Synthesis of a novel analytical reagent for the determination of active sites for conjugation on a catalytic aldolase monoclonal antibody

Article abstract of DOI:10.1016/j.tetlet.2012.01.016

An optimized and scalable synthesis of a novel analytical reagent for the determination of the number of active sites available for conjugation on a catalytic aldolase monoclonal antibody (mAb) is described. The original conditions suffered from lack of reproducibility, incomplete reactions, and required several chromatographies and lyophilizations that afforded material of low purity. A redesigned route and optimized protocols have been developed that eliminate the use of toxic and unsafe reagents such as HMPA and HATU. In addition, the number of chromatographies has been reduced to only one and time-consuming and energy-intensive lyophilizations are no longer required. The overall yield has been considerably improved from the original 4% to 20% after telescoping the last two steps of the synthesis and this new approach allowed for the preparation of material with higher chemical purity (≥99% vs the initial 90%) to meet specifications.

Full text of DOI:10.1016/j.tetlet.2012.01.016

Tetrahedron Letters 53 (2012) 1385–1389
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of a novel analytical reagent for the determination of active sites
for conjugation on a catalytic aldolase monoclonal antibody
Javier Magano a, , Douglas Farrand b, Jeffrey P. Haase a, Michael Lovdahl b, Mark T. Maloney a,
⇑
Mark J. Pozzo c, John J. Teixeira a, David C. Whritenour a, John Rizzo d, David Tumelty d, Abhijit Bhat d,
Curt Bradshaw e
a Chemical Research & Development, Pfizer Worldwide Research & Development, Eastern Point Road, Groton, CT 06340, USA
b Analytical Research & Development, Pfizer Worldwide Research & Development, Eastern Point Road, Groton, CT 06340, USA
c Biotherapeutics Pharmaceutical Sciences, Pfizer Worldwide Research & Development, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
d CovX Research, 9381 Judicial Drive, San Diego, CA 92121, USA
e Traversa Therapeutics, 10480 Wateridge Circle, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An optimized and scalable synthesis of a novel analytical reagent for the determination of the number of
active sites available for conjugation on a catalytic aldolase monoclonal antibody (mAb) is described. The
original conditions suffered from lack of reproducibility, incomplete reactions, and required several chro-
matographies and lyophilizations that afforded material of low purity. A redesigned route and optimized
protocols have been developed that eliminate the use of toxic and unsafe reagents such as HMPA and
HATU. In addition, the number of chromatographies has been reduced to only one and time-consuming
and energy-intensive lyophilizations are no longer required. The overall yield has been considerably
improved from the original 4% to 20% after telescoping the last two steps of the synthesis and this
new approach allowed for the preparation of material with higher chemical purity (P99% vs the initial
90%) to meet specifications.
Received 3 December 2011
Revised 4 January 2012
Accepted 5 January 2012
Available online 13 January 2012
Keywords:
Analytical reagent
Bioconjugation
Enaminone
Biopharmaceuticals
Ó 2012 Elsevier Ltd. All rights reserved.
O
O
Introduction
O
O
+
Me3N
Biotechnology-based pharmaceuticals, or biopharmaceuticals,
have seen a considerable expansion during the past decade.
‘Bioconjugation’, which refers to the covalent derivatization of bio-
molecules, can be employed to bring together two or more mole-
cules to form a complex that displays the combined properties of
each one of its individual components.1 This technique has been
applied to stabilize substances in blood, for the protection of sub-
stances prone to enzymatic degradation, and for decreasing the
immunogenicity of polypeptides, among others.2 An interesting
application is in drug delivery systems, such as in some oncology
treatments, where the drug is attached by means of a linker or
spacer to a carrier that can bind to its specific receptor contained
on the cancer cell. As a result, the therapeutic molecule can be di-
rected to the desired site of action.3 Both monoclonal antibodies
(mAbs)4 and synthetic polymers5 have been used as carriers.
In conjunction with a project to develop therapeutic complexes
that employ mAbs as carriers, our group undertook the preparation
of analytical reagent 1 on multi gram-scale (Fig. 1).
N
H
N
H
Cl−
1
Figure 1. Structure of analytical reagent 1.
The bioconjugation reaction in this case is mediated by an elec-
trophilic linker that is designed to react exclusively with a specific
lysine residue (Lys H93) in the Fab region of the carrier antibody.
