2290 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Stauffer et al.
1H), 7.32 (s, 1H), 7.22 (d, J ) 8.15 Hz, 1H), 7.12 (d, J ) 10.07
Hz, 1H), 4.8 (m, 1H), 4.44 (m, 1H), 4.15 (s, 1H), 4.05 (d, J )
12.64 Hz, 1H), 2.64 (m, 2H), 2.04-1.98 (m,1H), 1.75-1.71
(m, 2H), 1.56-1.53 (m, 1H); HRMS (ES/FTMS) C26H25ClN4O3
calcd 477.1688 (M + 1), found: 477.1683. Second-eluting
diastereomer 17b (0.25 g, 17% yield) HPLC tR ) 2.62 min;
>99% purity, LC-MS m/z ) 477; 1H NMR, CDCl3: δ 8.49-
8.41 (m, 3H), 8.27 (s, 1H), 8.07 (d, J ) 8.97 Hz, 1H), 7.71 (d,
J ) 8.24 Hz, 1H), 7.52-7.42 (m, 4H), 7.35 (s, 1H), 7.32 (s, 1H),
4.68-4.62 (m, 1H), 4.53-4.50 (m, 1H), 4.15-4.11 (m, 2H), 2.55
(s, 1H), 2.43 (m, 1H), 2.05-1.95 (m, 1H), 1.84-1.77 (m, 1H),
1.72-1.65 (m, 1H), 1.57-1.50 (m, 1H); HRMS (ES/FTMS)
C26H25ClN4O3 calcd 477.1688 (M + 1), found: 477.1683.
retention time of 4.72 min (18b). Each diastereomer was then
run through a preparative reverse phase HPLC using a TFA
buffered water:acetonitrile gradient to give, after lyophiliza-
tion, solid TFA salts of 2-aza-9-hydroxy-9-fluorenylcarbonyl-
L-prolyl-2-aminomethyl-5-chlorobenzylamide. 18a (40 mg, 18%
yield) HPLC tR ) 2.25 min; >99% purity, LC-MS m/z ) 477;
1H NMR, CD3OD: δ 8.80-8.76 (m, 2H), 8.27 (d, J ) 5.68 Hz,
1H), 8.12-8.09 (m, 1H), 7.67-7.63 (m, 2H), 7.62-7.55 (m, 1H),
7.49 (d, J ) 2.01 Hz, 1H), 7.44-7.38 (m, 2H), 4.56 (d, J ) 15.2
Hz, 1H), 4.46-4.42 (m, 1H), 4.30 (d, J ) 15.39 Hz, 1H), 4.26
(s, 2H), 3.19-3.09 (m, 1H), 2.98-2.95 (m, 1H), 2.10 (m, 1H),
1.82-1.68 (m, 3H). HRMS (ES/FTMS) C26H25ClN4O3 calcd
477.1688 (M + 1), found: 477.1680. 18b (34 mg, 13% yield)
HPLC tR ) 2.28 min; >99% purity, LC-MS m/z ) 477); 1H
NMR, CD3OD: δ 8.76 (d, J ) 5.5 Hz, 1H), 8.64 (s, 1H), 8.16
(d, J ) 5.68 Hz, 1H), 8.08-8.06 (m, 1H), 7.66-7.60 (m, 2H),
7.59-7.57 (m, 1H), 7.50 (d, J ) 1.83 Hz, 1H), 7.45-7.39 (m,
2H), 4.60 (d, J ) 15.2 Hz, 1H), 4.47-4.44 (m, 1H), 4.30-4.26
(m, 3H), 2.72 (s, 2H), 2.08-2.04 (m, 1H), 1.73-1.65 (m, 2H),
1.64-1.56 (m, 1H). HRMS (ES/FTMS) C26H25ClN4O3 calcd
477.1688 (M + 1), found: 477.1658.
2-Aza-9-hydroxy-9-fluorenylcarbonyl-L-prolyl-2-ami-
nomethyl-5-chlorobenzylamide (18a,b). Step 1. 4-Phe-
nylpyridine-3-carboxylic acid methyl ester13 (3.8 g, 17.8 mmol)
was hydrolyzed to 4-phenylpyridine-3-carboxylic acid using the
procedure given in Step 2 for 17a,b (4.7 g, theoretical yield )
3.6 g, balance is NaCl). HPLC tR ) 1.80 min; LC-MS m/z )
1
200; H NMR, CD3OD: δ 8.91 (s, 1H), 8.66 (d, J ) 5.22 Hz,
1H), 7.48 (d, J ) 5.22 Hz, 1H), 7.46-7.41 (m, 5H).
