A scalable synthesis of MN-447, an antagonist for integrins αvβ3 and αIIbβ 3

Article abstract of DOI:10.1021/op800073z

(2S)-Benzeneslfonylamino-3-[3-methoxy-4-{4-(1,4,5,6-tetrahydropyrimidin-2- ylamino)piperidin-1-yl}benzoylamino]propionic acid, MN-447, is a potent antagonist of the integrins α_vβ₃3 and α_IIbβ₃ Herein, we report a novel synthetic protocol that produces MN-447 in an overall yield of 45%. This protocol, when compared with the original synthetic route for MN-447, is more cost-effective, requires fewer steps, does not require chromatographic purification of intermediates and MN-447, and increases the overall yield by 35%. This report focuses on the synthetic strategies that were developed for this protocol. Now, the large quantities of MN-447 that are needed for preclinical and toxicological studies can be readily obtained.

Full text of DOI:10.1021/op800073z

Organic Process Research & Development 2008, 12, 596–602
A Scalable Synthesis of MN-447, an Antagonist for Integrins rvꢀ3 and rIIbꢀ3
Minoru Ishikawa,* Masaki Tsushima, Dai Kubota, Yumiko Yanagisawa, Yukiko Hiraiwa, Yasuo Kojima, Keiichi Ajito, and
Naomichi Anzai
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
Abstract:
(2S)-Benzenesulfonylamino-3-[3-methoxy-4-{4-(1,4,5,6-tetrahydro-
pyrimidin-2-ylamino)piperidin-1-yl}benzoylamino]propionic acid,
MN-447, is a potent antagonist of the integrins rvꢀ3 and rIIbꢀ3.
Herein, we report a novel synthetic protocol that produces MN-
Figure 1. MN-447 / CP4715 (1).
447 in an overall yield of 45%. This protocol, when compared
with the original synthetic route for MN-447, is more cost-effective,
requires fewer steps, does not require chromatographic purifica-
tion of intermediates and MN-447, and increases the overall yield
by 35%. This report focuses on the synthetic strategies that were
developed for this protocol. Now, the large quantities of MN-447
that are needed for preclinical and toxicological studies can be
readily obtained.
methoxy-4-{4-(1,4,5,6-tetrahydropyrimidin-2-ylamino)piperidin-
1-yl}benzoylamino]propionic acid (1), named at the time,
CP4715 (Figure 1).7 The compound is a strong antagonist of
both integrins Rvꢀ3 and RIIbꢀ3. Its water solubility and prelimi-
nary ADMET profiles suggest that it might be an infusible drug
candidate. In a canine ischemia-reperfusion model, 1 showed
superior cardioprotective efficacy, e.g., reduction in infarct size
after reperfusion, and also showed a low risk of bleeding.10 In
2006, 1 was licensed to MediciNova, Inc., which is a develop-
ment-stage biopharmaceutical company focusing on acquiring,
Introduction
The vitronectin receptor, integrin Rvꢀ3, mediates the adhesion
developing, and commercializing high-quality small-molecule
and migration of vascular smooth muscle cells and leukocytes.1
therapeutics.11 CP4715, renamed MN-447, is currently in
Another member of the integrin family, integrin RIIbꢀ3, functions
at the convergence point for the various pathways that lead to
preclinical development.
The original medicinal chemistry-based route7 of 1 is
platelet aggregation because, as a platelet surface receptor, it
outlined in Scheme 1. Commercially available fluorobenzoic
binds fibrinogen.2 Antagonists that block integrin RIIbꢀ3-
fibrinogen binding prevent platelet aggregation; therefore,
integrin RIIbꢀ3 antagonists3,4 are used to treat coronary throm-
bosis. The Fab fragment of a human-murine monoclonal
antibody, Abciximab,5 which binds to both integrins Rvꢀ3 and
acid 2 is simultaneously methylated at the hydroxyl and
carboxyl groups to give the 4-fluorobenzoate derivative 3.
Nucleophilic substitution of 3 with 4-hydroxypiperidine gives
the bicyclic alcohol 4 that is then converted after three
stepssone of which involves reaction with sodium azide and
RIIbꢀ3, is used to treat ischemic diseases. Small, nonproteina-
produces an azide intermediatesto the amine 5. Introduction
ceous, water-soluble molecules that are inexpensive to synthe-
of a pyrimidine moiety gives 6. Base hydrolysis of 6 gives the
size and act as antagonists of integrins Rvꢀ3 and RIIbꢀ3 would
carboxylic acid 7, that is then coupled with the diaminopropi-
likely be of therapeutic value in the treatment of acute ischemic
onate 812 to afford the amide 9. Finally, removal of the tert-
diseases. During the course of our efforts to develop such
butyl group with TFA followed by hydrogenation of the
antagonists,6–9 we discovered (2S)-benzenesulfonylamino-3-[3-
pyrimidine ring gives the desired molecule 1 as a noncrystalline
solid. The synthesis outlined above is ill-suited for large-scale
* Corresponding author. Telephone: +81-45-541-2521. Fax: +81-45-541-
production of 1. The overall yield of 1 (10.5% yield13) and
0667. E-mail: minoru_ishikawa@meiji.co.jp.
(1) Byzova, T. V.; Rabbani, R.; Douza, S. E.; Plow, E. F. Thrombosis
the need for expensive chemicals prohibitively increase the
and Haemostasis 1998, 80, 726.
production cost. The products of steps a, b, f, h, and j must be
purified chromatographically. Additionally, hazardous azides
are involved. As an alternative, we have developed and report
(2) Philips, D. R.; Charo, I. F.; Parise, L. V.; Fitzgerald, L. A. Blood
1988, 71, 831.