The apparent pKa of Lys H93 is approximately 5 under equilibrium
condition, which is substantially lower than the typical value for
the lysine e-amino group. The bioconjugation reactions are per-
formed at pH 6.5 wherein all the surface lysines except for Lys
H93 are protonated and hence non-reactive and thus the bioconju-
gation reaction occurs specifically with the e-amino group of Lys
H93. The analytical reagent 1 is employed for the determination
of the number of active sites (Lys H93) available for conjugation
on a catalytic aldolase mAb and thus allows the monitoring of
the progress of the bioconjugation reaction. The reagent reacts
with Lys H93 on the mAb (2) to form an enaminone 3 that absorbs
at 316 nm (Scheme 1). The absorption can be measured by UV/vis
spectroscopy and normalized to protein concentration to quantify
⇑
Corresponding author. Tel.: +1 860 6869021; fax: +1 860 4413342.
E-mail address: Javier.Magano@Pfizer.com (J. Magano).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.01.016

1386
J. Magano et al. / Tetrahedron Letters 53 (2012) 1385–1389
required the use of highly toxic HMPA as cosolvent and chromatog-
raphy to isolate intermediate 6. The major impurity (up to 10%)
came from the dimerization of benzyl bromide 5 under the reaction
conditions.8 The diketal formation step provided the transient
monoketal quickly but further reaction to the diketal did not go
to completion, even upon adding a large excess of 1,2-bis(trimeth-
ylsiloxy)ethane (10 equiv). In addition, the bis-ketal easily hydro-
lyzed to the monoketal, which made its isolation difficult, and a
lower yield (30%) was obtained in our hands on several gram-scale.
When we attempted to prepare the diketal by the standard method
using ethylene glycol and p-TsOHꢁH2O in toluene under Dean–Stark
conditions, a complex mixture of products was obtained with very
little product present that could not be purified without additional
decomposition and deketalization. The nitro reduction and in situ
coupling of the resulting aniline with glutaric anhydride worked
well and we envisioned that little optimization would be required
to obtain satisfactory results. The subsequent amide bond forma-
tion between acid 10 and amine 11 employed expensive and
high-energy HATU as coupling reagent and diisopropylethylamine
(DIPEA) as base. Intermediate 12 was isolated as an aqueous solu-
tion and time-consuming and energy-intensive lyophilization was
subsequently needed for its isolation. The final ketal cleavage under
acidic conditions gave crude 1 in low chemical purity (ꢂ25% by
HPLC). This material was then subjected to extensive reverse-phase
chromatography that required low column loading followed by a
second lyophilization to obtain 1 in a 28% yield (2 steps) and 90%
purity by HPLC. The overall yield for this route was 4%.
In view of the challenges described above, it became clear that
either considerable optimization of the current route or an alterna-
tive synthetic approach would be required to allow for the prepa-
ration of gram-quantities of 1. In addition, a specification of P98%
chemical purity had to be met for future deliveries. As a result of
our synthetic efforts, a modified route to 1 was devised, which is
shown in Scheme 3.
The preparation of 1,3-diketone 6 was completely redesigned
and a two-step protocol was developed.9 Thus, commercially avail-
able 3-(4-nitrophenyl)propionic acid (13) was converted into acid
chloride 14 with oxalyl chloride and catalytic 4-(dimethyl-
amino)pyridine (DMAP) in CH2Cl2. In parallel, tert-butyl acetoace-
tate (15) was treated with lithium hexamethyldisilazide
(LiHMDS, 1 M in THF) and, to the resulting enolate, was added a
solution of crude 14 in 2-MeTHF to provide intermediate 16. Since
16 is an oil, it was carried through directly into the next step,
which involved tert-butyl ester cleavage followed by decarboxyl-
ation in toluene and catalytic p-TsOHꢁH2O at 65–80 °C.10 After an
aqueous workup, the resulting 1,3-diketone 6 was purified via
1:1 heptane/2-propanol slurry in a 58% yield from acid 13. Despite
the additional step compared to the direct coupling between 4 and
5, this was the route of choice due to the higher overall yield and
HN
O
O
1
+
NH
2
NH2
mAb
HN
O
O
NH
NH
O
O
O
+
NMe3
N
H
N
H
Cl−
3
Scheme 1. Enaminone 3 formation via reaction between reagent 1 and mAb 2.
the number of active sites available to link to therapeutically active
agents.
