Microwave-enhanced nucleophilic fluorination in the synthesis of fluoropyridyl derivatives of [3,2-c]pyrazolo-corticosteroids, potential glucocorticoid receptor-mediated imaging agents

Article abstract of DOI:10.1016/j.bmcl.2006.04.008

Fluoropyridyl derivatives of [3,2-c]pyrazolo-corticosteroids have high affinity for the glucocorticoid receptor (GR) and are highly active glucocorticoids. They are thus considered to be excellent candidates for PET imaging of GR containing tissues when labeled with fluorine-18 (t_1/2 = 110 min). Previously reported syntheses of these fluorinated glucocorticoids were accomplished by conventional thermal nucleophilic halogen exchange reactions with chloropyridyl precursors. These reactions were found to proceed at rates too slow for feasible application to radiosynthesis using [¹⁸F]fluoride. We have applied microwave-heating methods to these reactions and found that significant rate enhancements can be realized. Kinetic experiments showed an average relative rate ratio of 3/1 for microwave versus conventional heating and preparative experiments showed an average relative conversion ratio of 4.5/1 during the initial 120 min, a period approximating one half-life of the isotope. The microwave method described was used to prepare previously unreported 2′-(2-fluoro-4-pyridyl)-11β,17,21-trihydroxy-16α-methyl-20-oxo-pregn-4-eno-[3,2-c]-pyrazole, which was evaluated for biological activity.

Full text of DOI:10.1016/j.bmcl.2006.04.008

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3454–3458
Microwave-enhanced nucleophilic fluorination in the synthesis
of fluoropyridyl derivatives of [3,2-c]pyrazolo-corticosteroids,
potential glucocorticoid receptor-mediated imaging agents
Michael G. C. Kahn,a Emmanuel Konde,a Francis Dossou,a David C. Labaree,b
Richard B. Hochbergb and Robert M. Hoytea,b,
*
aDepartment of Chemistry, State University of New York, Old Westbury, NY 11568, USA
bDepartment of Obstetrics, Gynecology and Reproductive Sciences and The Yale Cancer Center, Yale University School of Medicine,
New Haven, CT 06520, USA
Received 21 February 2006; revised 31 March 2006; accepted 3 April 2006
Available online 27 April 2006
Abstract—Fluoropyridyl derivatives of [3,2-c]pyrazolo-corticosteroids have high affinity for the glucocorticoid receptor (GR) and
are highly active glucocorticoids. They are thus considered to be excellent candidates for PET imaging of GR containing tissues
when labeled with fluorine-18 (t1/2 = 110 min). Previously reported syntheses of these fluorinated glucocorticoids were accomplished
by conventional thermal nucleophilic halogen exchange reactions with chloropyridyl precursors. These reactions were found to pro-
ceed at rates too slow for feasible application to radiosynthesis using [18F]fluoride. We have applied microwave-heating methods to
these reactions and found that significant rate enhancements can be realized. Kinetic experiments showed an average relative rate
ratio of 3/1 for microwave versus conventional heating and preparative experiments showed an average relative conversion ratio of
4.5/1 during the initial 120 min, a period approximating one half-life of the isotope. The microwave method described was used to
prepare previously unreported 20-(2-fluoro-4-pyridyl)-11b,17,21-trihydroxy-16a-methyl-20-oxo-pregn-4-eno-[3,2-c]-pyrazole, which
was evaluated for biological activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
It has been demonstrated that fluoropyridyl derivatives
of [3,2-c]pyrazolo-corticosteroids of structural types I
and II (Fig. 1) are active glucocorticoids and have
great potential as high affinity positron emission
tomography (PET) imaging agents of glucocorticoid
receptor (GR) containing tissues when labeled with
fluorine-18.1 We previously reported receptor binding
and biological activity data for Ia and Ib. We now
report the synthesis (Scheme 1)and comparative biolog-
ical evaluation (Table 1) of compound II. Effective
radiosyntheses of these ligands are best accomplished
by nucleophilic displacement of appropriately placed
leaving groups using [18F]fluoride. Typical conditions
involve heating the substrate with K18F in DMSO in
the presence of complexing agent kryptofix-222.2
While pyridine rings are generally activated to nucleo-
philic substitution, our experience with pyridylpyrazolo
systems such as these shows that they take place at slow
rates even at high temperatures. This is likely due to the
electron-donating pyrazolo substituent, which deacti-
vates the pyridine ring. Also, since leaving groups at
position 4 relative to the pyridine ring nitrogen are more
reactive than those at position 2,3 slower rates are
expected for reactions leading to compounds Ia, Ib,
and II. The advent of microwave-heating methods, espe-
cially the introduction of focused microwave chemical
reactors, has led to several reports of efficient micro-
wave-assisted nucleophilic substitutions in heteroaro-
matic systems. These have been discussed in a recent
review.4 Microwave heating has also been demonstrated
to be advantageous over conventional heating in nucle-
ophilic radiohalogenation.5–7
In our reported synthesis of fluoropyridyl deriva-
tives of [3,2-c]pyrazolo-corticosteroids we employed
nucleophilic displacement of chloride using KF/krypto-
fix/DMSO under conventional-heating conditions. Not
surprisingly these reactions, while successful in producing
Keywords: Microwave enhancement; Nucleophilic fluorination;
Glucocorticoid imaging agents; Steroid pyrazoles.
*
Corresponding author. Tel: +1 516 876 2754; fax: +1 516 876 2757;
e-mail: hoyter@oldwestbury.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.008