(3) Egbertson, M. S.; Chang, C. T.-C.; Duggan, M. E.; Gould, R. J.;
Halczenko, W.; Hartman, G. D.; Laswell, W. L.; Lynch, J. J., Jr.;
Lynch, R. J.; Manno, P. D.; Naylor, A. M.; Prugh, J. D.; Ramjit, D. R.;
Sitko, G. R.; Smith, R. S.; Turchi, L. M.; Zhang, G. J. Med. Chem.
1994, 37, 2537.
(9) Kubota, D.; Ishikawa, M.; Ishikawa, M.; Yahata, N.; Murakami, S.;
Fujishima, K.; Kitakaze, M.; Ajito, K. Bioorg. Med. Chem. 2006, 14,
4158.
(4) Goa, K. L.; Noble, S. Drugs 1999, 57, 439.
(5) Tam, S. H.; Sassoli, P. M.; Jordan, R. E.; Nakada, M. T. Circulation
1998, 98, 1085.
(10) (a) Sakuma, T.; Sari, I.; Goodman, C. N.; Lindner, J. R.; Klibanov,
A. L; Kaul, S. CardioVasc. Res. 2005, 66, 552. (b) Kaul, S. Basic
Res. Cardiol. 2006, 101, 391.
(6) Kubota, D.; Ishikawa, M.; Yamamoto, M.; Murakami, S.; Hachisu,
M.; Katano, K.; Ajito, K. Bioorg. Med. Chem. 2006, 14, 2089.
(7) Ishikawa, M.; Kubota, D.; Yamamoto, M.; Kuroda, C.; Iguchi, M.;
Koyanagi, A.; Murakami, S.; Ajito, K. Bioorg. Med. Chem. 2006, 14,
2109.
(11) http://www.medicinova.com/.
(12) Askew, B. C.; Bednar, R. A.; Bednar, B.; Claremon, D. A.; Cook,
J. J.; McIntyre, C. J.; Hunt, C. A.; Gould, R. J.; Lynch, R. J.; Lynch,
J. J., Jr.; Gaul, S. L.; Stranieri, M. T.; Sitko, G. R.; Holahan, M. A.;
Glass, J. D.; Hamill, T.; Gorham, L. M.; Prueksaritanont, T.; Baldwin,
J. J.; Hartman, G. D. J. Med. Chem. 1997, 40, 1779.
(13) The yields are uncorrected for purity.
(8) Ishikawa, M.; Hiraiwa, Y.; Kubota, D.; Tsushima, M.; Watanabe, T.;
Murakami, S.; Ouchi, S.; Ajito, K. Bioorg. Med. Chem. 2006, 14,
2131.
596
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Vol. 12, No. 4, 2008 / Organic Process Research & Development
10.1021/op800073z CCC: $40.75
2008 American Chemical Society
Published on Web 06/21/2008

Scheme 1. Medicinal chemistry route of 1a
Scheme 3. Second generation route for 1a
a Reagents: (a) MeI, K2CO3, DMF, rt, 17 h, chromatography, 82%;13 (b)
4-hydroxypiperidine, DMSO, 90 °C, 13 h, chromatography, 47%;13 (c) MsCl,
Et3N, CH2Cl2 rt, 15 min; (d) NaN3, DMF, 90 °C, 4.5 h; (e) Pd/C, H2, 1,4-dioxane,
rt, 6 h; (f) 2-bromopyrimidine, i-Pr2EtN, DMSO, 120 °C, 15 h, chromatography,
64%13 in 4 steps; (g) NaOH, THF, MeOH, H2O, 40 °C, 5 h, 68%;13 (h) EDC,
HOBt, N-methylmorpholine, DMF, rt, 12 h, chromatography, 100%;13 (i) TFA,
CH2Cl2, rt, 16 h; (j) 10% Pd/C, H2, 1,4-dioxane, H2O, rt, 5 h, chromatography,
63%13 in 2 steps.
a Reagents: (a) MeI, K2CO3, DMF, rt, 95%; (b) 10% Pd/C, H2, 1,4-dioxane,
MeOH, H2O, 35 °C, 96%; (c) TsOH H2O, MeOH 60°C; (d) HCO2H, H2O, rt,
2 h, chromatography, 58%13 in 2 steps; (e) NaBH4, MeOH, rt, chromatography,
95%.13
are less expensive than are the corresponding fluoro derivatives.
Therefore, the inexpensive 3-hydroxy-4-nitrobenzoic acid (12)
(289 yen/g) was selected as a starting material.
Scheme 2. Retrosynthesis of 4
A second generation route for the synthesis of 1, is shown
in Scheme 3. Introduction of two methyl groups into 12 gave
methyl 3-methoxy-4-nitrobenzoate (13) as a pale yellow solid
in 95% yield at 98.3% purity. Hydrogenation of 13 gave the
aniline 11 as a pale yellow solid in 96% yield at 100% purity.
The reaction time for the hydrogenation was shorten from 20 h
to 8-12 h by increasing the reaction temperature from room
temperature to 35 °C. We isolated 13 and 11 as solids, without
chromatographic purification, by simply pouring the product
solutions into water and filtering.
We then used the piperidine ring-closure method to synthe-
size the ketone 15. By following this method,15 reaction of 11
with 10 and Na2CO3 gave 15 in 25% yield.13 While attempting
to improve the yield, we identified another condition that
incorporated TsOH16 gave the acetal 14. The cleavage of the
acetal16 afforded 15 in 58%13 (two steps). Reducing the carbonyl
group of 15 with NaBH4 gave 4, which is the product of the
second step of the original route. While further investigating
the piperidine ring-closure, we found that the addition of TsOH
was unnecessary and that during the workup 14 converted
spontaneously to 15 when the water solution containing 14 was
concentrated. Consequently, the workup of 14saddition of
water, filtration, partial concentration by evaporation, neutraliza-
tion, and filtrationsproduced 15 directly. Thus, starting with
0.95 kg of 12, 1 kg of 15 was synthesized in 76.6% yield at
97.2% purity and isolated without any chromatographic puri-
fications (Scheme 4).
herein a more efficient means for the synthesis of 1 that avoids
the aforementioned shortcomings of the original protocol.