Step 2. The compound from the previous step (4.7 g, 23.8
mmol) was converted to 2-aza-9-fluorenone using the procedure
given in Step 3 for 20a,b except that extraction of the product
into CH2Cl2 required prior neutralization of the polyphosphoric
acid with 50% aqueous NaOH to pH 4 (3.1 g, 71% yield). HPLC
tR ) 2.16 min; LC-MS m/z ) 182); 1H NMR, CDCl3: δ 8.87 (s,
1H), 8.75 (d, J ) 4.95 Hz, 1H), 7.75 (d, J ) 7.33 Hz, 1H), 7.65
(d, J ) 7.50 Hz, 1H), 7.62-7.55 (m, 1H), 7.51-7.46 (m, 2H);
HRMS (ES/FTMS) C12H7NO calcd 182.0601 (M + 1), found:
182.0613.
Step 3. The compound from the previous step (3.1 g, 17.2
mmol) was converted to 2-aza-9-hydroxy-9-fluorene carboxylic
acid methyl ester using the procedure given in Step 4 for
20a,b, except that additional amounts of zinc iodide and
trimethylsilyl cyanide and a longer reaction time were required
for complete conversion of the ketone to the trimethylsilyl
cyanohydrin. The product was purified on a silica gel column
using 95:5 CH2Cl2:MeOH as the mobile phase (1.3 g, 31%
yield). HPLC tR ) 2.06 min; LC-MS m/z ) 242); 1H NMR,
CDCl3: δ 8.67-8.65 (m, 2H), 7.75 (d, J ) 6.96 Hz, 1H), 7.57
(d, J ) 5.04 Hz, 1H), 7.51-7.44 (m, 3H), 4.50 (s, 1H), 3.64 (s,
3H); HRMS (ES/FTMS) C14H11NO3 calcd 242.0812 (M + 1),
found: 242.0780.
3-Aza-9-hydroxy-9-fluorenylcarbonyl-L-prolyl-2-ami-
nomethyl-5-chlorobenzylamide (19a,b). Step 1. To a solu-
tion of methyl 3-bromopyridine-4-carboxylate14 (5.9 g) in
toluene (140 mL) were added phenylboronic acid (4.3 g, 35.2
mmol), potassium carbonate (4.9 g, 35.3 mmol), and tetrakis-
(triphenylphosphine)palladium (1.4 g, 1.18 mmol). The mixture
was heated to reflux under nitrogen for 4 h, cooled to ambient
temperature, and filtered. The filtrate solvent was removed
in vacuo, and the residue was purified on a silica gel column
using 1:2 EtOAc:hexanes as the mobile phase. Pure fractions
were combined and the solvents were removed in vacuo to yield
3-phenylpyridine-4-carboxylic acid methyl ester (3.6 g, 80%
yield). HPLC tR ) 2.70 min; LC-MS m/z ) 214; 1H NMR,
CDCl3: δ 8.74 (overlapping s and d, 2H); 7.65 (d, J ) 8 Hz,
1H); 7.4-7.5 (m, 3 H), 7.30-7.35 (m, 2H); 3.80 (s, 3H).
Step 2. The product from the previous step (3.6 g, 16.7 mmol)
was converted to 3-phenylpyridine-4-carboxylic acid using the
procedure given in Step 2 for 17a,b (4.5 g, theoretical yield )
3.3 g, balance is NaCl). HPLC tR ) 1.22 min; LC-MS m/z )
200; HRMS (ES/FTMS) C12H9NO2 calcd 200.0706 (M + 1),
found: 200.0695.
Step 3. The product from the previous step (4.5 g, 18.2 mmol
theoretical) was converted to 3-aza-9-fluorenone using the
procedure given in Step 3 for 20a,b except that extraction of
the product into CH2Cl2 required prior neutralization of the
polyphosphoric acid with 50% aqueous NaOH to pH 4 (2.9 g,
95% yield over two steps). HPLC tR ) 2.41 min; LC-MS m/z )
Step 4. The product from the previous step (1.4 g, 5.8 mmol)
was converted to 2-aza-9-hydroxy-9-fluorene carboxylic acid
hydrazide using the same procedure as given in Step 5 for
17a,b (1.4 g, 99% yield). HPLC tR ) 0.8 min; LC-MS m/z )
1
242; H NMR, CD3OD: δ 8.58 (s, 1H), 8.55 (d, 1H), 7.9-7.85
1
182; H NMR, CD3OD: δ 8.94 (s, 1H), 8.65 (d, J ) 4.76 Hz,
(m, 1H), 7.78 (d, 1H), 7.59-7.54 (m, 1H), 7.52-7.47 (m, 2H).