M. G. C. Kahn et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3454–3458
3455
OH
O
OH
OH
O
OH
HO
HO
CH3
CH3
N
N
N
Z
N
X
Y
N
F
N
Ia, X=Y=H, Z=F
Ib, X=F, Y=Z=H
Ic, X=Z=H,Y=F
II
Figure 1.
I
NHNH
2
H NNH
2
2
N
Cl
N
Cl
2
1
O
O
O
O
O
O
O
NHNH
2
O
HO
HO
H
O
CH
N
Cl
3
CH
3
HO
N
N
N
3
4
Cl
OH
O
OH
O
O
O
O
HO
HO
60%HCOOH,
KF, Kryptofix
DMSO, heat
CH
CH
3
3
o
90 C
N
N
15 min
N
N
N
N
II
5
F
F
Scheme 1.
the desired product, were too slow to be applicable to the
synthesis of 18F-labeled compounds. Fluorine-18 has a
half-life of 110 min. Ideally, following the production of
the isotope, synthetic incorporation into the compound
of interest should not extend much beyond one half-life.
Thus, conversions need to approach about 50% within
about 120 min (2 h) for fluorine-18. Reaction times to this
level of conversion using conventional-heating methods
were typically 24–48 h. Herein we report the results of
our application of controlled microwave-heating meth-
ods to these halogen exchange reactions and demonstrate
that sufficient rate enhancements are realized. Since com-
pound II, 20(2-fluoro-4-pyridyl)-11b,17,21-trihydroxy-
16a-methyl-20-oxo-pregn-4-eno[3,2-c]pyrazole, has not
been previously reported, its synthesis, chemical charac-
terization, and biological activity are reported here. Com-
pound II was prepared as shown in Scheme 1 by methods
analogous to those we have used for compounds Ia, Ib
and similar pyrazolo steroids.1 Briefly, 2-chloro-4-
hydrazinopyridine 2 was prepared in 85% yield by selec-
tive displacement of iodine by anhydrous hydrazine in
2-chloro-4-iodopyridine (Aldrich Chemical Co.). It was
then condensed with 11b-hydroxy-2-hydroxymethylene-
16a-methyl-17, 20:20, 21-bis-(methylenedioxy)-pregn-4-
en-3-one, 3, to give compound 4 in 57% yield. Reaction
of 4 with KF, kryptofix-222, in DMSO under microwave
heating gave a mixture of 65% 5 and 35% unreacted
4(Table 3), from which 5 was separated using flash

