Synthesis of 5-(1-H or 1-alkyl-5-oxopyrrolidin-3-yl)-8-hydroxy-[1,6]-naphthyridine-7-carboxamide inhibitors of HIV-1 integrase

Article abstract of DOI:10.1016/j.bmcl.2008.08.038

HIV-1 integrase catalyzes the insertion of viral DNA into the genome of the host cell. Integrase inhibitor N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide selectively inhibits the strand transfer process of integration. 4-Substituted pyrrolidinones possessing various groups on the pyrrolidinone nitrogen were introduced at the 5-position of the naphthyridine scaffold. These analogs exhibit excellent activity against viral replication in a cell-based assay. The preparation of these compounds was enabled by a three-step, two-pot reaction sequence from a common butenolide intermediate.

Full text of DOI:10.1016/j.bmcl.2008.08.038

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5307–5310
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 5-(1-H or 1-alkyl-5-oxopyrrolidin-3-yl)-8-hydroxy-[1,6]-
naphthyridine-7-carboxamide inhibitors of HIV-1 integrase
a
a
a
c
Jeffrey Y. Melamed a, , Melissa S. Egbertson , Sandor Varga , Joseph P. Vacca , Greg Moyer ,
Lori Gabryelski c, Peter J. Felock b, Kara A. Stillmock b, Marc V. Witmer b, William Schleif c,
Daria J. Hazuda b, Yvonne Leonard d, Lixia Jin d, Joan D. Ellis d, Steven D. Young a
*
a Department of Medicinal Chemistry, Merck Research Laboratories, 770 Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
b Department of Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA
c Department of Viral Vaccine Research, Merck Research Laboratories, West Point, PA 19486, USA
d Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2008
Accepted 12 August 2008
Available online 14 August 2008
HIV-1 integrase catalyzes the insertion of viral DNA into the genome of the host cell. Integrase inhibitor
N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide selectively inhibits the strand transfer
process of integration. 4-Substituted pyrrolidinones possessing various groups on the pyrrolidinone
nitrogen were introduced at the 5-position of the naphthyridine scaffold. These analogs exhibit excellent
activity against viral replication in a cell-based assay. The preparation of these compounds was enabled
by a three-step, two-pot reaction sequence from a common butenolide intermediate.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
HIV
Integrase
Antiviral
Pyrrolidinone synthesis
Butenolide
1,6-Naphthyridine
The HIV-1 integrase enzyme is responsible for the insertion of the
viral genetic code into the host genome. Integrase assembles onto
double-stranded viral DNA in the cytoplasm and processes the 30
ends to prepare for viral DNA integration. The resultant pre-integra-
tion complex then enters the nucleus, where integrase catalyzes vir-
al DNA insertion (strand transfer) into the host DNA.1 Many
laboratories have pursued discovery of integrase inhibitors suitable
for dosing in patients. Recently, raltegravir was approved as the first
integrase inhibitor to treat HIV-infected patients.1d–f
R'
N
O
R
O
N
N
N
F
F
N
H
N
H
N
N
O
OH
O
OH
1
2
O
R
Earlier reports from our laboratories have described a series of
8-hydroxyl-7-carboxamide-naphthyridine inhibitors. Compound
12 (Fig. 1) is a potent inhibitor of the strand transfer process of
HIV-1 integration with an IC50 of 33 nM in the enzyme inhibition
assay.3 It also inhibits the replication of HIV-1 in cell culture with
an IC95 of 1250 nM in the presence of 10% fetal bovine serum
(FBS).4 Addition of 50% normal human serum (NHS) to the cell
culture results in a fourfold loss in efficacy (IC95 = 5000 nM), as a
result of the compound’s high affinity for serum protein (99.2%
protein bound).
N
F
N
H
N
N
O
OH
3
Figure 1. HIV-integrase naphthyridine inhibitors.
HIV-1 replication in cell culture compared to 1.5 The authors sug-
gested that sp3 hybridized linkages attached at the 5-position of
the naphthyridine-based inhibitors were important for HIV-1 inhi-
bition in cells. Among a number of alternative substituents pro-
posed, the 4-substituted pyrrolidinone moiety of naphthyridine 3
A series of 5-dihydrouracil 8-hydroxy-[1,6]-naphthyridine-7-
carboxamides 2 (Fig. 1) was reported to more potently inhibit
* Corresponding author. Tel.: +1 215 652 9101; fax: +1 215 652 7310.