Results and discussion
The original synthetic route developed by the Medicinal Chem-
istry group is shown in Scheme 2.6 The synthesis started with the
reaction between the dianion of 2,4-pentanedione (4), generated
with lithium diisopropylamide (LDA, 2 equiv) at ꢀ78 °C in the
presence of hexamethylphosphoramide (HMPA), and 4-nitro-
benzylbromide (5) to provide intermediate 6 in low yield after
chromatography. Protection of the 1,3-diketone functionality as
the diketal with 1,2-bis(trimethylsiloxy)ethane (7) and catalytic
trimethylsilyl trifluoromethanesulfonate (TMSOTf) followed by
reduction of the nitro group with H2 and 10% Pd/C as catalyst in
the presence of glutaric anhydride (9) afforded acid 10 in a 55%
yield (two steps). Amide coupling between 10 and (2-aminoethyl)
trimethylammonium chloride hydrochloride (11) with O-7-(aza-
benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophos-
phate (HATU) as coupling reagent7 followed by lyophilization
generated amide 12. The diketal was cleaved with aqueous trifluo-
roacetic acid (TFA) and reverse phase chromatography followed by
lyophilization provided 1 in 28% for the last two steps combined.
This protocol was implemented to produce 350 mg of material.
When our group tried to reproduce this route with the goal of
delivering multi gram-quantities of material to meet the project’s
needs, several problems were identified that made it impractical
for further scale-up. The first step was low yielding (30–40%) and
H2, 10% Pd/C
OTMS
1. LDA (2 eq), −78 °C,
THF/HMPA
O
O
TMSO
O
O
O
OH
7
THF, rt
O
2.
TMSOTf, CH2Cl2
Br
9
O2N
−5 °C, 6 h
O2N
6
4
O
O
O
O
71%
5
8
O2N
77%
3. Chromatography
30-40%
+
1.
NMe3
Cl−
H2N
HCl
11
O
O
O
O
1. TFA, H2O, rt
O
O
O
O
+
1
O
O
Me3N
2. Reverse-phase
chromatography
3. Lyophilization
HATU, DIPEA,
DMF, rt, 1 h
N
N
H
HO
Cl−
O
N
H
H
O
2. Lyophilization
12
10
28% (2 steps)
Scheme 2. Medicinal Chemistry synthesis of 1.

J. Magano et al. / Tetrahedron Letters 53 (2012) 1385–1389
1387
O
O
(COCl)2, DMAP (cat.)
CH2Cl2, 0 °C to rt
OH
Cl
O2N
O
O2N
13
14
OH
O
O
O
1) LiHMDS (1 M in THF)
O
O
O
2-MeTHF, −20 °C
p-TsOH•H2O, PhMe
65−80 °C
58% (from 13)
O
2) 14, 0 °C to rt
O2N
O2N
O
15
6
16
Cl-
HCl
H2N
+
NH3
9
O
O
OH
17
O
O
O
HO
11
O
O
O
O
O
O
TsOH, 4 Å mol. sieves
CH3C(OCH3)3, reflux
O
T3P, Et3N,
CH3CN, 25 °C
44%
H2 (10 psi), 10% Pd/C
HO
N
H
O2N
THF 20−25 °C
8
70%
10
64%
1) 4 M HCl
2) 2-MeTHF extraction
3) NaHCO3 (solid)
O
O
O
O
+
1
O
4) 2-MeTHF extraction
5) Normal-phase
chromatography
68%
Me3N
N
N
H
O
Cl−
H
12
Scheme 3. Optimized route to 1.
the elimination of both cryogenic conditions and chromatographic
purification.
using anhydrous ethylene glycol gave a cleaner but incomplete
conversion to a mixture of mono- and diketals. Additional experi-
ments with different solvents and water scavengers led to a better
understanding and control of the process. The use of trimethylsilyl
chloride (TMSCl) as both the dehydrating agent and acid catalyst
gave mainly the monoketal. Adding an orthoester (trimethyl ortho-
formate) as water scavenger11 helped drive the reaction forward
and suppressed the retro-Aldol side-reaction, but molecular sieves
were still required for good conversion. Although the conversion
was slow in CH2Cl2 at reflux (days), it was significantly accelerated
in 2-MeTHF at reflux (hours) without impacting the purity profile.