3456
M. G. C. Kahn et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3454–3458
Table 1. Glucocorticoid action of steroidal pyridylpyrazoles
binding affinity for GR, it is not surprising that II would
have weaker biological potency in the HeLa cell assay
when compared to Ia (35% vs 237%). Contrariwise,
while II, as would be expected, is more potent than cor-
tisol, it is less potent than dexamethasone even though II
has a slightly greater affinity than dexamethasone for
GR. One likely explanation for this difference in GR
binding and glucocorticoid action may be that II is
metabolized and inactivated in the HeLa cell faster than
either dexamethasone or Ia. Nevertheless, compound II
is a potent glucocorticoid more powerful than cortisol
and thus it has potential as an in vivo-imaging agent
when labeled with fluorine-18.
GRa (RBA) HeLa AlkPb (RSA) logPo/w
c
Compound
Dexamethasone 100
Cortisol
100
19
1.87 0.63
1.43 0.47
3.62 1.08
3.62 1.09
3.97 1.09
6
129
1
6
5
2
II
Ia
Ib
35
250 30
93 31
237 68
121 51
a GR, glucocorticoid receptor; RBA, relative binding affinity where
dexamethasone Ka 4.6 0.1 nM = 100%.
b HeLa AlKP, HeLa cell alkaline phosphatase bioassay;
RSA, relative stimulatory activity where dexamethasone EC50
5.4 1.2 nM = 100%. Values are averages of three experiments per-
formed in duplicate SD. The results for Ib are taken from our
previous publication1 and are presented for illustrative purposes.
c logPo/w, octanol–water partition coefficients based on ACDLabs
computed predictions.
Table 1 also includes data (logPo/w) for compounds Ia,
Ib, and II, which show they are between one and two or-
ders of magnitude more lipophilic than cortisol and
dexamethasone. This effect of introducing the arylpyraz-
olo moiety into cortisol-related structures, which has
been previously observed,9 enhances the likelihood that
these compounds will cross the blood–brain barrier in
imaging studies utilizing the appropriate fluorine-18
labeled radiotracers.
chromatography (2:3 toluene–ethyl acetate on flash grade
silica gel). Deprotection of 5 (60% formic acid, 90 ꢁC,
15 min) gave 20-(2-fluoro-4-pyridyl)-11b,17,21-trihy-
droxy-16a-methyl -20-oxo-pregn-4-eno-[3,2-c]-pyrazole,
II, in 44% yield. An analytical sample was purified by
semi-preparative HPLC (25 · 1 cm Ultrasphere-ODS
column eluted at 3 mL/min with 70% methanol–water,
tR = 21 min). Compound II was characterized by its spec-
tral properties.8
Nucleophilic fluorination reactivity studies were carried
out on the isomeric side-chain protected chloropyridyl
precursors of compounds Ia–c and II. These are shown
in Figure 2. The reactions were typically conducted as
described.10 Under the conditions used these reactions
were found to follow pseudo-first order kinetics based
on the concentration of the starting chloropyridyl com-
pounds. The concentration of the nucleophile (fluoride)
was essentially constant due to the excess of insoluble
KF present throughout the reactions. Kinetic data are
summarized in Table 2.
As shown in Table 1 compound II was found to bind to
the GR (RBA 129%) slightly better than dexamethasone
and less than Ia (RBA = 250%). While the results of
both assays are somewhat lower than we previously
reported for 1a, they are not statistically different. We
had previously reported that compound 1b had about
the same glucocorticoid receptor binding affinity and
biological potency as dexamethasone. On the basis of
O
O
O
O
O
O
O
O
HO
HO
CH3
CH3
N
N
N
N
Cl
6
N
4
Cl
N
Precursor of Ib
Precursor of II
O
O
O
O
O
O
O
O
HO
HO
CH3
CH3
N
N
N
N
Cl
N
N
7
8
Cl
Precursor of Ia
Precursor of Ic
Figure 2.