E-mail address: jeffrey_melamed@merck.com (J.Y. Melamed).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.038

5308
J. Y. Melamed et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5307–5310
appeared to be an attractive target due to its low molecular weight
relative to other polar heterocyclic systems. In this letter, we
describe a series of 5-(1-H or 1-alkyl-5-oxopyrrolidin-3-yl)-8-hy-
droxy-[1,6]-naphthyridine-7-carboxamides, which inhibit HIV-1
viral replication in cell culture.
Depicted in Scheme 1 is our synthetic route developed to allow
for efficient introduction of various substituents on the pyrrolidi-
none nitrogen toward the end of the synthesis, while circumvent-
ing protection and deprotection of the 1,6-naphthryridine –OH.
Another key consideration in development of the synthesis was
that the 1,6-naphthyridine core does not tolerate strongly basic,
oxidizing, or reducing conditions.
The synthesis of inhibitor 9 starts with treatment of 5-bro-
monaphthyridine 36 (Scheme 1) with 4-tributylstannylfuran-
2(5H)-one7 4 and PdCl2(PPh3)2, to give 5 in excellent yield. It was
anticipated that treatment of 5 with methylamine would provide
compound 6, which could be cyclized to the desired unsaturated
pyrrolidinone 7. However, after treating 5 with methylamine, hy-
droxy amide 6 was present in only minute quantities. Detailed
NMR analysis revealed that the major products were trans and
cis aminopyrrolidinones 8a and 8b (8a:8b, 2:1).8 The cis isomer
8b converted on standing in d6-DMSO at room temperature to
the trans diastereomer 8a following re-running the NMR after 4
days. The trans diastereomer 8a is active against replication of
HIV with an IC95 of 94 nM (NHS). Another minor component iso-
lated from the reaction mixture was identified to be 8c.9 Com-
pound 8c is potent against integrase in the strand transfer
enzyme assay (IC50 = 40 nM), but shows ꢀ20-fold less potency in
cell culture (IC95 = 1000 nM NHS).
Pyrrolidinone 9 (Table 1) is a potent inhibitor of strand transfer
with an IC50 of 20 nM in the enzyme inhibition assay. It inhibits the
replication of HIV-1 in cell culture with an IC95 of 31 nM in the
presence of 10% fetal bovine serum (FBS). Addition of 50% normal
human serum (NHS) to the cell culture results in a fourfold loss
in potency (IC95 = 125 nM).13
It is worth noting that the reaction of 5 with ammonia and other
alkyl amines, including the more sterically hindered isopropyl-
amine, provided pyrrolidinones 10–12 in good yields. All of the com-
pounds are intrinsically potent against the Integrase enzyme
(IC50 6 40 nM). Compound 10 is the most active compound against
replication of HIV-1 in cell culture with an IC95 of 16 nM (FBS). Due
to the relatively high plasma protein binding (98.9%), this compound
shifts eightfold to 125 nM in the presence of 50% NHS. The unsubsti-
tuted analog 12 also exhibits good cell potency (FBS) and shows only
a twofold loss in potency in the presence of NHS. We were unable to
determine the protein binding of 12 due to low solubility.
The improved cell activity of 12 relative to 9–11 prompted us to
introduce polar substituents to reduce plasma protein binding. The
synthetic route that was developed was found to tolerate more func-
tionalized amines as exemplified by glycine dimethylamide, thus
enabling the synthesis of 13. Compound 13 exhibited significantly
lower protein binding (94.8%), and proved to be among the most po-
tent inhibitors in this class of compounds in the presence of NHS
with an IC95 of 63 nM (twofold loss of potency when assayed in
10% FBS) compared to compound 9 (fourfold loss in potency from
FBS).
We recently reported that substitution at the 2-position of the
benzyl moiety with polar substituents led to integrase inhibitors
with improved physical properties and increased antiviral potency
in the presence of NHS.2 Application of this finding led to pyrrolid-
inones 14 and 15. Addition of a methyl sulfone16 to 9 provides 14,
which shows very good antiviral activity in the presence of NHS
with an IC95 of 63 nM (twofold loss of potency from FBS). Similar
activity was observed with the ethyl analog 15. These results cor-
relate well with decreased protein binding and log P (see Table 1).
