Practical syntheses of a CXCR3 antagonist

Article abstract of DOI:10.1021/jo102399a

Two new, reliable syntheses of a pyrido[2,3-d]- pyrimidine inhibitor of the CXCR3 receptor are described. A nine-step synthesis of the CXCR3 inhibitor (1) from 2-aminonicotinic acid was demonstrated on a multikilogram scale and incorporates a classic reso

Full text of DOI:10.1021/jo102399a

ARTICLE
pubs.acs.org/joc
Practical Syntheses of a CXCR3 Antagonist
Johann Chan,*,§ Brenda J. Burke,*,§ Kyle Baucom,§ Karl Hansen,‡ Matthew M. Bio,‡ Evan DiVirgilio,§
Margaret Faul,§ and Jerry Murry§
§Chemistry Process Research and Development, Amgen, Inc., Thousand Oaks, California 91320, United States
‡Chemistry Process Research and Development, Amgen, Inc., 360 Binney St., Cambridge, Massachusetts 02141, United States
S Supporting Information
b
ABSTRACT: Two new, reliable syntheses of a pyrido[2,3-d]-
pyrimidine inhibitor of the CXCR3 receptor are described. A
nine-step synthesis of the CXCR3 inhibitor (1) from 2-ami-
nonicotinic acid was demonstrated on a multikilogram scale
and incorporates a classic resolution to deliver the enantioen-
riched active pharmaceutical ingredient (API). A second synth-
esis of the CXCR3 inhibitor starts from (þ)-(D)-Boc alanine
and 2-chloronicotinic acid and utilizes a Goldberg coupling.
This second synthesis, performed on a gram scale, intersects
the former route at a common intermediate thereby complet-
ing a formal synthesis of the enantioenriched API in higher
overall yield without the need for a resolution.
’ INTRODUCTION
Herein we report two novel syntheses of 1. The first entails a
racemic synthesis of rac-2, which proceeds by way of a 2-ethyl
pyrido[2,3-d]pyrimidine and incorporates a classic resolution of a
primary amine intermediate. The second begins with commercially
available (þ)-(D)-Boc alanine and preserves the stereocenter through
four transformations to intersect the former route at (þ)-2.
Identified by Loetscher1 in the mid-1990s, the CXCR3
chemokine receptor can be found predominantly in activated
T cells (CD4þ and CD8þ). The overexpression of this receptor
is linked to several autoimmune disorders including psoriasis,
rheumatoid arthritis, multiple sclerosis, and solid organ trans-
plantations. CXCR3 animal knockout mice studies indicate that
an antagonist of this receptor may inhibit T-cell migration and
thereby attenuate the incidence and severity of these diseases. As
part of our research program toward discovering effective
inhibitors of this receptor,2 1 was identified as a potent inhibitor
of the CXCR3 receptor (Scheme 1). To support further tox-
icological studies, a practical and efficient synthesis was required.
Initial synthetic efforts to provide small quantities of preclini-
cal candidate 1 and analogues followed the strategy outlined in
Scheme 1: disconnection of 1 at the tertiary amide reveals
primary amine 2 along with phenylacetic acid 3 and sulfone
aldehyde 4. Amine 2 is derived from nucleophilic addition of
4-chloroaniline to oxazinone 5. Oxazinone 5 arises from a
condensation of commercially available 2-aminonicotinic acid 6
and D-Boc-alanine 7. Key problems identified in this synthesis
were the enantiomeric erosion observed during the low-yielding
conversion of 7 to primary amine 2 as well as the propensity of
oxazinone 5 to hydrolyze upon exposure to mildly basic aqueous
wash to give ring-opened products. Although this route was
sufficient for the small-scale preparation of 1, attempts to provide
quantities for further studies via this route resulted in amplifica-
tion of the issues mentioned above. Moving forward, we aimed to
develop a reliable, scalable synthesis of 1 via primary amine 2 that
did not proceed by way of unstable oxazinone intermediate 5.
’ RESULTS AND DISCUSSION
Synthesis of rac-2. Having identified limitations in scaling up
known protocols for the synthesis of pyrido[2,3-d]pyrimidines,3
we considered alternate entries to access this heterocyclic core
that would satisfy our scale-up needs. Cognizant of the tendency
of the secondary stereocenter to undergo epimerization during
the synthesis of 2, we devised a strategy toward rac-2 that hinged
on its successful resolution. Such an approach to efficiently access
rac-2 was envisioned to go by way of pyrido[2,3-d]pyrimidine 8,
which could arise from a condensation of 2-aminonicotinamide 9
with a propionic acid equivalent (Scheme 2).
Toward that end we first set out to synthesize the appropriate
2-aminonicotinamide precursor with which to condense with a
propionic acid equivalent. 2-Aminonicotinic acid 6 was coupled
with 4-chloroaniline by using EDC HCl to give a 72% yield of
amide 9 (Scheme 3).4 After screening a variety of conditions, we
found that reacting amide 9 with a mixture of triethylorthopro-
pionate and acetic acid was most effective in promoting the
desired transformation. Ultimately this procedure was used to
provide >2 kg of pyrido[2,3-d]pyrimidine 8 from 9 in 93% yield.5
Received: December 3, 2010
Published: February 07, 2011
r
2011 American Chemical Society
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Scheme 1. First Generation Route to 1
Scheme 4. Functionalization of 8 To Give rac-2
Scheme 5. Classic Resolution of rac-2
Scheme 2. Approach to the Synthesis of rac-2
Scheme 3. Synthesis of Pyrido[2,3-d]pyrimidine 8
displacement of the chloride to provide 11. Employing triethyl-
phosphite to reduce azide 11 produced the desired phosphor-
amide 12 as determined by LC/MS.7 Cleavage of the
phosphoramide was accomplished in situ with concentrated
HCl to afford the desired primary amine rac-2 in 68% yield over
the three steps. With the desired racemic amine in hand, efforts to
develop a chiral resolution commenced.