HRMS (ES/FTMS) C13H11N3O2 calcd 242.0924 (M + 1).
Found: 242.0916.
1H), 7.82 (d, J ) 7.33 Hz, 1H), 7.71 (d, J ) 7.32 Hz, 1H), 7.64-
7.62 (m, 1H), 7.56 (d, J ) 4.40 Hz, 1H), 7.46-7.42 (m, 1H);
HRMS (ES/FTMS) C12H7NO calcd 182.0601 (M + 1), found:
182.0592.
Step 5. The product from the previous step (1.4 g, 5.8 mmol)
was converted to 2-aza-9-hydroxy-9-fluorenylcarbonyl-L-prolyl-
2-(tert-butyloxycarbonylaminomethyl)-5-chlorobenzylamide
using the procedure given in Step 6 for 20a,b (0.27 g, 8% yield).
Step 4. The product from the previous step (2.9 g, 16 mmol)
was converted to 3-aza-9-hydroxy-9-fluorene carboxylic acid
methyl ester using the procedure given in Step 4 for 20a,b
except that neat trimethylsilyl cyanide was used in the
conversion of the ketone to the trimethylsilyl cyanohydrin.
The product was purified on a silica gel column using 1:1
EtOAc:hexanes as the mobile phase (2.2 g, 57% yield). HPLC
1
HPLC tR ) 2.75 min; LC-MS m/z ) 577; H NMR, CDCl3: δ
8.90-8.66 (m, 2H), 7.98-7.90 (m, 2H), 7.68-7.54 (m, 2H), 7.49
(d, J ) 7.15 Hz, 1H), 7.30-7.23 (m, 2H), 7.20 (s, 1H), 5.45 (s,
1H), 4.63-4.54 (m, 2H), 4.31 (s, 2H), 2.46-2.26 (m, 2H), 2.03-
1.87 (m, 2H), 1.81-1.71 (m, 1H), 1.58-1.49 (m, 1H), 1.42 (s,
9H); HRMS (ES/FTMS) C31H33ClN4O5 calcd 577.2212 (M + 1),
found: 577.2185.
1
tR ) 2.00 min; LC-MS m/z ) 242; H NMR, CDCl3: δ 8.93 (s,
1H); 8.56 (d, J ) 5 Hz, 1H); 7.76 (d, J ) 7.5 Hz, 1H); 7.48 (t,
J ) 7 Hz, 2H); 7.38 (t, J ) 7 Hz, 2H); 4.60 (s, 1H), 3.62 (s,
3H); HRMS (ES/FTMS) C14H11NO3 calcd 242.0812 (M + 1),
found: 242.0818.
Step 6. The product from the previous step (0.27 g, 0.47
mmol) was deprotected using the same procedure as given in
Step 7 for 20a,b. The crude product was purified by prepara-
tive reverse phase HPLC using a TFA buffered water:aceto-
nitrile gradient. The diastereomers were then separated on a
Deltapak C18 column (40 × 100 mm, 3 in series) with a mobile
phase gradient of 85% A, 15% B to 60% A, 40% B (A ) 0.1%
NH4OAc in water, B ) acetonitrile) over 1 h, flow rate ) 50
mL/min. On an analytical column using a similar mobile
phase, the first-eluting diastereomer had a retention time of
3.50 min (18a) and the second-eluting diastereomer had a
Step 5. The product from the previous step (2.2 g, 9.1 mmol)
was converted to 3-aza-9-hydroxy-9-fluorene carboxylic acid
hydrazide using the procedure given in Step 5 for 17a,b (2.0
g, 90% yield). HPLC tR ) 0.7 min; LC-MS m/z ) 242; 1H NMR,
DMSO-d6: δ 9.51 (br s, 1H); 9.04 (s, 1H); 8.51 (d, J ) 4 Hz,
1H); 7.89 (d, J ) 7 Hz, 1H); 7.4-7.5 (m, 3H), 7.35 (t, J ) 6 Hz,
1H); 6.78 (s, 1H); HRMS (ES/FTMS) C13H11N3O2 calcd 242.0924
(M + 1), found: 242.0939.