The rat pharmacokinetic (PK) profiles of representative com-
pounds are shown in Table 2. Compound 9 exhibits low clearance
and good oral bioavailability. The more potent analogs 13 and 14
displayed higher clearance and low oral bioavailability.
Diastereomers 8a and 8b were taken up in TFA and heated for
2 h to effect elimination of methylamine, providing a 4:1 insepara-
ble mixture of alkene isomers 7a and 7b.10 The mixture exhibits
potent antiviral activity in cells with an IC95 of 78 nM, but shows
a >10-fold decrease in potency in the presence of NHS. The reduc-
tion in potency is consistent with the high protein binding of 7a
and 7b (99.7% protein bound). Treatment of the crude mixture of
8a and 8b with triethylsilane in the presence of TFA at 75 °C for
1.5 h provides the desired racemic naphthyridine 9 in a two-step,
one-pot sequence.11 Further exploratory work on
a model
system showed that the reaction works well with other aryl
systems.12
O
O
O
Me
O
N
NH
Me
Br
N
H
O
N
N
F
O
F
F
F
b
a
N
N
N
N
+
H
H
+
H
H
N
N
N
N
N
N
N
SnBu3
O
OH
O
OH
O
OH
O
OH
3
4
5
8c
8a: trans
8b: cis
d
c
O
Me
O
N
O
N
Me
Me
N
N
N
O
Me
N
OH
F
F
F
+
N
H
H
H
N
N
N
N
N
N
7b
O
OH
O
OH
O
OH
e
9
6
7a
Scheme 1. Reagents: (a) PdCl2(PPh3)2, toluene, reflux (92%); (b) MeNH2, MeOH, 70 °C, sealed tube (48% of a 2.1/1 mixture of 8a:8b); (c) TFA, sealed tube, 105 °C (76%); (d) TFA
105 °C, sealed tube, 2 h followed by TFA, Et3SiH, 75 °C, sealed tube (48%, one pot, 2 steps); (e) TFA, Et3SiH, sealed tube, 75 °C.

J. Y. Melamed et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5307–5310
5309
Table 1
Inhibition of HIV-1 integrase catalytic activities and HIV-1 replication in cells by
carboxamides
a series of 5-(1-H or 1-alkyl-5-oxopyrrolidin-3-yl)-8-hydroxy-[1,6]-naphthyridine-7-
O
R
N
F
N
H
N
N
R'
O
OH
Compound
R
R0
Inhibition of strand transfer IC50
(nM)a
Antiviral activity IC95 (nM) (10%
FBS)b
Antiviral activity IC95 (nM) (50%
NHS)b
%Prot.Bndc Log
Pd
9
Me
Et
i-Pr
H
CH2C(O)NMe2
Me
Et
H
H
H
H
H
20
14
40
24
33
31 (n = 3)
16 (n = 2)
31 (n = 2)
42 (n = 3)
31 (n = 3)
31 (n = 3)
38 (n = 5)
125 (n = 3)
125 (n = 3)
125 (n = 3)
83 (n = 3)
63 (n = 3)
63 (n = 3)
81 (n = 3)
98.8
98.9
97.8
insol.
94.8
97.0
96.0
2.3
2.7
2.7
n.d.
1.8
1.6
1.9
10
11
12
13
14
15
SO2Me 123
SO2Me 26
a
The strand transfer assay was performed with recombinant HIV-integrase (100 nM) preassembled on immobilized oligonucleotides using 0.5 nM DNA.2 Values are means
of at least two experiments with a lower limit of accuracy ꢀ5 nM, standard deviation is approximately twofold.
b
Antiviral activity was assessed by measuring the decrease in HIV-1 p24 core antigen in MT-4 human T-lymphoid cells/HIV-1 IIIb cultured in the presence of increasing
concentrations of inhibitor. Antiviral activity in cell culture (IC95) is the drug concentration, which inhibits 95% viral growth relative to control. FBS: run with 10% fetal bovine
serum, NHS: run with 50% normal human serum, see Ref. 4
c
Measure of the percentage of test compound bound to human serum proteins, see Ref. 14.
Log P measurement (partition coefficient), see Ref. 15.
d
Marchand, C. Nat. Rev. Drug Disc. 2005, 4, 236; (c) Hazuda, D. J.; Felock, P.;
Witmer, M.; Wolfe, A.; Stillmock, K.; Grobler, J. A.; Espeseth, A.; Gabryelski, L.;
Schleif, W.; Blau, C.; Miller, M. D. Science 2000, 287, 646; (d) Markowitz, M.;
Table 2
Rat pharmacokinetics
O
R
Morales-Ramirez, J. O.; Nguyen, B. Y.; Kovacs, C. M.; Steigbigel, R. T.; Cooper, D.