Classic Resolution of rac-2. Initial screening of enantiopure
acids for the resolution of rac-2 led to the identification of
di-(þ)-(p-anisoyl)-D-tartrate (DAT). It was found that stirring
rac-2 with 0.5 equiv of this tartrate in acetonitrile afforded a 19%
yield of the desired diastereomer of a 2:1 amine:DAT salt having
a 95% ee at the amine stereocenter. Further investigations into
solvent combinations identified IPA:water, and developmental
work ensued that entailed measuring the mother liquor enantio-
meric excesses of a range of IPA:water concentrations at 15
volumes.8 The mother liquor had an enrichment of 60-63% ee
in the undesired amine DAT salt using 7:3 v/v IPA:water
Installation of the amino group was then accomplished via a
two-step process. Reacting 8 with N-chlorosuccinimide (NCS)
in acetic acid cleanly afforded chloride 10, which was crystallized
from solution by the addition of water to achieve a 95% isolated
yield (Scheme 4). Reacting 10 with sodium azide6 effected
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Scheme 6. Final Steps to 1
to the corresponding cyclohexyl amide by HPLC analysis. A
solution of secondary amine 14 in acetonitrile was charged to the
solution of activated 3 over 1 h and the mixture was aged for 20 h,
after which time 99% conversion of 14 was observed. After
seeding the reaction mixture with 2 wt % of a suspension of 1 in
water, addition of water continued until a 59 wt % aqueous
mixture resulted. After holding for 16 h, product 1 was isolated by
filtration in 83% yield.
However, we observed a higher than expected amount (7 wt %)
of a single impurity, 15, whose structure was determined by
direct synthesis of the product formed upon treating 1 with
K2CO3 in MeOH (Figure 1). Stress studies conducted prior to
the campaign in which 1 was stirred with 1 equiv of imidazole
resulted in the formation of only 1.4 wt % of 15 over 18 h.
Product 15 was presumably formed due to the excess of
imidazole in the water/acetonitrile mixture which facilitated a
base-mediated prototropic rearrangement of 1 (see Figure 1).
This hypothesis was later confirmed by additional stress studies
that demonstrated the propensity of 1 to rearrange to 15 at
higher imidazole levels (see the Supporting Information). To
reduce the levels of 15 in 1 a purification procedure was
implemented that involved suspending 1 in 5 volumes of IPA,
which reduced the amount of 15 to approximately 3 wt % after
filtration. Following the IPA suspension, 1 was recrystallized
from 27 wt % aqueous ethanol at 20 volumes. This protocol
allowed us to deliver 2.6 kg of 1 in overall 70% yield containing
<0.5 wt % of impurity 15.
Synthesis of 1 from (þ)-D-Boc-Alanine. Concomitant with
the multikilo delivery of 1, we developed a second route to this
molecule with several goals in mind. First, we were interested in
replacing 2-amino nicotinic acid with a less expensive substituted
pyridine starting material. Also, making use of a D-alanine
derivative as the source of chirality in the molecule was appealing
in that a chiral resolution could be circumvented. Finally, we
recognized that a long-term route would need to eliminate the
use of azide chemistry for safety considerations. We ultimately
developed an approach to primary amine (þ)-2 that addressed
each of these concerns.14
(Scheme 5). Interestingly, a sharp drop in liquor concentration
and ee was observed when the concentration of IPA in the
aqueous mixture exceeded 70% v/v. The outcome of a resolution
of rac-2 having a mother liquor ee of 63% enriched in the
undesired enantiomer provides a maximum yield of 37.5-
38.5%, or 75-77% of theoretical, of resolved 2 (13). In practice,
the resolution of rac-2 was performed as described above with 5
kg of rac-2 to ultimately deliver 3.9 kg (38% yield, 99.7% ee)
of amine:DAT salt 13 for use in the subsequent reductive
amination step.
Reductive Amination of Aldehyde 4 and Amine-DAT Salt
13. With technology in place to access large quantities of
aldehyde 49 via its corresponding bisulfate adduct,10 we turned
our attention to the reductive amination. Early protocols called
for preformation of the imine by reacting aldehyde 4 with amine-
DAT salt 13 for 1 h in THF at 0 °C prior to addition of the
reducing agent, NaBH(OAc)3, and acetic acid. After further
agitating the solution for 1 h at 0 °C, up to 90% conversion of
the amine-DAT salt 13 to secondary amine 14 was observed by
HPLC. However, two impurities arise from this protocol11 that
increase in concentration with prolonged aging of the preformed
imine or if the reaction is carried out above 0 °C for any extended
length of time. To eliminate the formation of these impurities, the
order of addition was altered such that 1.4 equiv of aldehyde 4 in
dichloromethane was charged to a solution of amine-DAT salt 13,
HOAc, and NaBH(OAc)3 in dichloromethane over ∼3 h while
maintaining the internal temperature below -5 °C (Scheme 6).12
Under these improved conditions, quantitative conversions of 13
and assay yields of up to 99.7% of 14 were observed.
Given the advantages of employing 2-chloronicotinic acid
instead of 2-aminonicotinic acid as a starting material, the
development of a C-N coupling strategy with a derivative of the
former was attractive.15 A survey of the literature revealed that
palladium catalysis was competent at promoting such a coupling
between primary amides and 2-chloropyridines.16 Thus, alanine
amide 1717 was reacted with methyl 2-chloronicotinate 1618,19 in
the presence of 2 mol % of Pd(OAc)2, 4 mol % of Xantphos, and 2
equiv of potassium carbonate. Stirring these reagents in toluene at
80 °C resulted in a >95% assay yield of the desired coupled product
18 (Scheme 7). Although employing THF as solvent gave slightly
lower assay yields of 18 (92%), this was the solvent of choice in
order to facilitate a distillative solvent switch to isopropyl acetate
(IPAc) at the completion of the reaction. Following aqueous
washes, the IPAc solution was subjected to a crystallization protocol
using heptane as antisolvent to achieve a 70% isolated yield of 18
with the remaining material being in the aqueous washes (3%),
mother liquor (8%), and filter cake washes (7%). The enantiomeric
integrity of the alanine starting material was retained through the
transformation to 18 (>99:1 er).