Results and Discussion
We first developed an alternative route for the synthesis of
4 that circumvents the nucleophilic substitution reaction, thereby
eliminating the expensive compound 2 (14,800 yen/g), and the
chromatographic separation of the complex product mixture.
A retrosynthesis of 4 is outlined in Scheme 2. Reactions of
anilines with 1,5-dichloro-3-pentanone14 (10) produce piperidine
rings.15,16 We thought that such a reaction might be a suitable
step during the large-scale production of 1 because, generally,
anilines (e.g., 11) and their precursor nitro-derivatives (e.g., 12)
(14) Owen, G. R.; Reese, C. B. J. Chem. Soc. 1970, 2401.
(15) Bowden, K.; Green, P. N. J. Chem. Soc. 1952, 1164.
(16) Faja, M.; Reese, C. B.; Song, Q.; Zhang, P.-Z. J. Chem. Soc., Perkin
Trans. 1 1997, 191.
Subsequent attempts to decrease the number of steps required
to synthesize 1 focused on the synthesis of 5 from 15. We first
tried a reductive amination of 15 with benzylamine. The reaction
Vol. 12, No. 4, 2008 / Organic Process Research & Development
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597

Scheme 4. Practical route for 5a
a Reagents: (a) 10, MeOH, 60 °C, 84%; (b) BnNH2, NaBH(OAc)3, MeOH, 4 °C, 97%;13 (c) 10% Pd/C, H2, MeOH, rt, 100%;13 (d) 10% Pd/C, H2, conc. NH4OH,
1,4-dioxane, H2O, rt, 90%.
Scheme 5. Optimized synthetic procedure for scalable synthesis of 1a
a Reagents: (a) MeI, K2CO3, DMF, rt, 22 h, 95%; (b) 10% Pd/C, H2, 1,4-dioxane, MeOH, H2O, 35 °C, 12 h, 96%; (c) 10, MeOH, 60 °C, 4 h, 84%; (d) 10% Pd/C,
H2, conc. NH4OH, 1,4-dioxane, H2O, rt, 12 h, 90%; (e) 2-chloropyrimidine, NaHCO3, n-BuOH, 100 °C, 27 h; (f) NaOH, MeOH, H2O, 50 °C, 2 h, 80% in 2 steps; (g)
8, BOP, HOBt, N-methylmorpholine, DMF, 4 °C, 23 h, 96%; (h) i) 0.5 M HCl, 60 °C, 5.5 h, ii) 10% Pd/C, H2, 0.25 M HCl, 30 °C, 7.5 h, 84%.
proceeded smoothly to give 16. The benzyl group was removed
by hydrogenation to give 5. Thus, 5 was synthesized from 15
in two steps and in 97% yield.13 Additionally, the number of
steps required to synthesize 5 from 15 was reduced by two.
We also considered that an even shorter route might be
possible if 15 could be converted to 5 directly by a reductive
amination. Therefore, reactions using various ammonia sources
(NH4OAc, NH4CO2H, (NH4)2SO4, (NH4)2CO3, NH2OH hy-
drochloride, or NH4OH) and various reducing reagents (NaB-
H(OAc)3, NaBH3CN, NaBH4, or Pd/C under H2) were exam-
ined. The amination using NH4OH and Pd/C under H2 gave
the most promising results. While the reaction did not produce
4 as a byproduct, it did produce the contaminating secondary
amine 17, which resulted when 5 further reacted with 15. We
tried to reduce the amount of 17 produced by modifying the
reaction conditions. Table 1 summarizes the findings of this
investigation. The relative amounts (reported as percentages)
of 5 and 17 reported in Table 1 were determined after measuring
the areas under their respective HPLC peaks. With MeOH, as
the solvent, 23% of the product mixture was 17 (entry 1). A
solution containing equal volumes of 1,4-dioxane and MeOH
was next tried because 15 was not completely soluble in MeOH.
With 1,4-dioxane and MeOH present, the reaction produced a
smaller amount of 17 (15%; entry 2). Increasing the pressure
had no effect (entry 4), but decreasing the concentration of 15
reduced the amount of 17 produced (compare entries 5 and 6).
Finally, the amount of 17 decreased to 8% of the product
mixture when 1,4-dioxane was the solvent (entry 8). Compound
17 could be selectively removed from the product mixture by
adjusting the pH of the aqueous solution to 7. In this pH, 17
was precipitated, whereas 5 was soluble in the aqueous solution.
After extracting the precipitated 17 with AcOn-Bu, 5 was
extracted from the aqueous solution (pH 10) with AcOEt. The
yield of 5 was 90%, and its assay purity was 99.4%.
Having established a practical synthesis for 5, we focused
on optimizing the conversion of 5 to 1 via the intermediates of
Scheme 1. First, we attempted to minimize the cost of the
synthesis of 6 from 5, which originally added a pyrimidine
598
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Vol. 12, No. 4, 2008 / Organic Process Research & Development

Table 1. Preparation of 5 by reductive amination of 15
entry
solventsa
MeOH
conc. of 15 (M)
Pd/C/15 (wt %)
aqueous NH4OH (equiv)
time (h)
5b (%)
17b (%)
1
2
3
4c
5
6
7
8
0.11
0.22
0.22
0.22
0.33
0.11
0.22
0.11
30
30
10
10
10
10
10
10
6
8
8
8
8
8
8
8
3
4
12
3
16.5
25
23.5
20.5
70.0
80.0
76.4
74.2
71.9
79.7
85.0
87.3
23.0
15.2
16.8
15.8
19.2
11.9
11.2
8.4
MeOH/dioxane (1/1)
MeOH/dioxane (1/1)
MeOH/dioxane (1/1)
MeOH/dioxane (1/1)
MeOH/dioxane (1/1)
dioxane
dioxane
a Water (4%) was also added. b HPLC area %. c H2 pressure was 2 kg/cm3.