The final conditions for the conversion of 6 to 8 involved the use
of ethylene glycol (8 equiv) in the presence of p-TsOHꢁH2O
(0.05 equiv), trimethyl orthoformate (0.1 equiv), and molecular
sieves in 2-MeTHF at reflux. The orthoester acts as an in situ dehy-
drating agent to prevent side-product formation and, in combina-
tion with refluxing over an addition funnel packed with activated
molecular sieves, provided greater than 95% conversion to product.
After workup, the product was crystallized from 2-propanol and
isolated in 65–70% yield and 96% purity by HPLC (area %).
The protection of diketone 6 as the ethylene glycol diketal was
surprisingly challenging (Scheme 4). Monoketal 18 formed quickly
while the second ketal formation required prolonged reaction
times. In addition, the diketal was sensitive to hydrolysis to the
monoketal even under neutral aqueous conditions. The starting
material was also sensitive to trace amounts of water and, under
acid-catalyzed conditions, a retro-Aldol reaction took place that
cleaved off the terminal acetyl group to produce compound 19. With
the goal of replacing 7 with inexpensive ethylene glycol (17), a
screen of conditions was carried out (Table 1). Thus, attempts to
drive the reaction by the removal of water as an azeotrope with tol-
uene gave a complex mixture of products. Removing water by
refluxing a THF or CH2Cl2 solution through activated sieves and
O
O
O
O
O
O
The nitro reduction via catalytic hydrogenation (10 psi) with 10%
Pd/C in the presence of glutaric anhydride (9) and THF as solvent to
afford acid 10 proved reproducible and required little optimiza-
tion.12 Upon reaction completion, the catalyst was filtered off and
the solvent was removed under vacuum. The purity of the resulting
crude material was upgraded via slurry in 9:1 MTBE/n-BuOH to pro-
vide 10 in 60–70% yield and >95% purity by HPLC (area %).
O2N
O2N
8
O
O
O
O2N
6
18
The amide bond formation step to generate 12 was originally
performed in the presence of HATU in either MeCN or DMF. How-
ever, the reactions were not clean and due to the cost and safety is-
sues associated with this reagent, an alternative was investigated.
Thus, 1,10-carbonyldiimidazole (CDI) afforded the corresponding
glutarimide as a consequence of competing intramolecular cycliza-
tion, which suggested that activation of the carboxylic acid step
prior to addition of the diamine salt was not feasible. On the other
O
O
O2N
19
Scheme 4. Ketal-formation step products

1388
J. Magano et al. / Tetrahedron Letters 53 (2012) 1385–1389
Table 1
Screen of conditions for the preparation of diketal 8
Reagent
Catalyst
Solvent
Product
1,2-Bis(trimethylsiloxy)ethane
Ethylene glycol
Ethylene glycol, sieves
Ethylene glycol
Ethylene glycol, sieves
Ethylene glycol, sieves, HC(OCH3)3
TMSOTf
p-TsOH
p-TsOH
TMSCl
p-TsOH
p-TsOH
CH2Cl2
Toluene
CH2Cl2
CH2Cl2
THF
8 (30%); lower yield on scale
Complex mixture of products
8 (60%) + 18 (20%)
18, traces of 8
19, traces of 8 and 18
8 (>95%) + 18 (3%)
2-MeTHF
1.
+
NMe3
Cl−
H2N
11
O
O
T3P, Et3N, CH3CN, rt
O
O
+
O
10
Me3N
2. NaHCO3 (solid)
3. Filter and concentrate
N
H
N
H
O
X−
12a: X− = Cl−
12b: X− = P,P',P''-tripropyltriphosphonic acid bis-anion
1. 4 N HCl, rt, 4 h
2. 2-MeTHF extraction
3. NaHCO3 (solid) to pH = 7
1
4. 2-MeTHF extraction
5. Chromatography
75% (2 steps)
Scheme 5. Telescoped process for the preparation of 1.
hand, when propanephosphonic anhydride (T3P, 50% in EtOAc)7
was evaluated, a clean reaction was observed at ambient tempera-
ture. The operational simplicity associated with the use of this re-
agent (addition to a mixture of the acid, amine, and triethylamine
in MeCN at rt) and the mild reaction conditions were the factors
that contributed to its selection for the preparation of amide 12.