Due to difficulties encountered in identifying and isolating
a crystalline salt of 14 with satisfactory properties, direct isolation
of the secondary amine freebase was required. This was accom-
plished by a distillative solvent switch of the dichloromethane
extracts into methanol, followed by concentration and crystal-
lization from a methanol-ethanol solution at 40 °C. Yields were
typically on the order of 90% isolated, with the balance of
material in the mother liquor and washes. The batches also
contained 1-2% of ent-14 due to racemization under the
reaction conditions, which was well rejected along with the
other chemical impurities leading to >99.5% LCAP (liquid
chromatography area percentage) isolated penultimate inter-
mediate.
Amide Bond Formation. Early methods utilized EDC HCl
3
to promote amide bond formation between secondary amine 14
and 4-trifluoromethyl(3-fluoro)phenyl acetic acid (3) to form 1.
Under these conditions the amide coupling reaction proceeded
in yields ranging from 80% to 85%. Upon further investigation we
identified 1,1-carbonyldiimidazole (CDI) as a more readily
available and cost-effective coupling agent to effect this
transformation.13 Thus, phenylacetic acid 3 was slowly added
to a slurry of CDI in acetonitrile, after which time quenching an
aliquot into cyclohexylamine revealed complete conversion of 3
To carry out the formation of the N-4-chlorophenyl nicotinamide
moiety, it was necessary to add a solution of methyl ester 18 to a
prestirred solution of excess 4-chloroaniline and isopropylmagne-
sium chloride in THF. After allowing the resulting solution to warm
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Figure 1. Proposed formation of impurity 15.
Scheme 7
to an acid-base extraction sequence in order to remove colored
impurities that formed during the dehydrative cyclization. The
organic extracts were concentrated in vacuo to approximately
three volumes, and then product (þ)-2 was precipitated with nine
volumes of MTBE and isolated by filtration in 82% yield over the
2 steps, with the remainder of material in the mother liquors.
’ CONCLUSION
We have developed two new practical syntheses of the
CXCR3 antagonist 1. The first, which begins with racemic
starting materials, constructs the pyrido[2,3-d]pyrimidine cores
from a 2-amino nicotinamide and triethyl orthopropionate. The
selective functionalization of the ethyl moiety of pyrido[2,3-
d]pyrimidine 8 provided access to the requisite primary amine
rac-2 in four steps and 72% overall yield. Furthermore, a classic
resolution of this primary amine (rac-2) was developed and
implemented on a 5 kg scale. A practical and reliable synthesis of
sulfone aldehyde 4 was developed that improves upon known
procedures to synthesize its precursor acid, 14, and then in-
corporates the formation of a crystalline bisulfite adduct 16 as a
purification and hold point. Coupling partners 13 and 4 were
then combined in a reductive amination to give secondary amine
14. A CDI-mediated coupling to form the final amide bond to
give 1 was employed in place of the existing EDC HCl technol-
ogy. Using these methods the synthesis of the CXCR3 antagonist
1 was demonstrated on a multikilogram scale.
Our second synthesis of 1, in which the stereocenter is derived
from the readily available enantiopure building block (þ)-D-
alanine, makes use of a challenging C-N coupling reaction that
allowed us to employ 2-chloronicotinic acid as a cheaper alter-
native to 2-aminonicotinic acid. By preserving the stereocenter
through the isopropylmagnesium chloride mediated amide bond
formation with 4-chloroaniline and the subsequent dehydrative
cyclization and Boc group cleavage, we accessed (þ)-2 in
enantioenriched form without the need for a resolution. The
free primary amine (þ)-2 is known to perform in the reductive
amination with 4 and amide bond coupling with 3 to arrive at 1,
thereby completing the formal synthesis of our target compound.
Although this route requires additional research to support large-
scale production of the API, it demonstrates a practical means to
access 1 as a starting point for future process development.
to ambient temperature and 2 h of stirring, an 87% assay yield of
bisamide 19 was observed. Bisamide 19 was isolated in 80% yield
and 98% ee after acidic workup and silica gel chromatography.
Next, we required a dehydrative cyclization and conditions for
Boc group cleavage to arrive at key intermediate primary amine 2.
A survey of conditions revealed that heating 19 in either HOAc
or isobutyric acid achieved the desired transformation to the
pyrido[2,3-d]pyrimidine core (N-Boc-2); however, chiral HPLC
analysis revealed that this product had undergone racemization.
Thus, milder conditions were sought that would not disrupt the
stereocenter. Following a report by Argade,20 we subjected
bisamide 19 to hexamethyldisilazane and iodine in dichloro-
methane and observed clean (>99%) conversion to the desired
cyclized product after 16 h of stirring. Pleasingly, no epimeriza-
tion occurred during this transformation. This product was
carried on without further purification to the deprotection in
which a solution of crude N-Boc-2 in dichloromethane was treated
with TFA. After 1 h of stirring, HPLC analysis revealed full
conversion to primary amine (þ)-2. The product was subjected
’ EXPERIMENTAL SECTION
(R)-2-(N-1-(3-(4-Chlorophenyl)pyrido[2,3-d]pyrimidin-4(3H)-
one)-N-(1,1-dioxohexahydro-1λ6-thiopyran-4-yl)methyl)-2-(3-
fluoro-4-(trifluoromethyl)phenyl)acetamide (1). To a 100 L re-
actor was charged 14 (2595 g, 92.4 wt %, 5.8 mol) followed by acetonitrile
(32 L, 12 vol). To remove the residual EtOH from the previous step,
approximately half of the acetonitrile was vacuum distilled at 25-30 °C.
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Additional acetonitrile was charged (16 L, 6 vol) and distillation was repeated
to afford a solution of 14 (141 mg/mL) that contained 0.2 wt % EtOH. The
solution was cooled to 15 °C and held overnight.