Table 2. Preparation of 6
entry
Xa
base (equiv)
solvent (M)
temp (°C)
time (h)
yieldb (%)
1
2
3
4
5
6
7
Br
Br
Cl
Br
Br
Br
Cl
i-Pr2EtN (5.6)
i-Pr2EtN (2.0)
i-Pr2EtN (2.0)
NaHCO3 (2.0)
NaHCO3 (2.0)
NaHCO3 (2.0)
NaHCO3 (2.0)
DMSO (0.1)
DMSO (0.1)
DMSO (0.1)
DMSO (0.1)
EtOH (0.1)
n-BuOH (0.76)
n-BuOH (0.76)
120
100
100
100
78
100
100
20
20
20
20
26
19
26
38
51
42
25
47
72
73
a 2-X-pyrimidine. b The yield is not corrected for purity.
substituent to 5, using the expensive 2-bromopyrimidine. We
varied the base, solvent, halogenated pyrimidine, and temper-
ature, and ran the reactions in Nautilus, an automated synthe-
sizer. The information of Table 2 summarizes the conditions
for some of the reactions investigated. Entry 1 lists the
conditions used in the original synthesis, which include an
excess of i-Pr2EtN and a temperature of 120 °C. We discovered
that only two equivalents of i-Pr2EtN and a temperature of 100
°C were necessary (entry 2). However, when 2-bromopyrimi-
dine was replaced with the inexpensive reagent, 2-chloropyri-
midine, with all other conditions of entry 2 remaining the same,
the yield decreased (entry 3). Replacing i-Pr2EtN with NaHCO3,
and DMSO with EtOH, which necessitated a decrease in the
reaction temperature, decreased the formation of byproduct, but
the reaction then proceeded more slowly (compare entries 2
and 5). To improve the reaction speed, n-BuOH, which has
the higher boiling point, replaced EtOH. Starting with either
2-bromopyrimidine or the inexpensive 2-chloropyrimidine now
provided 6 in more than 70% yield,13 although the methyl group
that protected the carboxyl was partially replaced with an n-Bu
group (entries 6 and 7). We isolated 6 and the n-Bu ester
derivative by the addition of MeOH, poured the mixture into
water and filtration. Their ester moieties were removed by
hydrolysis to give 7 in 80% yield (two steps) at 95.8% purity.
For the subsequent amidation of 7, which gives 9, the
following changes were made. 1) Chromatographic purification
was eliminated. Instead the product mixture was poured into
water and solid 9 was isolated by filtration. 2) To decrease the
reaction time, BOP17 (7 h at rt) replaced EDC (12 h at rt). 3)
The reaction temperature was kept below 8 °C at all times to
avoid coprecipitating a sticky yellow material with 9. The yield
of 9 was 96% and its assay purity was 94.4%.
If the t-Bu group could be removed in an acidic environment,
the pyrimidine moiety of 9 would be in its acid salt, which is
required for the final hydrogenation reaction. Therefore, we
designed a procedure that combined these two reactions so that
one purification step could be eliminated. During the original
synthesis, acid (TFA) hydrolysis was used to remove the t-Bu
group of 9. To further decrease the cost of the synthesis, we
replaced TFA with HCl, which is inexpensive, and removed
the t-Bu group in 0.5 M HCl at 60 °C. (Not only did this
procedure remove the t-Bu group, but it also left the pyrimidine
moiety of the product as the HCl salt, which is required for the
hydrogenation.) After cooling the mixture, we added Pd/C and,
under an H2 atmosphere, hydrogenated the product of the
preceding step. Thus, a one-pot, one-step procedure consisting
(17) Bates, A. J.; Galpin, I. J.; Hallett, A.; Hudson, D.; Kenner, G. W.;
Ramage, R. HelV. Chim. Acta 1975, 58, 688.
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of the hydrolysis of the t-Bu group and the hydrogenation was
achieved. An unidentified byproduct, with a molecular weight
that is 16 Da greater than that of 1, accumulated to a level of
about 10%. After several trials, we found that production of
this byproduct could be suppressed if, before adding Pd/C, the
pH of the solution was adjusted to 1.7.
similar to those found when 0.95 kg of 12 was used. Samples
of compound 1, which have been synthesized using this new
route have already been used for preclinical and toxicological
studies.
Experimental Section
Our final task was to purify 1. No crystallization method
was known for 1. We first surveyed many crystallization
conditions. A chromatographically purified sample of 1 crystal-
lized from an i-PrOH-water mixture. We next tried to crystallize
1 directly from the reaction mixture. First Pd/C was removed
by filtration, then after adjusting the pH of the solution to 5,
crystals of 1 were obtained in 56% yield13 with 87 area % by
HPLC. Further investigation indicated that the maximum yield
of 1, achievable by crystallization, was obtained when the pH
of the filtrate was pH 7. However, as the pH of the filtrate was
increased, sometimes a gummy form of 1 precipitated. This
problem was prevented by first adjusting the pH of the filtrate
to 2.8, and then adding MeOH before neutralizing the solution.
Crystals of 1, obtained by this method, were isolated in 84%
yield and were 96.5% pure. The value, 96.5%, was calculated
using the purity of a standard (95.7%), the relative area of the
HPLC peak found for 1 (98.3%), and the relative amount of
residual water (2.6%) associated with the crystal. If crystalline
1 is the monohydrate form of the compound, then, theoretically,
water accounts for 3.1% of the weight, a number that is very
similar to 2.6%, and, in that case, the assay purity of 1 is 99%.
The optical purity of 1 was >99%, no racemization was
apparent.
