 41.8, 28.1, 23.4.
tert-Butyl {4-[2-(1-Fluoro-2-methyl-1-oxopropan-2-yl)-
hydrazinyl]phenyl}acetate (12). Carboxylic acid 11 (205.4 mg,
0.67 mmol, 1 equiv) was dissolved in deoxygenated CH2Cl2 (2 mL)
and cooled to 0 °C. A solution of diethylaminosulfur trifluoride (0.09
mL) in deoxygenated CH2Cl2 (1 mL) was added dropwise. Reaction
was allowed under N2 gas for 1 h 10 min. The mixture was diluted
with CH2Cl2 and washed with H2O. The extract was dried over
anhydrous MgSO4 powder, filtered, and solvent was evaporated to
leave a dark-brown residue. 1H NMR (CDCl3, 400 MHz, δ): 7.01 (d, J
= 8.48 Hz, 2H), 6.8 (d, J = 8.52 Hz, 2H), 3.35 (s, 2H), 1.36 (s, 9H)
(methyl groups not assigned).
Analysis. Purity and retention time were assessed by reverse-phase
HPLC with Varian 5 μm, C-18, 110 Å, 4.6 mm × 250 mm analytical
column, with a 30 min elution gradient of H2O (0.1% TFA)/CH3CN
(0.1% TFA) 95:5−35:65, followed by isocratic elution. Identity of each
peptide was confirmed by MALDI-MS or ES±-MS. Absorbance
maxima were determined by photodiode array detector.
General Procedure for Preparation of Protected Peptide.
Protected peptide on Sieber amide resin was placed over a frit filter
and rinsed with a solution of 1% v/v TFA in CH2Cl2, with the filtrate
being collected in a 12% v/v solution of N,N-diisopropylethyl amine in
MeOH. The filtrate solvent was evaporated, and the remaining liquid
was diluted with CHCl3 and washed with deionized H2O. The solvent
was removed from the chlorinated extract by rotary evaporation and
tert-Butyl {4-[(1-Fluoro-2-methyl-1-oxopropan-2-yl)-
diazenyl]phenyl}acetate (13). Hydrazo 12 was allowed to oxidize
in air. 1H NMR (CDCl3, 400 MHz, δ): 7.61 (d, J = 8.36 Hz, 2H), 7.31
(d, J = 8.40 Hz, 2H), 3.51 (s, 2H), 1.61 (s, 6H), 1.36 (s, 9H).
Nα-[α-({4-[(tert-Butoxycarbonyl)methyl]phenyl}diazenyl)-
isobutyryl]-Phe-OMe (14). Acid fluoride 13 (crude, maximum 0.9
mmol, ∼1 equiv) and H-Phe-OMe·HCl (135 mg, 0.63 mmol, 1 equiv)
were treated with N,N-diisopropylethyl amine (0.22 mL, 1.26 mmol, 2
equiv) and dissolved in anhydrous DMF (0.5 mL). The mixture was
stirred for 21 h 44 min. The mixture was diluted with CH2Cl2 and
washed with 1 M HCl aqueous solution (3 × 10 mL) and H2O (2 ×
15 mL). The product was purified by column chromatography through
silica. Yield: 12.4 mg, 0.03 mmol, 29% from 4a. 1H NMR (CDCl3, 400
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Schlenk vacuum. The residual crude solid was used without
purification.
(14) Jain, A.; Gupta, Y.; Jain, S. K. Crit. Rev. Ther. Drug Carrier Syst.
2006, 23, 349.
Leu-Phe-Leu-Leu-Gly-Arg(Pbf)-Val-Leu-Ser(t-Bu)-Gly-Leu-Leu-
NH2. A sample was deprotected and analyzed. ES+-MS 626 [M + 2]2+.
General Procedure for Preparation of α-[4-(Carboxymethyl)-
phenyl]azo Peptides. Carboxylic acid 15 (1 equiv), N,N′-
dicyclohexylcarbodiimide (1 equiv), and 1-hydroxybenzotriazole (1
equiv) were stirred in anhydrous DMF (10.3 mL/mmol carboxylic
acid) under argon for 10 min. Protected peptide (1 equiv) was added,
and the mixture was stirred overnight for 1 day. The mixture was then
deprotected, purified, and analyzed.
(15) McVey, D. C.; Liddle, R. A.; Riggs-Sauthier, J.; Ekwuribe, N.;
Vigna, S. R. Dig. Dis. Sci. 2005, 50, 565.
(16) Kennedy, D. A.; Devocelle, M. In Breaking Away: Proceedings of
the 21st American Peptide Symposium; Lebl, M., Ed.; Prompt Scientific
Publishing: San Diego, CA, U.S.A., 2009; p 290.
(17) Kennedy, D. A. PhD Thesis, The Royal College of Surgeons in
Ireland, 2010.
(18) Devocelle, M.; Kennedy, D. J. Pept. Sci. 2010, 16, 148.
(19) Degenkolb, T.; Kirschbaum, J.; Bruckner, H. Chem. Biodiversity
̈
Nα-(α-[4-(Carboxymethyl)phenyl]diazenyl-isobutyryl)-temporin
(17). Yellow solid. Rt 31.39 min.; λmax 197 nm; ES+-MS 551 [M + 2 +
Na]3+, 557 [M + 2 + K]3+, 816 [M + 2]2+, 826 [M + 1 + Na]2+, 835
[M + 1 + K]2+; HRMS ES− TOF Calcd for C80H128N19O17:
1626.9736. Found: 1626.9747.
2007, 4, 1052.
(20) Duclohier, H. Chem. Biodiversity 2007, 4, 1023.
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1309.
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1994, 289.
ASSOCIATED CONTENT
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(27) Takeda, K.; Akiyama, A.; Nakamura, H.; Takizawa, S.-i.;
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■
S
* Supporting Information
Characterization spectra for compounds; crystallographic
information files for compounds 2a, 3a, 4a, 4b, and 10a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: +353 01 403 2247. E-mail: d_kennedy@hotmail.
co.uk.
(32) Kamin
Chelli, M.; Rovero, P.; Błaszczyk, M.; Głow
Am. Chem. Soc. 2005, 127, 16912.
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Am. Chem. Soc. 1990, 112, 9651.
́ ́
ski, Z. J.; Kolesin
ACKNOWLEDGMENTS
■
́
This publication has emanated from research conducted with
the financial support of Science Foundation Ireland (SFI
Programme Codes 06/RFP/CHO024 and 06/RFP/CHO024/
EC07). We thank Adam Coburn for all HRMS; Suzanne
Donnelly for combustion elemental analyses; and Celine Petit
for the ES+-MS of compound 17.
́
(35) Nguyen, H. H.; Imhof, D.; Kronen, M.; Schlegel, B.; Hartl, A.;
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Grafe, U.; Gera, L.; Reissmann, S. J. Med. Chem. 2002, 45, 2781.
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