(6500 mL). The reaction mixture was maintained at 20 °C for 23 h, then
poured into water (40 L). The mixture was filtered then the filtrate was
washed with water (3 ꢀ 2.0 L) and taken directly to the next step
without drying. Diagnostic data for intermediate azide 11 (mp (DSC)
149-163 °C, minimum 160 °C): 1H NMR (400 MHz, DMSO-d6) δ
9.03 (d, J = 3.0 Hz, 1H), 8.53 (dd, J = 8.0, 2.0 Hz, 1H), 7.66 (m, 5H), 4.00
(q, J = 6.5 Hz, 1H), 1.55 (d, J = 6.5 Hz, 3H); signals for 2 NH protons not
observed; 13C (100 MHz, DMSO-d6) δ 161.8, 157.5, 156.6, 156.1,
136.1, 134.7, 134.2, 131.0, 130.6, 129.6 (2C), 123.1, 116.4, 55.5, 17.0; IR
3445, 3259, 2097, 1680, 1599, 1584, 1566, 1488, 1432, 1367, 1256, 1088,
796 cm-1; exact mass m/z calcd for C15H12ClN6O [M þ H]þ
327.07611, found 327.07524.
A solution of 3 (1547 g, 7.0 mol) and CH3CN (3.2 L) was added to
a slurry of CDI (1226 g, 7.6 mol) and CH3CN (2.6 L, 1 vol) over 10
min, and the mixture was stirred for an additional 10 min at which time
1.4% of 3 remained by HPLC analysis. A solution of 14 (141 mg/mL in
CH3CN) was added over 1 h followed by additional CH3CN (3.2 L) to
rinse the vessel. The reaction mixture was maintained for 20 h at which
point 0.1% 14 remained. Water (2.6 L, 1 vol) was charged to the reactor
over 0.5 h followed by a slurry of 2 wt % 1 (55 g) in water (4.4 L) to
achieve a 59 wt % aqueouws mixture. The reaction mixture was aged for
16 h, then the solids were filtered and washed with 7:3 water:acetonitrile
(12.7 L) and water (12.9 L) and then dried in the vacuum oven at 40 °C.
The solids (3570 g) were then slurried in IPA (26 L, 0.016 wt % water)
for 2 h, filtered, washed with IPA (8.6 L), and dried in a vacuum oven at
40 °C overnight. The solids (3685 g) were thendissolved inEtOH (46L),
heated to 65 °C, and filtered through a 5 μL polypropylene filter. Water
(13.5 L) was charged to the solution at 61 °C followed by 2 wt % of 1
(109 g) suspended in water (0.5 L). Water (0.3 L) was charged into the
vessel to wash the remaining seeds into the reactor to give a 27 wt %
water mixture. The slurry was cooled to 22 °C and agitation continued
for an additional 16 h before the solids were isolated by filtration and
dried in a vacuum oven at 40 °C overnight to afford 2585 g of 1 (99.7 wt %
70.9% corrected isolated yield) as a white solid (mp (DSC) 206-257 °C,,
Triethylphosphite (1880 g, 11.3 mol, 2.3 equiv) was slowly added to a
mixture of 11 (1607 g, 4.9 mol, 1.0 equiv), THF (8.0 L), and water (6.5
L). The reaction mixture was maintained at 60 ( 5 °C for 20 h then
cooled to 0 °C. Concentrated HCl (3.0 L) was added slowly keeping the
internal temperature between 0 and 5 °C, and the resulting mixture was
allowed to warm to ambient temperature and stirred for 15 h. The
reaction mixture was concentrated in vacuo and to the residue was added
water (3.5 L) and DCM (12.0 L). The biphasic mixture was cooled to
5 °C, and NaOH (2.6 kg) in water (3.0 L) was added slowly to keep the
internal temperature at 10 ( 5 °C to basify the aqueous layer to pH 9.
The layers were separated, and the DCM layer was dried with Na2SO4,
concentrated in vacuo. The residue was washed with MTBE (3 ꢀ 1 L)
and EtOAc (2 ꢀ 1 L) and dried under vacuum to afford 1010 g of rac-2
(68%, 3 steps) as a light yellow solid: 1H NMR (400 MHz, CDCl3) δ
9.00 (dd, J = 4.7, 2.0 Hz, 1H), 8.56 (dd, J = 7.9, 2.0 Hz, 1H), 7.56 (dd,
J = 7.4, 1.6 Hz, 2H), 7.44 (dd, J = 8.1, 4.7 Hz, 1H), 7.25 (m, 2H), 3.74 (q,
J = 6.6 Hz, 1H), 1.35 (d, J = 6.6 Hz, 3H); 13C (100 MHz, CDCl3) δ
165.2, 162.7, 157.7, 156.6, 137.0, 136.2, 134.6, 130.7, 130.5, 130.1, 129.8,
122.7, 116.2, 49.2, 23.6; IR 3446, 3260, 2990, 2097, 1682, 1599, 1584,
1566, 1432, 1256, 1088, 795 cm-1; exact mass m/z calcd for
C15H14ClN4O [M þ H]þ 301.0856, found 301.0837.
1
exothermic decomposition, maximum 236 °C): H NMR (400 MHz,
CDCl3) δ 1.43 (d, J = 7.24 Hz, 3H), 1.65 (d, J = 6.65 Hz, 1H), 1.71 (s,
2H), 1.80-2.01 (m, 3 H), 2.05-2.13 (m, 1H), 2.50 (d, J = 2.93 Hz, 1H),
2.78 (d, J = 13.11 Hz, 1H), 3.07-3.21 (m, 3H), 3.43-3.60 (m, 2H),
3.66-3.75 (m, 1H), 3.78 (s, 2H), 4.96 (q, J = 7.24 Hz, 1H), 6.79-6.88
(m, rotamer), 6.98-7.06 (m, 2H), 7.14 (dd, J = 8.61, 2.54 Hz, rotamer),
7.22 (dd, J = 8.41, 2.54 Hz, 1H), 7.35 (dd, J = 8.51, 2.45 Hz, rotamer),
7.44 (dd, J = 7.82, 4.69 Hz, 1H), 7.49-7.60 (m, 3H) 7.66-7.72 (m,
rotamer), 7.75 (dd, J = 8.41, 2.54 Hz, 1H), 8.56 (dd, J = 7.92, 2.05 Hz,
1H), 8.65 (dd, J = 8.02, 1.76 Hz, rotamer), 8.94 (dd, J = 4.70, 19.6 Hz,
1H), 9.09 (dd, J = 4.50, 1.96 Hz, rotamer); 13C NMR (100 MHz,
CDCl3) δ 16.1, 28.0, 28.2, 37.4, 40.3, 49.4, 51.0, 51.1, 54.2, 77.2, 116.3,
117.8, 122.6, 125.0, 127.3, 129.7, 130.6, 130.7, 134.0, 136.0, 136.7, 141.0,
141.1, 156.4, 157.2, 161.8, 162.5, 170.8; IR 3264, 2929, 1624, 1584,
1514, 1491, 1395, 1367, 1320, 1290, 1253, 1110, 1093, 1093, 1046, 828,
773, 712, 662 cm-1; exact mass m/z calcd for C30H28ClF4N4O4S [M þ
H]þ 651.14504, found 651.14540.