A.; Liporace, R.; Schwartz, R.; Isaacs, R.; Gilde, L. R.; Penning, L.; Zhao, J.;
Teppler, H. J. Acquired Immune Deficiency Syndromes 2006, 43, 509; (e) Summa,
V.; Petrocchi, A.; Scarpelli, R.; Gardelli, C.; Muraglia, E.; Nizi, E.; Laufer, R.;
Gonzalez, P.; Fiore, F.; Jones, P.; Monteagudo, E.; Hazuda, D.; Witmer, M.;
Rowley, M. J. Med. Chem. 2008; f Crescenzi, B.; Gardelli, C.; Mruaglia, E.; Nizi, E.;
Orvieto, F.; Pace, P.; Pescatore, G.; Petrocchi, A.; Poma, M.; Rowley, M.; Sarpelli,
R.; Summa, V. Preparation of N-substituted hydroxypyrimidinone carboxamide
inhibitors of HIV integrase. Merck and Co, Inc.: USA. 02-GB04753[03035077],
217, 05-01-0003. WO 10-21-2002.
N
F
N
H
N
N
R'
O
OH
Compound
R
R’
Cl (mL/min/kg)b
T1/2 (h)b
% Fc
9a
Me
H
H
2.5
23.4
17.2
1.3
3.4
0.67
65
5
4
2. Egbertson, M. S.; Moritz, H. M.; Melamed, J. Y.; Han, W.; Perlow, D. S.; Kuo, M.
S.; Embrey, M.; Vacca, J. P.; Zrada, M. M.; Cortes, A. R.; Wallace, A.; Leonard, Y.;
Hazuda, D. J.; Miller, M. D.; Felock, P. J.; Stillmock, K. A.; Witmer, M. V.; Schleif,
W.; Gabryelski, L. J.; Moyer, G.; Ellis, J. D.; Jin, L.; Xu, W.; Braun, M. P.; Kassahun,
K.; Tsou, N. N.; Young, S. D. Bioorg. Med. Chem. Lett. 2007, 17, 1392.
3. Hazuda, D. J.; Felock, P.; Hastings, J. C.; Pramanik, B.; Wolfe, A. J. Virol. 1997, 71,
7005.
4. Vacca, J. P.; Dorsey, B. D.; Schleif, W. A.; Levin, R. B.; McDaniel, S. L.; Darke, P. L.;
Zugay, J.; Quintero, J. C.; Blahy, O. M.; Roth, E.; Sardana, V. V.; Schlabac, A. J.;
Graham, P. I.; Condra, J. H.; Gotlib, L.; Holloway, M. K.; Lin, J.; Chen, I.-W.;
Vastag, K.; Ostovic, D.; Anderson, P. S.; Emini, E. E.; Huff, J. R. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 4096.
5. Embrey, M. W.; Wai, J. S.; Funk, T. W.; Homick, C. F.; Perlow, D. S.; Young, S. D.;
Vacca, J. P.; Hazuda, D. J.; Felock, P. J.; Stillmock, K. A.; Witmer, M. V.; Moyer, G.;
Schleif, W. A.; Gabryelski, L. J.; Jin, L.; Chen, I. W.; Ellis, J. D.; Wong, B. K.; Lin, J. H.;
Leonard, Y. M.; Tsou, N. N.; Zhuang, L. Bioorg. Med. Chem. Lett. 2005, 15, 4550.
6. Anthony, N. J.; Gomez, R. P.; Young, S. D.; Egbertson, M.; Wai, J. S.; Zhuang, L.;
Embrey, M.; Tran, L.; Melamed, J. Y.; Langford, H. M.; Guare, J. P.; Fisher, T. E.;
Jolly, S. M.; Kuo, M. S.; Perlow, D. S.; Bennett, J. J.; Funk, T. W. Preparation of
(poly)azanaphthalenyl carboxamides as HIV integrase inhibitors. PCT Int. Appl.,
2002, 434 pp. CODEN: PIXXD2 WO 0230930 A2 20020418.
13a
14
CH2C(O)NMe2
Me
SO2Me
a
Compound dosed as Na salt.