Even though in early experiments we employed crude 12 in the fi-
nal ketal-cleavage step, the difficulty to bring 1 to acceptable purity
levels forced us to look at ways to purify this intermediate. Because
of the unusual properties of this molecule due to its ionic nature, we
had to develop a chromatographic protocol that employed an
ammonium chloride solution in methanol (10 g/L) as mobile phase.
After concentration of the fractions containing desired product, the
residue was slurried in MeCN and the inorganic salts were filtered
off. The filtrates were concentrated and amide 12 was isolated from
a 1:1 heptane/MTBE mixture in a 44% yield. This product was a mix-
ture of chloride and P,P0,P00-tripropyltriphosphonic acid salts 12a/
12b (Scheme 5) from the T3P reagent by 1H NMR and it also con-
tained residual TEA salts.
In the initial experiments to cleave the diketal to prepare 1 with
aqueous HCl in MeCN or EtOH, some conversion back to the
monoketal and partial decomposition was observed when the
acidic reaction mixture was concentrated under vacuum even un-
der freeze-drying conditions. In addition, slow conversion to 1 was
observed (24–48 h). However, it was found that hydrolysis in ace-
tone was significantly faster (30–60 min) and that the conversion
back to the mono-ketal could be prevented by neutralizing the
mixture with solid sodium bicarbonate upon reaction completion.
The neutral solutions were stable at 50 °C while being concen-
trated under vacuum even as completely aqueous solutions. After
concentration of the reaction mixture, crude 1 was purified by nor-
mal phase chromatography (MeCN/MeOH, gradient elution), which
was able to separate the two different salts of the final product. The
chloride salt came off between 20–40% methanol and the propyl-
phosphonate between 60–80% methanol.
process was developed (Scheme 5). After performing the amide
coupling with T3P as described above, solid NaHCO3 was added
to the reaction to neutralize the triethylamine salts and the mix-
ture was filtered, concentrated, and directly treated with 4 N HCl.
The ketal deprotection was complete within 4 h at ambient tem-
perature and an extraction with 2-MeTHF effectively removed
the P,P0,P00-tripropyltriphosphonic acid byproduct. The addition of
solid NaHCO3 neutralized any residual triethylamine salts and an
additional extraction with 2-MeTHF removed the basic impurities.
After concentration, the residue was triturated in MeCN to remove
inorganic salts and the resulting crude was chromatographed to
provide 1 as the chloride salt exclusively in a 75% yield for the
two steps combined and 99.1% chemical purity on 10 g-scale.
Conclusion
In conclusion, we have developed an optimized protocol for the
preparation of quaternary salt 1 on multi gram-scale.13 The unu-
sual properties of this material due to its ionic nature and water
solubility required the development of efficient purification proto-
cols. The outcomes of this work are: (a) elimination of unsafe re-
agents, one chromatography, and two lyophilizations; (b) an
overall yield that has been considerably improved from the origi-
nal 4% to 20% after telescoping the last two steps of the synthesis;
and (c) a new approach that allowed for the preparation of material
on multi gram-scale with higher chemical purity (P99% vs the ini-
tial 90%) to meet stringent specifications.
Acknowledgment
The authors thank Ronald Morris for collecting high-resolution
mass spectrometry data.
References and notes
1. Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Academic Press: San Diego,
CA, 2008.
2. Veronese, F. M.; Morpurgo, M. Farmaco 1999, 54, 497.
With the goal of simplifying the experimental and purification
protocols for the last two steps of the synthesis, a telescoped

J. Magano et al. / Tetrahedron Letters 53 (2012) 1385–1389
1389
3. Le Sann, C. Nat. Prod. Rev. 2006, 23, 357.
3H), 3.21 (s, 9H), 3.49 (t, J = 6.53 Hz, 2H), 3.67 (t, J = 6.40 Hz, 2H), 5.51 (s, 1H),
7.17 (m, J = 8.28 Hz, 2H), 7.49 (m, J = 8.03 Hz, 2H). 13C NMR (100 MHz, CD3OD)
d ppm 22.75, 24.78, 29.79, 30.96, 31.98, 34.67, 36.10, 37.02, 45.90, 121.48,
129.74, 137.96, 138.03, 138.17, 173.55, 175.89, 192.33, 194.61. ESI-HRMS calcd
for C22H34N3O4 (M+H)+ 404.25438, found 404.25436.