3-(4-Chlorophenyl)-2-ethylpyrido[2,3-d]pyrimidin-4(3H)-
one (8). HOAc (100 g, 1.5 mol, 0.25 equiv) was added to a mixture of 9
(1530 g, 6.2 mol, 1 equiv) and 1,1,1-triethoxypropane (4350 g, 24.8 mol,
4 equiv). The reaction mixture was heated to 60 °C, maintained for 18 h,
and then cooled to 20 °C and stirred for an additional 48 h. The mixture
was filtered, then the filtrate was washed with hexane (2 ꢀ 1.0 L) and
dried under vacuum to afford 1490 g of 8 (84%) as an off-white solid
1
(mp (DSC) 172-183 °C, minimum 179 °C): H NMR (400 MHz,
DMSO-d6) δ 8.98 (dd, J = 4.5, 2.0 Hz, 1H), 8.48 (dd, = 7.6, 2.1 Hz, 1H),
7.66 (d, J = 8.6 Hz, 2H), 7.54 (m, 3H), 2.38 (q, J = 7.6 Hz, 2H), 1.15 (t, J
= 7.6 Hz, 3H); 13C (100 MHz, DMSO-d6) δ 162.0, 160.9, 157.2, 155.9,
136.0, 135.9, 133.8, 130.6 (2C), 129.7 (2C), 122.2, 115.6, 28.9, 10.4; IR
3445, 3259, 2990, 1682, 1599, 1584, 1566, 1487, 1432, 1367, 1254, 1087,
1015, 820 cm-1; exact mass m/z calcd for C15H14ClN3O [M þ H]þ
286.07471, found 286.07395.
(þ)-2-(1-(R)-Aminoethyl)-3-(4-chlorophenyl)pyrido[2,3-
d]pyrimidin-4(3H)-one ((þ)-2). Hexamethyldisilylazide (7.6 mL,
36.5 mol, 3.0 equiv) was added to a solution of 19 (5.1 g, 12.18 mol, 1.0
equiv), I2 (4.6 g, 18.3 mol, 1.5 equiv), and CH2Cl2 (77 mL). The
reaction mixture was maintained for 18 h, then washed with 10 wt %
aqueous Na2S2O3 (2 ꢀ 50 mL) and brine (50 mL), dried over
Na2SO4, and concentrated in vacuo to a brown oil, which was
dissolved in CH2Cl2 (50 mL) and treated with trifluoroacetic acid
(25 mL). The reaction mixture was maintained for 1 h, then poured
into HCl (1 N aqueous, 50 mL). The layers were separated, and then
the aqueous layer was basified to pH 10 with NaOH (5 N aqueous)
and extracted with CH2Cl2 (2 ꢀ 25 mL). The combined organic
extracts were reduced in vacuo to 10 mL, then MTBE (50 mL) was
added slowly to precipitate the product. The mixture was filtered and
washed with MTBE (5 mL) to yield 3.0 g of (þ)-2 (82%) as a pale
yellow solid (mp (DSC) 175-206 °C, exothermic decomposition,
maximum 182 °C). All spectral data for (þ)-2 match that of rac-2, and
the enantiomeric ratio was determined to be 99:1 by using the chiral
SFC method described for 13.
2-Amino-N-(4-chlorophenyl)nicotinamide (9). Et3N (1993 g,
19.7 mol, 2 equiv) was added to a mixture of 6 (1360 g, 9.9 mol, 1 equiv),
4-chlorobenzenamine (1257 g, 9.9 mol, 1 equiv), EDCI HCl (2070 g, 10.8
3
mol, 1.1 equiv), HOBt (122 g, 0.9 mol, 0.1 equiv), and DCM (8.0 L). The
mixture was stirred for 21 h then concentrated in vacuo. The residue was
triturated with water (2.5 L) and the resulting mixture was filtered. The filtrate
was washed with water (2.5 L), 5% Na2CO3 (2ꢀ2.0 L), water (2ꢀ2.5 L),
and MTBE (2ꢀ2.0 L) and dried under vacuum overnight to afford 1540 g of
9 (72%) as a white solid (mp (DSC) 207-218 °C, minimum 214 °C): 1H
NMR (400 MHz, DMSO-d6) δ 10.27 (br s, 1H), 8.13 (dd, J = 6.5, 2.0 Hz,
1H), 8.04 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 9.1 Hz, 2H), 7.40 (d, J = 9.1 Hz,
2H), 6.99 (br s, 2H), 6.67 (dd, J = 7.5, 4.5 Hz, 1H); 13C (100 MHz, DMSO-
d6) δ 165.3, 157.4, 150.4, 136.8, 135.9, 127.1, 125.8, 120.8, 110.0, 108.6; IR
3444, 3258, 1639, 1611, 1515, 1491, 1441, 1397, 1309, 1251, 1089, 1014, 821
cm-1; exact mass m/z calcd for C12H11ClN3O [M þ H]þ 248.05906, found
248.05842.