Dosed iv in DMSO at 2 mg/kg.
Dosed po in 1% methylcellulose at 10 mg/kg.
b
c
In summary, we have developed an efficient route for the prepa-
ration of 4-[5-(1,6-naphthyridyl)]pyrrolidinones. The synthetic se-
quence permits late-stage variation of the lactam nitrogen
substituent without tedious protection/deprotection steps. This
methodology can also be used to prepare various 4-aryl substituted
pyrrolidinones. Pyrrolidinone 9 is a potent inhibitor of HIV-1 integr-
ase in cell culture. The compound exhibited low clearance and good
oral bioavailability in rat. Installation of polar substituents on the
pyrrolidinone nitrogen or the benzylamide provides compounds
with lower protein binding and reduced shift in antiviral activity.
7. Compound 4 was prepared by the procedure described by Hollingworth, G. J.;
Perkins, G.; Sweeney, J. J. Chem. Soc., Perkin Trans. 1, 1996, 1913. Compound 5
was prepared as follows: To
hydroxy-5-bromo-1,6-naphthyridine-7-carboxamide 3 (2.0 g, 5.32 mmol) in
anhydrous toluene (35 mL) under nitrogen were added
dichloro[bis(triphenylphosphine)]palladium (II) (0.374 g, 0.53 mmol) and 4-
(tributylstannyl)furan-2(5H)-one (1.98 g, 5.32 mmol). The solution was
heated to reflux for 4.5 h, cooled, and the solids that precipitated from the
solution were collected by vacuum filtration (1.85 g, 92%). The compound was
taken onto the next step without further purification. 1H NMR (400 MHz,
a stirring solution of N-(4-fluorobenzyl)-8-
Acknowledgments
The authors thank Carl Homnick for chiral HPLC support, Harri
Ramjit, Charles Ross, and Arthur Coddington for mass spectroscopy
analysis and Ken Anderson, Matt Zrada, and Bang-Lin Wan for pro-
viding protein binding and log P data. We thank David Evans and
Steve Pitzenberger for helpful discussions.
4
DMSO-d6),
d 9.77 (1H, t, J = 6.4 Hz), 9.23 (1H, d, J = 3.4 Hz), 8.92 (1H, d,
J = 8.0 Hz), 7.91–7.88 (1H, dd, J = 8.6, 4.2 Hz), 7.46–7.42 (2H, dd, J = 8.6, 5.6 Hz),
7.18 (2H, t, J = 8.9 Hz), 6.99 (1H, s), 5.69 (2H, s), 4.60 (2H, d, J = 6.4 Hz). ES HRMS
calcd for C20H14N3O4F (M+H): 380.1041, Found: 380.1050.
References and notes
8. To
a heavy wall glass pressure vessel was added 5 (0.83 g, 1.93 mmol)
dissolved in a saturated solution of methylamine in MeOH (15.0 mL). The
solution was heated to 55 °C for 2 h, cooled, and concentrated under reduced
pressure. The crude material was dissolved in DMF (2 mL) and injected on the
1. (a) Dayam, R.; Al-Mawsawi, L. Q.; Zawahir, Z.; Witvrouw, M.; Debyser, Z.;
Neamati, N. J. Med. Chem. 2008, 51, 1136; (b) Pommier, Y.; Johnson, A. A.;

5310
J. Y. Melamed et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5307–5310
Gilson autoprep using a Waters 6
18 min gradient of 95:5 to 5:95, H2O/CH3CN/0.1% TFA. The desired fractions
were combined and concentrated to afford 0.45 g (48%) of mixture of
diastereomers 8a and 8b, which were taken onto the next step. (NMR of
analytically pure trans diastereomer): 1H NMR (400 MHz, DMSO-d6), d 13.45
(1H, br s), 9.28 (1H, br s), 9.45–9.25 (1H, br s), 9.21 (1H, dd, J = 4.2, 1.4 Hz),
8.78–8.75 (1H, dd, J = 8.7, 1.3 Hz), 7.93–7.90 (1H, dd, J = 8.6, 4.2 Hz), 7.44–7.41
(2H, dd, J = 8.6, 5.6 Hz), 7.20 (2H, t, J = 8.9), 5.95 (1H, br s), 4.71–4.57 (3H, br s),
3.29–3.22 (1H, dd, J = 17.2, 9.6 Hz), 2.92 (3H, s), 2.64 (3H, s), 2.38–2.34 (1H, d,
l
M Nova Pak reverse phase column with an
room temperature (once it was determined by LC–MS that elimination of
methylamine had occurred). After cooling, triethylsilane (0.62 g, 5.31 mmol)
was added to the solution, and the reaction mixture was heated to 75 °C for
1.5 h. The mixture was cooled and concentrated under reduced pressure. The
crude mixture was re-dissolved in DMF (2 mL) and injected on the Gilson auto
a
prep using a Waters 6 lM Nova Pak reverse phase column with an 18 min
gradient of 95:5 to 5:95, water/CH3CN/0.1% TFA. The desired fractions were
combined and concentrated to afford 0.20 g, (48%) of 9 as an off-white solid.1H
NMR (400 MHz, DMSO-d6), d 13.55.