1H and 13C NMR spectra were recorded on JNM-LA400
spectrometers with chemical shifts reported in ppm with internal
tetramethylsilane as a standard. Electron ionization (EI) mass
spectra were recorded on a Hitachi M-80B instrument. Fast-
atom bombardment (FAB) mass spectra were recorded on a
JEOL JMS-700 instrument. Thermospray electrospray ionization
(ESI) mass spectra were recorded on a Hewlett-Packard 5989A
instrument. High-resolution mass spectra (HRMS) were re-
corded under FAB conditions. Optical rotation was obtained
on JASCO DIP-370 polarimeter. Melting point was determined
on a Yanaco micro melting point apparatus. 3-Hydroxy-4-
nitrobenzoic acid (12) was purchased from Acros Organics.
Diaminopropionate 8 was purchased from Peptide Institute, Inc.
Elemental analyses were performed by the Toray Research
Center and were within ( 0.5% of calculated values. All assay
yields were obtained by HPLC, using pure and characterized
standards. HPLC purities were determined with Inertsil ODS-2
reversed phase column (4.6 mm × 250 mm).
Methyl 3-methoxy-4-nitrobenzoate (13). DMF (9.5 L) and
K2CO3 (1.44 kg, 10.5 mol) were added to 3-hydroxy-4-
nitrobenzoic acid (12) (0.95 kg, 98% purity, 5.08 mol). To this
suspension, was added methyl iodide (2.20 kg, 15.5 mol). The
reaction mixture was stirred for 22 h at a temperature range of
25-30 °C. The mixture was poured slowly into cooled water
(17 L), while maintaining the temperature between 13 and 25
°C. Resulting solids were collected, rinsed with water (6.8 L),
and dried under reduced pressure for 16 h at room temperature
to give 13 (1.04 kg, 95.3% yield, HPLC assay 98.3% (area))
Conclusions
A scalable synthesis of 1, which is an antagonist for both
integrins Rvꢀ3 and RIIbꢀ3, has been developed. We first
developed a piperidine ring-closure route that, unlike the one
used in the original procedure, does not use the expensive
reagent 2. Instead, we used the inexpensive reagent 12. Many
different efforts were made to decrease the number and types
of steps required to synthesize 1 from 12 and some of these
efforts resulted in the optimized route. Incorporating the direct
reductive amination of 15 decreased the number of steps
required to synthesize 5 from 15 by three, because steps d, e of
Scheme 3 and steps c, d, e of Scheme 1 are no longer used.
This reductive amination also did not use hazardous azides. To
prepare 6, the less expensive 2-chloropyrimidine could replace
the expensive 2-bromopyrimidine if the base and the solvent
were also changed. We discovered a crystallization condition
that allowed us to isolate 1 directly from the reaction mixture
in 84% yield and at 96.5% purity. By following our optimized
procedure, 1 can be synthesized in eight steps, which are two
steps less than the original synthesis. The overall yield of 1 is
45%, which is a 35% improvement compared with the yield
obtained from the original synthesis. The new protocol does
not involve chromatographic purifications, but instead relies only
simple procedures, i.e. precipitations or crystallizations, that are
more suited to the large-scale processes necessary for drug
synthesis. Further, 1 is now available with a 98% cost saving
relative to the original protocol. The overall yield (55%) and
the purity (96.4%) of 1 synthesized from 50 g of 12 are very
1
as a pale-yellow solid: H NMR (400 MHz, CDCl3) δ 3.97
(3H, s, OMe), 4.02 (3H, s, OMe), 7.70 (1H, dd, J ) 1.6, 8.3
Hz, C6H3), 7.76 (1H, d, J ) 1.6 Hz, C6H3), 7.84 (1H, d, J )
8.3 Hz, C6H3); EIMS m/z 211 M+. HPLC method: detector
wavelength, 260 nm; flow rate, 0.8 mL/min; eluent, CH3CN/
H2O ) 50/50; temp, rt.
An analytical sample was purified with Inertsil ODS-2
column chromatography: Anal. Calcd for C9H9NO5: C, 51.19;
H, 4.30; N, 6.63. Found: C, 51.11; H, 4.28, N, 6.66.
Methyl 4-Amino-3-methoxybenzoate (11). To a solution
of 13 (1.00 kg, 98.3% purity, 4.66 mol), 1,4-dioxane (5 L),
and MeOH (5 L) was added 10% Pd/C (50 g) in water (1.0 L),
and the mixture was hydrogenated under balloon pressure of
hydrogen for 12 h at a temperature range of 35-40 °C. The
mixture was filtered through Celite, and solids were washed
with 1,4-dioxane/MeOH (1:1) (2 L). The combined filtrate and
washings were concentrated to a final volume that was ap-
proximately 2 L. To the solution was added water (6.7 L), and
resulting suspension was cooled to 12 °C for 1 h. The solids
were collected by filtration, washed with water (5.0 L), and
dried under reduced pressure at 35 °C for 18 h to provide 11
(811 g, 96.4% yield, HPLC assay 100.3% (area)) as a pale-
yellow solid: 1H NMR (400 MHz, CDCl3) δ 3.86 (3H, s, OMe),
3.90 (3H, s, OMe), 6.66 (1H, d, C6H3), 7.46 (1H, d, C6H3),
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7.55 (1H, dd, C6H3); EIMS m/z 181 M+. HPLC method:
detector wavelength, 260 nm; flow rate, 1.0 mL/min; eluent,
CH3CN/H2O ) 50/50; temp, rt.