2-(1-Aminoethyl)-3-(4-chlorophenyl)pyrido[2,3-d]pyrimi-
din-4(3H)-one (rac-2). Sodium azide6 (480 g, 7.38 mol, 1.5 equiv)
was added to a solution of 10 (1575 g, 4.92 mol, 1.0 equiv) and DMF
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2-(1-Chloroethyl)-3-(4-chlorophenyl)pyrido[2,3-d]pyri-
midin-4(3H)-one (10). N-Chlorosuccinimide (765 g, 5.72 mol, 1.1
equiv) was added over 3 h to a solution of 8 (1484 g, 5.20 mol, 1.0 equiv)
and HOAc (5600 mL) at 40 °C, maintaining the internal temperature
between 50 and 53 °C, then maintained at 40 °C for an additional 19 h
before it was cooled to 20 °C and poured into water (15 L). The mixture
was filtered, then the filtrate was washed with water (2 ꢀ 7.5 L), 1% NaOH
(10 L), and water (2 ꢀ 5 L) and dried under vacuum to afford 1580 g of 10
(95%) as a white solid (mp (DSC) 201-219 °C, minimum 217 °C): 1H
NMR (400 MHz, CDCl3) δ 9.05 (dd, J1 = 2.0 Hz, J2 = 4.5 Hz, 1H), 8.61
(dd, J1 = 2.1 Hz, J2 = 8.1 Hz, 1H), 7.55 (m, 4H), 7.16 (dd, J1 = 2.5 Hz, J2 =
8.5 Hz, 1H), 4.59 (q, J = 7.0 Hz, 1H), 1.95 (d, J = 7.0 Hz, 3H); 13C (100
MHz, CDCl3) δ 162.7, 158.0, 157.3, 156.8, 137.1, 136.6, 134.0, 131.4,
130.6, 130.5, 126.5, 123.6, 116.9, 52.7, 22.0; IR 3444, 3260, 1682, 1601,
1587, 1568, 1433, 1367, 1259, 1088, 798, 719 cm-1; exact mass m/z calcd
for C15H12Cl2N3O [M þ H]þ 320.03574, found 320.03467.
J = 8.61, 2.54 Hz, 1H), 7.24 (dd, J = 8.61, 2.54 Hz, 1H), 7.48 (dd, J = 7.83,
4.0 Hz, 1H), 7.57-7.61 (m, 2H), 8.59 (dd, J = 7.83, 1.96 Hz, 1H), 9.01
(dd, J = 4.60, 2.05 Hz, 1H), NH not visible in spectrum; 13C NMR (100
MHz, CDCl3) δ 21.2, 28.1, 28.2, 36.4, 50.7, 50.8, 52.2, 53.4, 55.7, 115.9,
122.5, 129.3, 129.6, 130.4, 134.2, 136.0, 136.7, 156.3, 157.4, 162.3, 164.0;
IR 3563, 2927, 1684, 1597, 1583, 1569, 1491, 1433, 1279, 1255, 1123,
1092, 851, 799 cm-1; exact mass m/z calcd for C21H23ClN4O3S [M þ
H]þ447.12576, found 447.12475.
(R)-Methyl 2-(2-(tert-Butoxycarbonyl)propanamido)nico-
tinate (18). 16 (35.1 g, 206 mmol, 1.0 equiv), 17 (42.6 g, 226 mmol,
1.1 equiv), Pd(OAc)2 (924 mg, 4.1 mmol, 0.02 equiv), Xantphos (4.8 g,
8 mmol, 0.04 equiv), K2CO3 (56.9 g, 412 mmol, 2.0 equiv), and THF
(265 mL) were combined and heated to reflux and stirred for 16 h. The
reaction mixture was filtered through Celite and the filter cake was
washed with IPAC (600 mL). The combined organic solutions were
then washed with H2O (300 mL) and brine (300 mL) before a full
solvent switch to IPAC was performed. The slurry in IPAC (130 mL)
was heated to 72 °C, and then heptane (51 mL) was added to the
solution, which was seeded with 18 (575 mg) and cooled to rt before
additional heptane (339 mL) was added. The slurry was stirred over-
night and then filtered and the filter cake was washed with 75% heptane/
IPAC (102 mL) and dried overnight in the oven at 40 °C to afford 46.1 g
of 18 (70%) as a pale yellow solid (mp (DSC) 180-196 °C, minimum
193 °C): 1H NMR (400 MHz, CDCl3) δ 11.23 (br s, 1H, NH), 8.64 (dd,
J = 4.7, 1.3 Hz, 1H, ArH), 8.33 (dd, J = 8.0, 1.5 Hz, 1H, ArH), 7.09 (dd, J
= 8.0, 5.0 Hz, 1H, ArH), 5.19 (br s, 1H, NH), 4.64 (br s, 1H, CH), 3.95
(s, 3H, CH3), 1.51 (d, J = 7.5 Hz, 3H, CH3), 1.47 (s, 9H, CH3); 13C
NMR (100 MHz, CDCl3) δ 171.5, 166.9, 155.3, 153.1, 152.0, 139.9,
118.5, 111.8, 79.9, 52.8, 51.6, 28.3 (3C), 18.8; IR 3292, 2928, 1701, 1687,
1595, 1581, 1531, 1494, 1366, 1279, 1251, 1163, 1124, 1091, 1065, 1017,
858, 789 cm-1; exact mass m/z calcd for C15H22N3O5 [M þ H]þ
324.1560, found 324.1543.
(R)-tert-Butyl-1-(3-((4-chlorophenyl)carbamoyl)pyridin-2-
ylamino)-1-oxopropan-2-ylcarbamate (19). A solution of iso-
propylmagnesium chloride and THF (61 mL of a 2.0 M solution in THF,
121 mmol, 4 equiv) was added via dropwise addition funnel to a solution
of 18 (9.8 g, 30 mmol, 1.0 equiv), 4-chloroaniline (5.4 g, 42 mmol, 1.4
equiv), and THF (40 mL) at 2 °C maintaining the internal temperature
<25 °C. After 10 min of stirring, the cooling bath was removed and the
solution was allowed to warm to ambient temperature and maintained
for 2 h. The solution was poured into 1 N HCl (125 mL) and the layers
were separated. The aqueous layer was extracted with EtOAc (125 mL)
and the combined organic extracts were dried (Na2SO4), filtered, and
concentrated in vacuo. The crude residue was purified by silica gel
chromatography (1% MeOH/DCM) to afford 10.2 g of 19 (80% yield)
as a light yellow solid (mp (DSC) 233-244 °C, minimum 227 °C): 1H
NMR (400 MHz, DMSO-d6) δ 10.59 (br s, 1H, NH), 10.40 (br s, 1H,
NH), 8.50 (d, J = 4.5 Hz, 1H, ArH), 8.01 (d, J = 4.0 Hz, 1H, ArH), 7.70
(d, J = 8.5 Hz, 2H, ArH), 7.37 (d, J = 8.5 Hz, 2H, ArH), 7.32 (dd, J = 7.5,
4.5 Hz, 1H, ArH), 4.22-4.13 (m, 1H, CH), 1.32 (s, 9H, CH3), 1.16
(d, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 171.8, 165.