4. (a) Hermentin, P.; Seiler, F. R. Behing Inst. Mitt. 1988, 197; (b) Hermentin, P.;
Doenges, R.; Gronski, P.; Bosslet, K.; Kraemer, H. P.; Hoffmann, D.; Zilg, H.;
Streinstraesser, A.; Schwartz, A.; Kuhlmann, L.; Lüben, G.; Seuler, F. R.
Bioconjugate Chem. 1990, 1, 100.
5. (a) Pang, Y.; Liu, J.; Wu, J.; Li, G.; Wang, R.; Su, Y.; He, P.; Zhu, X.; Yan, D.; Zhu, B.
Bioconjugate Chem. 2010, 21, 2093; (b) Voit, B. I.; Lederer, A. Chem. Rev. 2009,
109, 5924; (c) Carlmark, A.; Hawker, C.; Hult, A.; Malkoch, M. Chem. Soc. Rev.
2009, 38, 352; (d) Saha, A.; Ramakrishnan, S. Macromolecules 2008, 41, 5658; (e)
Gao, C.; Yan, D. Y. Prog. Polym. Sci. 2004, 29, 183; (f) Tomalia, D. A.; Fréchet, J. M.
J. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2719; (g) Bosman, A. W.; Janssen,
H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 1665.
6. The first three steps have been previously disclosed. See: (a) Doppalapudi, V. R.;
Tryder, N.; Li, L.; Aja, T.; Griffith, D.; Liao, F.; Roxas, G.; Ramprasad, M. P.;
Bradshaw, C.; Barbas, C. F., III Bioorg. Med. Chem. Lett. 2007, 17, 501; (b)
Bradshaw, C.; Doppalapudi, V. R. WO 2006/094269 A2. Chem. Abstr. 145,
315273.
Compound 6: pale yellow solid. Mp: 49–51 °C. 1H NMR (400 MHz, CDCl3) d ppm
2.04 (s, 3H), 2.65 (t, J = 7.65 Hz, 2H), 3.06 (t, J = 7.53 Hz, 2H), 5.48 (s, 1H), 7.36
(m, J = 8.53 Hz, 2H), 8.14 (m, J = 8.53 Hz, 2H), 15.36 (br s, 1H). 13C NMR
(100 MHz, CDCl3) d ppm 24.53, 30.96, 39.23, 100.10, 123.74, 129.22, 146.60,
148.57, 190.35, 192.77. ESI-HRMS calcd for C12H13NO4 (M+H)+ 236.09173,
found 236.09182.
Compound 8: White solid. Mp: 83–85 °C. 1H NMR (400 MHz, CDCl3) d ppm 1.44
(s, 3H), 2.05 (s, 2H), 2.08–2.18 (m, 2H), 2.76–2.93 (m, 2H), 3.87–4.15 (m, 9H),
7.37 (m, J = 8.28 Hz, 2H), 8.14 (m, J = 8.28 Hz, 2H). 13C NMR (100 MHz, CDCl3) d
ppm 24.58, 30.03, 38.95, 44.56, 64.32, 64.71, 108.59, 109.49, 123.62, 129.22,
146.26, 150.70. MS (APCI+): m/z 324 (M+H). Anal. Calcd for C16H21NO6: C,
59.43; N, 4.33; H, 6.55. Found: C, 59.48; N, 4.14; H, 6.59.
7. (a) El-Faham, A.; Albericio, F. Chem. Rev. 2011, 111, 6557; (b) Montalbetti, C. A.