An analytical sample was purified with Inertsil ODS-2
column chromatography: Anal. Calcd for C9H11NO3: C, 59.66;
H, 6.12; N, 7.73. Found: C, 59.66; H, 6.12, N, 7.73.
until the remaining volume was approximately 3.5 L. The
mixture was adjusted to pH of 6.9-7.1 with 2 M HCl (520
mL). Insoluble byproduct 17 was washed with AcOn-Bu (1.8
L) three times, maintaining the pH of the aqueous layer. To
the aqueous layer (4.5 L) was added NaCl (899 g) and the pH
adjusted to 9.8 with 2 M aqueous K2CO3 (980 mL). The mixture
was extracted with AcOEt (6.3 L, 2.6 L ×2) three times, while
the pH of the aqueous layer was maintained upper 9.8 with 2
M aqueous K2CO3. Combined organic layer was concentrated
and dried under reduced pressure for 16 h at 50 °C to give 5
(276 g, 90.4% yield, HPLC assay 99.4% (area)) as a colorless
solid: 1H NMR (400 MHz, CDCl3) δ 1.58 (2H, dddd, J ) 3.9,
10.5, 11.6, 12.7 Hz, piperidine), 1.95 (2H, br d, J ) 12.7 Hz,
piperidine), 2.70 (2H, ddd, J ) 2.4, 11.6, 12.4 Hz, piperidine),
2.82 (1H, tt, J ) 4.3, 10.5 Hz, piperidine), 3.57 (2H, br d, J )
12.4 Hz, piperidine), 3.88 (3H, s, OMe), 3.92 (3H, s, OMe),
6.91 (1H, d, J ) 8.3 Hz, C6H3), 7.50 (1H, d, J ) 1.8 Hz, C6H3),
7.62 (1H, dd, J ) 1.83, 8.3 Hz, C6H3). HPLC method: detector
wavelength, 300 nm; flow rate, 1.0 mL/min; eluent, CH3CN/
2% aqueous NH4OAc ) 45/55; temp, rt.
Methyl 3-Methoxy-4-{4-(pyrimidin-2-ylamino)piperidin-
1-yl}benzoate (6). To a solution of 5 (270 g, 99.4% purity,
1.01 mol) in n-BuOH (1.35 L), 2-chloropyrimidine (117 g, 1.02
mol) and NaHCO3 (171 g, 2.04 mol) were added. The reaction
mixture was heated to 100-110 °C for 27 h. The cooled (50
°C) mixture was added MeOH (2.7 L) and poured into water
(13.5 L). Resulting suspension was stirred for 2.5 h under
cooling with an ice bath. Solids were collected by filtration,
washed with water (1.0 L), and dried with P2O5 for 2 days to
give a mixture of 6 and n-butyl 4-{4-(pyrimidin-2-ylamino)pi-
peridin-1-yl}benzoate (297 g, 71.1% yield, HPLC assay of (6)
82.7%, HPLC area % of Bu ester: 8.4%) as a pale fellow solid:
HPLC method: detector wavelength, 300 nm; flow rate, 1.0 mL/
min; eluent, CH3CN/2% aqueousNH4OAc ) 50/50; temp, rt.
An analytical sample was purified with Inertsil ODS-2
column chromatography: 1H NMR of 6 (400 MHz, CDCl3) δ
1.75 (2H, dddd, J ) 3.7, 10.7, 11.0, 12.9 Hz, piperidine), 2.20
(2H, br d, J ) 12.9 Hz, piperidine), 2.86 (2H, ddd, J ) 2.3,
10.7, 12.8 Hz, piperidine), 3.58 (2H, br d, J ) 12.8 Hz,
piperidine), 3.89 (3H, s, CO2Me), 3.93 (3H, s, C6H3OMe),
3.96-4.07 (1H, m, piperidine), 6.54 (1H, t, J ) 4.9 Hz,
pyrimidine), 6.94 (1H, d, J ) 8.3 Hz, C6H3CO), 7.51 (1H, d,
J ) 1.9 Hz, C6H3CO), 7.64 (1H, dd, J ) 1.9, 8.3 Hz, C6H3CO),
8.29 (2H, d, J ) 4.9 Hz, pyrimidine); ESIMS of 6 m/z 343 (M
+ H)+; Anal. Calcd for C18H22N4O3: C, 63.14; H, 6.48; N,
16.36. Found: C, 63.34; H, 6.53, N, 16.42.
1,5-Dichloropentan-3-one (10). 3-Chloropropionyl chloride
(1.40 kg, 11.0 mol) was dissolved in dichloromethane (1.7 L),
and the solution was cooled to -30 °C. To this solution was
added AlCl3 (1.87 kg, 14.3 mol) slowly at a rate that kept the
internal temperature of the mixture below 0 °C. The mixture
was stirred at a temperature range of 10-20 °C under bubbling
ethylene gas for 6 h. The reaction mixture was poured into a
precooled (4 °C) mixture of water (5.8 L), conc. HCl (0.47 L),
and dichloromethane (1.4 L), while the internal temperature of
the mixture was maintained below 13 °C. Organic layer was
separated, and aqueous layer was washed with dichloromethane
(1.4 L) twice. Combined organic layer was washed with 1.6 L
of water once and 3.0 L of water twice, dried over anhydrous
MgSO4 (310 g), and concentrated to give 10 (1.74 kg) as a
black syrup: 1H NMR (400 MHz, CDCl3) δ 2.94 (4H, t, J )
6.5 Hz, COCH2CH2Cl), 3.76 (4H, t, J ) 6.5 Hz, COCH2-
CH2Cl).
Methyl 3-Methoxy-4-(4-oxopiperidin-1-yl)benzoate (15).
To 11 (804 g, 100% purity, 4.45 mol) was added MeOH (8.0
L), and the suspension was warmed to 60 °C. To the resulting
solution was added 10 (1.44 kg, 9.28 mol) and stirred for 4 h
at a temperature range of 60-67 °C. The mixture was poured
into water (16 L), and the resulting tar was filtrated with Celite.
The filtrate was concentrated until the total volume of the residue
was approximately 8.5 L. To the resulting water solution was
added activated carbon (80 g) and stirred for 30 min at 50 °C.