1, 155.0, 149.5, 148.2, 138.26, 137.7, 128.3 (3C), 126.8, 124.0, 121.2,
119.7, 77.8, 50.0, 28.0 (3C), 17.3; IR 3233, 2979, 2931, 1685, 1653,
1586, 1516, 1492, 1446, 1395, 1330, 1306, 1246, 1159, 1094, 1074, 823,
773 cm-1; exact mass m/z calcd for C20H24ClN4O4 [M þ H]þ
419.1486, found 419.1480.
2-(R)-(1-Aminoethyl)-3-(4-chlorophenyl)pyrido[2,3-d]py-
rimidin-4(3H)-one Dibenzoyl-(þ)-p-methoxy-D-tartrate Water/
Isopropanol Solvate (13). Di-(þ)-(p-anisoyl)-D-tartrate (3.95 kg,
92 wt %, 8.7 mol) was dissolved in aqueous IPA (39 L, 38.4 wt % water).
A total of 4.8 L of this DAT solution was added to a 100 L reactor
containing a solution of rac-2 (5.22 kg, 17.4 mol) and aqueous IPA (33
L, 38.4 wt % water) and seeded (266 g of 13). An additional 2 L of DAT
solution was added to wash the seed into the reactor, and the remaining
DAT solution was added over 3.5 h. The reaction mixture was stirred for
an additional 10 h at which time the mother liquor was assayed by chiral
HPLC at 64% ee, enriched in the undesired enantiomer. The reaction
mixture was heated to 42 °C for 1.5 h and cooled over 1.5 h to 22 °C. The
reaction mixture was filtered through a 25 μm filter at rate of approxi-
mately 2.2 L/min. The filter cake was washed with aqueous IPA (2 ꢀ
12.5 L, 38.4 wt % water) and the solids dried at 40 °C for 4 d to afford
3.93 kg (38%, 99.7% ee as determined on a Berger SFC, Chiralcel AD-H,
5 μm, 4.6 ꢀ 250 mm, 35 °C, 4 mL/min, 100 psi, isocratic mixture of
65:35 CO2:0.1% isopropylamine/MeOH) of IPA/water solvate 13 as a
white solid (mp (DSC) 131-149 °C, minimum 139 °C).
2-((R)-(1-(1,1-Dioxohexahydro-1λ6-thiopyran-4-yl)methyla-
mino)ethyl)-3-(4-chlorophenyl)pyrido[2,3d]pyrimidin-4(3H)-
one (14). Acetic acid (205 g, 3.4 mol, 0.1 equiv) and NaBH(OAc)3
(2893 g, 13.7 mol, 2.0 equiv) were charged to a solution of 13 (3740 g of
13 ≈ 2050 g of (þ)-2, 6.8 mol, 1.0 equiv) and DCM (18.5 L, 5 vol) at -
4 °C. The contents of the reactor were aged for 30 min, then a solution of
48 (1548 g, 9.5 mol, 1.4 equiv) and DCM (10900 g, 142 mg of 4/g of
solution) was charged over 1 h keeping the internal temperature
below -5 °C, at which time e5 LCAP of 13 was observed by HPLC
analysis. Saturated aqueous NaHCO3 (39.3 kg, 10 vol) was charged at a
rate to control gas evolution, and the reaction mixture was warmed to
24 °C. The phases were separated, and the organic phase was washed
sequentially with saturated aqueous NaHCO3 (41.0 kg, 11 vol) and
saturated aqueous NaCl (49.9 kg, 13 vol). A distillative solvent switch to
MeOH was performed, and the MeOH solution was concentrated to ∼2
volumes, diluted with EtOH (2 vol), and warmed to 40 °C. Holding this
solution resulted in self-seeding; however, 2 wt % of freebase 15 (58.9 g)
was charged as seed to control the process. The reaction mixture was
cooled to 25 °C over 2 h then diluted with EtOH (6 vol) over 1.5 h. The
reaction mixture was cooled to 0 °C and held until the supernatant
concentration was e6 mg/g then it was filtered. The wet cake was
washed with 1 volume of 0 °C ethanol and dried at 40 °C under vacuum
with an N2 sweep until e1 wt % loss on drying was observed to afford
2871 g of product 14 (92.4 wt %, 87.1% corrected isolated yield) was
collected as a white solid (mp (DSC) 124-138 °C, minimum 133 °C):
1H NMR (400 MHz, CDCl3) δ 1.30 (d, J = 6.65 Hz, 3H), 1.50-1.55 (m,
1H), 1.71- 1.83 (m, 2H), 2.03-2.12 (m, 1H), 2.17 (dd, J = 11.25, 7.34
Hz, 1H), 2.20-2.30 (m, 1H), 2.57 (dd, J = 11.25, 5.97 Hz, 1H), 2.88-
2.91 (m, 2H), 2.97-3.05 (m, 2H), 3.41 (q, J = 6.52 Hz, 1H), 7.19 (dd,
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details for com-
b
pound 15 and copies of 1H NMR and 13C NMR spectral data of
1, rac-2, 8, 9, 10, 11, 14, 15, 18, and 19. This material is available
free of charge via the Internet at http://pubs.acs.org.