G. N.; Falque, V. Tetrahedron 2005, 61, 10827.
Compound 10: white solid. Mp: 118–120 °C. 1H NMR (400 MHz, DMSO-d6) d
ppm 1.34 (s, 3H), 1.80 (quin, J = 7.65 Hz, 2H), 1.89–1.98 (m, 4H), 2.27 (t,
J = 7.40 Hz, 2H), 2.33 (t, J = 7.40 Hz, 2H), 2.49–2.60 (m, 3H), 7.09 (m, 2H), 7.48
(m, 2H), 9.80 (s, 1H), 12.08 (br s, 1H). 13C NMR (100 MHz, DMSO-d6) d ppm
20.47, 24.41, 28.93, 32.98, 35.32, 39.19, 43.74, 63.69, 64.01, 107.91, 108.95,
119.19, 128.25, 136.86, 136.93, 170.43, 174.14. ESI-HRMS calcd for C21H29NO7
(M+H)+ 408.20168, found 408.20177.
8. The formation of this dimeric impurity presumably proceeds via the formation
of a benzylic anion as a result of the reaction between the enolate of 4 and a
molecule of benzyl bromide 5. This anion is stabilized through conjugation
with the nitro group and can further react with a second molecule of 5 to afford
the dimer.
9. Minter, D. E.; Winslow, C. D. J. Org. Chem. 2004, 69, 1603.
Compound 12a: Amorphous solid. 1H NMR (400 MHz, CD3OD) d ppm 1.41 (s,
3H), 1.95–2.08 (m, 6H), 2.35 (t, J = 7.40 Hz, 2H), 2.44 (t, J = 7.28 Hz, 2H), 2.61–
2.70 (m, 2H), 3.20 (s, 9H), 3.45–3.54 (m, 2H), 3.63–3.72 (m, 2H), 3.94 (s, 4H),
3.95–4.01 (m, 4H), 7.15 (m, J = 8.41 Hz, 2H), 7.47 (m, J = 8.53 Hz, 2H).13C NMR
(100 MHz, CD3OD) d ppm 22.76, 25.13, 30.64, 34.67, 36.11, 37.01, 40.99, 45.28,
54.06, 65.36, 65.68, 65.86, 65.89, 65.92, 109.92, 110.93, 121.55, 129.67, 137.55,
139.92, 173.52, 175.88. ESI-HRMS calcd for C26H42N3O6 (M+H)+ 492.30681,
found 492.30660.
10. (a) Witschel, M. Bioorg. Med. Chem. 2009, 17, 4221; (b) Yoakim, C.; Deroy, P.;
Duplessis, M.; Gagnon, A.; Goulet, S.; Huecke, O.; Lemke, C.; Surprenant, S. WO
2008/067644 A1. Chem. Abstr. 149, 53990.; (c) Gahman, T. C.; Lang, H.; Herbert,
M. R.; Thayer, A. M.; Hassig, C. A.; Noble, S. A.; Cousins, R. D.; Zhuang, H.;
Santos, C. R.; Chen, X. U.S. Patent 20060116515 A1. Chem. Abstr. 145, 8185.
11. Caserio, F. F., Jr.; Roberts, J. D. J. Am. Chem. Soc. 1958, 80, 5837.
12. (a) Bensel, N.; Reymond, M. T.; Reymond, J.-L. Chem. Eur. J. 2001, 7, 4604; (b)
Clifford, T.; Boswell, C. A.; Biddlecombe, G. B.; Lewis, J. S.; Brechbiel, M. W. J.
Med. Chem. 2006, 49, 4297.
Compound 16: oil. 1H NMR (400 MHz, CDCl3) d ppm 1.38 (s, 9H), 2.17 (s, 3H),
2.91 (dq, J = 11.67, 5.90 Hz, 4H), 7.23 (m, J = 8.53 Hz, 2H), 8.00 (m, J = 8.28 Hz,
2H). 13C NMR (100 MHz, CDCl3) d ppm 25.19, 28.21, 30.30, 30.91, 38.58, 81.81,
110.41, 123.73, 129.22, 146.59, 148.70, 166.26, 194.13, 196.71. ESI-HRMS calcd
for C17H25N2O6 (ammonium adduct, M+H)+ 353.17071, found 353.17086.
13. Analytical data for compounds 1, 6, 8, 10, 12a, and 16: Compound 1: Amorphous
solid. 1H NMR (400 MHz, CD3OD) d ppm 1.95–2.07 (m, 4H), 2.18 (s, 1H), 2.34 (t,
J = 7.53 Hz, 2H), 2.44 (t, J = 7.28 Hz, 2H), 2.60 (t, J = 7.65 Hz, 1H), 2.82–2.93 (m,