Insolubles were removed by filtration and washed with water
(2.0 L). The combined filtrate and washings were adjusted to
pH of 6.8-7.2 with NaHCO3 (1.14 kg, 13.6 mol). Resulting
solids were collected by filtration, washed with water (3.2 L),
and dried under reduced pressure for 44 h at 40 °C to give 15
(1.01 kg, 83.8% yield, HPLC assay 97.2% (area)) as a colorless
solid: 1H NMR (400 MHz, CDCl3) δ 2.64 (4H, t, J ) 5.8 Hz,
piperidine), 3.46 (4H, t, J ) 5.8 Hz, piperidine), 3.90 (3H, s,
OMe), 3.96 (3H, s, OMe), 6.94 (1H, d, J ) 8.2 Hz, C6H3),
7.56 (1H, d, J ) 1.7 Hz, C6H3), 7.65 (1H, dd, J ) 1.7, 8.2 Hz,
C6H3); EIMS m/z 263 M+. HPLC method: detector wavelength,
300 nm; flow rate, 1.0 mL/min; eluent, CH3CN/H2O ) 50/50;
temp, rt.
An analytical sample was purified with Inertsil ODS-2
column chromatography: Anal. Calcd for C14H17NO4: C, 63.87;
H, 6.51; N, 5.32. Found: C, 64.00; H, 6.63, N, 5.35.
3-Methoxy-4-{4-(pyrimidin-2-ylamino)piperidin-1-
yl}benzoic Acid (7). To a solution of a mixture of 6 and the
butyl ester derivative (290 g, 82.7% purity, 0.701 mol) in MeOH
(1.45 L), was added 1.2 M NaOH (1.45 L). The reaction mixture
was stirred for 2 h at a temperature range of 50-60 °C. The
mixture was concentrated until the remaining volume was
approximately 1.45 L, water (580 mL) was added, and the
mixture was concentrated again until the remaining volume was
approximately 1.45 L. The pH of the residue was adjusted to
4.5 with 2 M HCl (855 mL), while the temperature was
maintained between 7 and 10 °C. To the mixture was added
water (595 mL). Resulting solids were collected by filtration,
Methyl 4-(4-Aminopiperidin-1-yl)-3-methoxybenzoate (5).
To a solution of 15 (311 g, 97.2% purity, 1.15 mol) and 1,4-
dioxane (10.5 L) was added 10% Pd/C (35.0 g) in water (0.4
L) and 28% aqueous ammonia (2.8 L). The mixture was
hydrogenated under balloon pressure of hydrogen for 12 h at a
temperature range of 21-28 °C. The mixture was filtered
through Celite, and solids were washed with 1,4-dioxane/MeOH
(1:1) (1.0 L). The combined filtrate and washings were
concentrated until the remaining volume was approximately 2.8
L, water was added (2.8 L), and this was concentrated again
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washed with water (600 mL), and dried under reduced pressure
for 16 h at the room temperature to give 7 (272 g, 113% yield,
HPLC assay 95.8% (area)) as a colorless solid: 1H NMR (400
MHz, DMSO-d6) δ 1.64 (2H, dddd, J ) 3.5, 10.5, 11.6, 12.7
Hz, piperidine), 1.93 (2H, br d, J ) 12.7 Hz, piperidine), 2.70
(2H, br dd, J ) 11.6, 12.2 Hz, piperidine), 3.50 (2H, br d, J )
12.2 Hz, piperidine), 3.83 (3H, s, C6H3OMe), 3.83-3.90 (1H,
m, piperidine), 6.55 (1H, t, J ) 4.7 Hz, pyrimidine), 6.93 (1H,
d, J ) 8.3 Hz, C6H3CO), 7.41 (1H, d, J ) 1.8 Hz, C6H3CO),
7.49 (1H, dd, J ) 1.8, 8.3 Hz, C6H3CO), 8.27 (2H, d, J ) 4.7
Hz, pyrimidine); ESIMS m/z 329 (M + H)+. HPLC method:
detector wavelength, 305 nm; flow rate, 1.0 mL/min; eluent,
CH3CN/2% aqueousNH4OAc ) 50/50; temp, rt.
(2S)-Benzenesulfonylamino-3-[3-methoxy-4-{4-(1,4,5,6-tetra-
hydropyrimidin-2-ylamino)piperidin-1-yl}benzoylamino] pro-
pionic Acid (1). A solution of 9 (200 g, 94.4% purity, 0.309
mol) in 0.5 M HCl (4.0 L) was warmed at a temperature range
of 60-61 °C and stirred for 5.5 h. The mixture was cooled to
35 °C and water was added (4.0 L). The pH of the mixture
was adjusted to 1.6-1.7 with 5 M aqueous NaOH (226 mL).
To the mixture was added 10% Pd/C (10.0 g) in water (400
mL), and this was hydrogenated under balloon pressure of
hydrogen for 7.5 h at a temperature range of 30-36 °C.
Insolubles were filtrated and washed with 0.1 M HCl (4.7 L).
The filtrate and washing were combined, and the solution was
adjusted to pH of 2.8-2.9 by adding 5 M NaOH (148 mL).
To the mixture was added MeOH (2.0 L) and pH adjusted to
5.0-5.2 with 5 M aqueous NaOH (96 mL). The pH of the
mixture was further adjusted to pH of 7.4-7.6 carefully with
1 M aqueous NaOH (22.5 mL). The resulting solution was
seeded with an authentic sample and stirred slowly for 20 h.
The solvents (about 1.6 L) were removed via evaporation. Solids
were collected by filtration and dried under reduced pressure
for 18 h at 35 °C to give 1 (149 g, 83.5% yield, HPLC assay
An analytical sample was washed with AcOEt, CH3CN and
H2O: Anal. Calcd for C17H20N4O3: C, 62.18; H, 6.14; N, 17.06.