J = 18.5 Hz). ES HRMS calcd for
424.1781.
C
22H22N5O3F (M+H): 424.1786, Found:
12. Examples of the synthesis of various substituted 4-aryl-pyrrolidinones from
the corresponding butenolides:
9. Tricyclic compound 8c is the major product obtained upon prolonged reactions
O
O
CH3
times and/or higher reaction temperatures.
A
proposed mechanism for
O
N
formation of 8c:
Me
NH
Me
NH
R2
O
O
O
R2
Me
N
H
N
R1
R1
CH3
F
R2
F
N
N
H
N
H
N
Yielda
R2
N
N
H
.
O
O
OH
55%
H
H
H
H
N(CH3)2
60%
20%
O2
m-2
m-1
NO2
F
H
8c
H
CH3
49%
98%
1H NMR of 8c: (400 MHz, DMSO-d6), d 13.25 (1H, br s), 9.68 (1H, t, J = 6.4 Hz),
8.44 (1H, d, J = 7.3 Hz), 8.10 (1H, m), 7.95 (1H, s), 7.43 (2H, m), 7.19 (3H, m),
4.64 (2H, d, J = 6.5 Hz), 3.87 (3H, s), 3.68 (2H, s), 2.48 (3H, d, J = 4.5 Hz).
10. A mixture of 8a and 8b (0.1 g, 0.24 mmol) in TFA (3 mL) in a glass pressure
tube was heated to 105 °C for 2 h. The reaction mixture was cooled and
concentrated, then redissolved in DMF (1 mL). The solution was injected on
a) Isolated yield for two step process, unoptimized.
b) For conditions see Scheme 1, reactions b and d.
13. Resolution of 9 by chiral HPLC provides optically pure (+) and (ꢁ) isomers
which are equipotent in the enzyme and cellular assays.
the Gilson autoprep using a Waters 6 lM Nova Pak reverse phase column
14. Analytical HPLC/UV detection-based assay that measures the ability of
a
with an 18 min gradient of 95:5 to 5:95, H2O/CH3CN/0.1% TFA. The desired
fractions were combined and concentrated to afford 0.076 g (76%) of an
inseparable 4:1 mixture of diastereomers 7a and 7b. Major component of
mixture (7a): 1H NMR (400 MHz, DMSO-d6), d 13.84 (1H, br s), 9.75 (1H, br
s), 9.20 (1H, d, J = 4.2 Hz), 8.85 (1H, d, J = 8.7 Hz), 7.85 (1H, dd, J = 8.6,
4.2 Hz), 7.44 (2H, m), 7.19 (2H, m), 6.76 (1H, s), 4.80 (2H, s), 4.60 (2H, d,
J = 6.4 Hz), 3.05 (3H, s). ES HRMS calcd for C21H17N4O3F (M+H): 393.1357,
Found: 393.1357.
compound to bind with human plasma (primarily Albumin) in pH 7.4 buffer at
room temperature. Each value is the result of the average of three
determinations.
15. UV detection method determining the relative concentration of a methanol
solution of a compound partitioned between an octanol and pH 7.4 (KH2PO4/
NaOH) water layer. Log P = log (Octanol HPLC area)(Octanol dilution)/(Buffer
HPLC area)(Buffer dilution).
16. Perlow, D. S.; Juo, S.; Moritz, H. M.; Wai, J. S.; Egbertson, M. S. Synth. Commun.
2007, 37, 1887.
11. A mixture of 8a and 8b (0.45 g, 1.06 mmol) in TFA (3 mL) in a glass pressure
tube was heated to 105 °C for 2 h. The reaction mixture was then cooled to