M. G. C. Kahn et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3454–3458
3457
Table 2. Kinetic data
of microwave heating to the synthesis of fluoropyridyl
derivatives of [3,2-c]pyrazolo-corticosteroids will in-
crease the feasibility of producing fluorine-18 labeled
versions of these important glucocorticoids for imaging
of GR containing tissues using positron emission
tomography.
Isomer
Temp
(ꢁC)
kc · 103 minꢀ1
km · 103 minꢀ1
km/c
(rel rate)
6
4
7
8
150
150
130
110
13
48
3.7
2
2.7
0.6
3.5
5.4
3.7
5.7
6.2
1.6
c, conventional heating.
m, microwave heating.
Acknowledgments
This work was supported by National Institutes of
Health Grants GM08180 and GM08722 (to R.M.H.)
and CA37799 (to R.B.H.).
Table 3. Percent conversion to fluorinated product
Isomer Temp (ꢁC) Method Time (min) % conversion
6
4
7
8
150
150
Conventional
Microwave
60
60
12
33
References and notes
150
150
Conventional 120
Microwave 120
11
65
1. Hoyte, R. M.; Zhang, J.; Lerum, R.; Oluyemi, A.;
Persaud, P.; O’Connor, C.; Labaree, D. C.; Hochberg,
R. B. J. Med. Chem. 2002, 45, 5397.
2. Kilbourn, M. R. In Flourine-18 labelling of radiopharma-
ceuticals; National Academy Press: Washington, DC,
1990; pp 22–35.
130
130
Conventional 120
Microwave 120
7
47
110
110
Conventional 120
Microwave 120
19
51
3. Liveris, M.; Miller, J. J. Chem. Soc. 1963, 3486.
4. Kappe, C. O. Angew. Chem., Int. Ed. 2004, 43, 6250.
5. Dolce, F.; Dolle, F.; Jubeau, S.; Vaufrey, F.; Crouzel, C.
J. Labelled Compd. Radiopharm. 1999, 42, 975.
6. Dolle, F.; Dolce, F.; Valette, H.; Hinnen, F.; Vaufrey, F.;
Guenther, I.; Fuseau, C.; Coulon, C.; Bottlaender, M.;
Crouzel, C. J. Med. Chem. 1999, 42, 2251.
In addition to the kinetic experiments we collected data
from preparative runs over extended times to demon-
strate the extent of conversion to product under both
conventional and microwave-heating conditions. In con-
sideration of the goal of achieving significant conver-
sions at approximately one half-life of the isotope (t1/2
18F = 110 min), we present in Table 3 the degree of con-
version to fluorinated products under conventional and
microwave heating. The kinetic data and the conver-
sions observed in preparative experiments demonstrate
that microwave heating enhanced these halogen ex-
change reactions. The best enhancement was a 6.2-fold
increase observed for isomer 7, the precursor to fluoro-
pyridyl compound Ia. Significantly, Ia has been shown
to be a high affinity and highly active glucocorticoid
(Table 1). Rate enhancements were also demonstrated
(Tables 2 and 3) for isomers 6 and 4 leading to the other
active glucocorticoids Ib and II.
7. Kumar, P.; Wiebe, L. I.; Asikoglu, M.; Tandon, M.;
McEwan, A. J. Appl. Radiat. Isot. 2002, 57, 697.
8. Data for II: mp 139–144 ꢁC; UV kmax = 270 nm; 1H
NMR (400 MHz, CDCl3) d 8.27 (d, 1H, J = 5.6 Hz,
pyridine H-6), 7.50 (s, 1H, pyrazole-H), 7.44 (d, 1H,
J = 5.6 Hz, pyridine H-5), 7.15 (s, 1H, pyridine H-3),
6.27 (s, 1H, H-4), 4.51 (m, 1H, H-11a), 4.62 & 4.30 (AB
quartet, J = 19.9 Hz, 2H, H-21), 1.31 (s, 3H, H-19), 1.07
(s, 3H, H-18), 0.94 (d, 3H, J = 7.4 Hz, 16a-CH3);
HRMS (M+H) calcd for C28H34FN3O4: 495.2606,
found: 495.2602.
9. Wust, F.; Carlson, K. E.; Katzenellenbogen, J. A. Steroids
2003, 68, 177.
10. Fluorination reactions were carried out using 5 mg
(0.009 mmol) of chloropyridyl compound, 10 mg
(0.027 mmol) kryptofix-222, and 5.4 mg (0.093 mmol)
KF in 0.5 mL DMSO. In reactions studied using conven-
tional heating the reactants were mixed in a screw-capped
test tube that was closed and then heated with stirring in
an oil bath at the indicated temperature (Table 2).
Reaction progress was monitored by withdrawing small
aliquots, which were diluted in methanol and analyzed for
fluorinated product by high-performance liquid chroma-
tography (HPLC): Beckman System Gold (Model 126
modular pumps and Model 168 diode-array detector) with
32Karat Software using a 25 cm · 4.4 mm Ultrasphere-
ODS column eluted at 1 mL/min with 70% or 85%
methanol–water. UV detection was at 270 nm. In reac-
tions studied using microwave heating the reactants were
mixed in a 10 mL conical flask fitted with a condenser and
mounted in the cavity of a CEM Discover microwave
reactor. The reactor was operated with stirring in open-
vessel mode under constant temperature conditions. The
temperature was maintained by a combination of com-
pressed air cooling and applied microwave power. For
example, to maintain a temperature of 130 ꢁC, typically
required about 80 W with compressed air at 40 psi.
These results demonstrate that significant rate enhance-
ments in nucleophilic fluorination of pyridyl substitu-
ents by halogen exchange can be realized using
microwave heating compared to conventional-heating
methods. The applicability of these results to no-carri-
er-added radiochemical synthesis has not yet been eval-
uated. However, the very low concentrations of
[18F]fluoride under such conditions would be counter-
balanced by an excess of the substrate, which can be
expected to favor successful radiolabeling. Indeed, expe-
rience with similar nucleophilic fluorinations shows that
even faster rates are often observed under no-carrier-
added conditions compared to those of routine chemical
synthesis.11 Finally, as indicated in the above discussion
and in Scheme 1, a deprotection step is required in each
case to obtain the biologically active product. As we
have shown1 this can be accomplished efficiently in
15 min and will not add significant time to the synthesis
of the radiolabeled material. Therefore, the application

3458
M. G. C. Kahn et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3454–3458
Reaction progress was monitored by withdrawing small
fluorinated products computed at various time points
using integration data from the HPLC analyses above.
11. Snyder, S. E.; Kilburn, M. R. In Handbook of Radiophar-
maceuticals: Radiochemistry and Applications; Welch, M.
J., Redvanly, C. S., Eds.; Wiley: West Sussex, UK, 2003;
pp 195–227.
aliquots by syringe through narrow bore Teflon tubing
extending through the top of the condenser into the
reaction vessel. The aliquots were diluted with methanol
and analyzed by HPLC as described above. Kinetic data
for these reactions were obtained from concentrations of