Found: C, 62.47; H, 6.25, N, 17.09.
tert-Butyl (2S)-Benzenesulfonylamino-3-[3-methoxy-4-{4-
(pyrimidin-2- ylamino)piperidin-1-yl}benzoylamino]propio-
nate (9). The carboxylic acid 7 (112 g, 95.8% purity, 0.326
mol) and N-methylmorpholine (41.3 g, 0.480 mol) were
suspended in DMF (1.12 L) and cooled to 4 °C; (benzotriazol-
1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP reagent) (165 g, 0.374 mol) was added slowly, while the
temperature was maintained at 4 °C. After 30 min, tert-butyl
(2S)-N-benzenesulfonyl-2,3-diaminopropionate 8 (102 g, 0.341
mol) was added to the mixture at a rate that kept the internal
temperature of the mixture below 7 °C. The mixture was stirred
an additional 23 h at a temperature range of 3-8 °C and then
was poured into water (5.6 L) over a 20-min period. The
reaction flask was washed with acetone (70 mL), and the
washing was also poured into the mixture. Resulting solids were
collected by filtration and washed with water (700 mL), and
5% aqueous NaHCO3 (10 L) was added. After stirring for 30
min, the solids were collected by filtration and washed with
water (1.15 L) until the pH of the filtrate was below 6.5. The
solid was dried under reduced pressure for 14 h at room
temperature, for 22 h at 40 °C, and for 23 h with P2O5 to give
9 (202 g, 96.0% yield, HPLC assay 94.4% (area)) as a colorless
solid: 1H NMR (400 MHz, CDCl3) δ 1.29 (9H, s, t-Bu), 1.76
(2H, br dddd, J ) 4.1, 10.4, 11.5, 12.9 Hz, piperidine), 2.20
(2H, br d, J ) 12.9 Hz, piperidine), 2.85 (2H, br dd, J ) 11.5,
11.5 Hz, piperidine), 3.47-3.59 (3H, m, piperidine and
CONHCH2CH), 3.88-4.06 (3H, m, piperidine and CONH-
CH2CH), 3.95 (3H, s, C6H3OMe), 6.54 (1H, t, J ) 4.8 Hz,
pyrimidine), 6.95 (1H, d, J ) 8.2 Hz, C6H3CO), 7.33 (1H, dd,
J ) 2.0, 8.2 Hz, C6H3CO), 7.41 (1H, d, J ) 2.0 Hz, C6H3CO),
7.50 (2H, br dd, J ) 7.1, 7.4 Hz, C6H5), 7.58 (1H, br t, J ) 7.4
Hz, C6H5), 7.86 (2H, br d, J ) 7.1 Hz, C6H5), 8.29 (2H, d, J )
4.8 Hz, pyrimidine); FABMS m/z 611 (M + H)+. HPLC
method: detector wavelength, 305 nm; flow rate, 1.0 mL/min;
eluent, CH3CN/phosphate buffer Na (pH) ) 40/60; temp,
40 °C.
1
96.5% (area), >99% ee) as a colorless solid: H NMR (400
MHz, DMSO-d6) δ 1.39-1.43 (2H, m, piperidine), 1.73-1.80
(4H, m, piperidine and tetrahydropyrimidine), 2.39-2.47 (2H,
m, piperidine), 3.08-3.15 (2H, m, CONHCH2CH and piperi-
dine), 3.19 (4H, m, tetrahydropyrimidine), 3.28 (2H, m,
piperidine), 3.48 (1H, m, CONHCH2CH), 3.60 (1H, m,
CONHCH2CH), 3.68 (3H, s, C6H3OMe), 6.77 (1H, d, J ) 8.7
Hz, C6H3CO), 7.26 (1H, d, J ) 8.7 Hz, C6H3CO), 7.28 (1H, s,
C6H3CO), 7.53-7.57 (2H, m, C6H5), 7.61 (1H, m, C6H5), 7.83
(1H, d, J ) 6.8 Hz, C6H5), 7.84 (1H, d, J ) 6.8 Hz, C6H5),
8.31 (1H, br t, J ) 5.0 Hz, CONHCH2CH), 8.71 (1H, br s,
NH), 9.42 (1H, br d, J ) 7.6 Hz, NH); 13C NMR (DMSO-d6)
δ 19.8, 31.8, 32.1, 37.8, 42.6, 47.3, 48.9, 49.0, 55.3, 55.4, 110.5,
117.2, 119.6, 126.6, 128.3, 129.0, 132.3, 140.8, 143.9, 151.3,
152.0, 165.4, 173.5; FABMS m/z 559 (M + H)+; FAB-HMS
(M + H)+ calcd for C26H34N6O6S: 559.2339, found: 559.2343.
HPLC methods: (A) Nonchiral HPLC conditions: detector
wavelength, 305 nm; flow rate, 1.0 mL/min; eluent, CH3CN/
10 mM sodium phosphate buffer ) 40/60 or 20/80 (for
hydrogenation); temp, 40 °C. (B) Chiral HPLC conditions:
column, sumichiral OA-3200; detector wavelength, 305 nm;
flow rate, 1.0 mL/min; eluent, MeOH/0.5 mM aqueous
NH4OAc; temp, rt.
An analytical sample was prepared by recrystallization from
EtOH/H2O ) 2/1: mp 162-164 °C; Anal. Calcd for
C26H34N6O6S 1.68 H2O: C, 53.03; H, 6.39; N, 14.27. Found:
C, 52.60; H, 5.95; N, 14.12; [R]23D +92° (c 0.31, CHCl3/MeOH
) 1:1).
Acknowledgment
We thank Shigeko Miki and Takako Miyara for mass
spectral analysis.
An analytical sample was purified with Inertsil ODS-2
column chromatography: Anal. Calcd for C30H40N6O7S (as
monohydrate): C, 57.31; H, 6.41; N, 13.37. Found: C, 57.40;
H, 6.47, N, 13.41.
Received for review March 27, 2008.
OP800073Z
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Vol. 12, No. 4, 2008 / Organic Process Research & Development