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ARTICLE
’ AUTHOR INFORMATION
(9) Initial quantities of sulfone aldehyde 4 (<100 g) were synthe-
sized by oxidizing the commercially available sulfide aldehyde:
Corresponding Author
*E-mail: johannc@amgen.com and bburke@amgen.com.
’ ACKNOWLEDGMENT
The authors wish to thank Jenny Chen for her help with
development of chiral HPLC methods and ee determinations.
Subsequent process development provided procedural improve-
ments and the use of the corresponding bisulfite adduct as a
control point allowed the delivery of >4 kg of this precursor to 4.
For details see: Bio, M. M.; Hansen, K.; Gipson, J. Org. Proc. Res.
Dev. 2008, 12, 892-895, along with references cited therein.
(10) At the time of this work, the use of bisulfite adducts in reductive
aminations without prior conversion to the aldehyde was not known.
However, since the completion of this work, a report has been published
describing this application. See: Chennagiri, R. P.; Neelakandha, S. M.
Synthesis 2009, 23, 4032–4036.
’ REFERENCES
(1) Loetscher, M.; Gerber, B.; Loetscher, P.; Jones, S. A.; Piali, L.;
Clark-Lewis, I.; Baggiolini, M.; Moser, B. J. Exp. Med. 1996, 184 (3), 963.
(2) For more information about the development of the medicinal
chemistry CXCR3 antagonist program at Amgen, see: (a) Du, X.; Chen, X.;
Mihalic, J. T.; Deignan, J.; Duquette, J.; Li, A. R.; Lemon, B.; Ma, J.; Miao, S.;
Ebsworth, K.; Sullivan, T. J.; Tonn, G.; Collins, T. L.; Medina, J. C. Bioorg.
Med. Chem. Lett. 2008, 18, 608–613. (b) Du, X.; Gustin, D. J.; Chen, X.;
Duquette, J.; McGee, L. R.; Wang, Z.; Ebsworth, K.; Henne, K.; Lemon, B.;
Ma, J.; Miao, S.; Sabalan, E.; Sullivan, T. J.; Tonn, G.; Collins, T. L.; Medina,
J. C. Bioorg. Med. Chem. Lett. 2009, 19, 5200–5204. (c) Chen, X.; Henne, K.;
Medina, J. C. Patent WO 2009094168, 2009.
(11) These impurities were isolated and characterized as cyclized
products A and B:
(3) Efforts to apply our recently developed aza-Wittig approach to
construct the pyrido[2,3-d]pyrimidine core were met with failure: Chan, J.;
Faul, M. Tetrahedron Lett. 2006, 47, 3361–3363. The requisite imide was
unstable and prone to hydrolysis. Therefore, this strategy was abandoned.
(4) No products arising from self-coupling of 2-aminonicotinic acid
were observed.
(5) Chan, J.; Gustin, D.; Divirgilio, E.; Guram, A.; Faul, M. M.
Synthesis 2007, 23, 3678–3682.
(6) Sodium azide is an extremely toxic reagent. For safety data
regarding its use, see: Urben, P. G. Bretherick’s Handbook of Reactive
Chemical Hazards, 7th ed.; Elsevier Ltd.: Amsterdam, The Netherlands,
2007; pp 1886-1888.
(7) Interestingly, attempts to reduce a related azide with trimethyl-
phosphine exclusively produced a triazole based on 1H and 13C NMR
spectra and the mass spectrum. This transformation presumably pro-
ceeds through the cyclization of phosphine bound azide onto the
electrophilic C2 of the pyrido[2,3-d]pyrimidine followed by loss of
trimethylphosphine oxide and tautomerization to give the triazole:
(12) Running the reductive animation under these conditions
mandated that the reaction temperature be kept under 0 °C to prevent
the formation of another impurity (observed by LC and supported by
mass spectrometry) that is speculated to be the boron complex:
(13) In looking at Schotten-Baumann approaches, the acyl chloride
derived from 4-trifluoromethyl-3-fluorophenyl acetic acid was found to
be unstable and as a result, this approach was abandoned. CDMT was
also an effective coupling reagent.
(14) During the long-term route selection, an unexpected oxidative
coupling of aldehydes and sulfonamides was discovered: Chan, J.;
Baucom, K. D.; Murry, J. A. J. Am. Chem. Soc. 2007, 129, 14106–14107.
(15) Although a formal cost comparison between 2-aminonicotinic
acid and 2-chloronicotinic acid was not performed, a preliminary
investigation indicates that the latter is more readily available from a
wider range of manufacturers and significantly less expensive.
(16) Klapars, A.; Antilla, J. C.; Huang, X.; Buchwald, S. L. J. Am.
Chem. Soc. 2001, 123, 7727–7729. Yin, J.; Buchwald, S. L. J. Am. Chem.
Soc. 2002, 124, 6043–6048.
(17) For Boc-protection, see: Lucet, D.; Le Gall, T.; Mioskowski, C.;
Ploux, O.; Marquet, A. Tetrahedron: Asymmetry 1996, 7, 985–988. For
amide formation see: Pozdnev, V. F. Tetrahedron Lett. 1995, 36, 7115–7118.
(18) 2-Chloronicotinonitrile also performed under similar condi-
tions to couple with alanine amide 17 in 38% isolated yield.
(19) Notably, it was necessary to synthesize the methyl nicotinate 16
as attempts to directly couple either 2-chloronicotinic acid or with 17
were met with failure. Very low conversions of the starting nicotinic acid
derivatives were observed at 5% palladium loadings. Furthermore, when
2-chloro-N-(4-chlorophenyl)nicotinamide was reacted with a full
equivalent of palladium acetate and either Xantphos or DPPF
(8) One volume (abbreviated “vol”) is defined as 1 mL of solvent per 1
g of solvate. In this case 15 volumes refers to 15 mL of IPA:water mixtures
per gram of rac-2.
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ARTICLE
conditions, insoluble solids formed within 5 min that were tentatively
assigned to be a mixture of palladacycles D and E based on 31P{1H}
NMR resonances:
(20) Kshirsagar, U. A.; Mhaske, S. B.; Argade, N. P. Tetrahedron Lett.
2007, 48, 3243–3246.
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