Orally bioavailable prodrugs of a BCS class 2 molecule, an inhibitor of HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.bmcl.2008.10.090

The N-2 position of pyridazinone 1, a potent HIV-1 NNRTI that has limited aqueous solubility, was derivatized into a series of hydroxymethyl esters and carbonates as well as one phosphate. The derivatives served as prodrugs to effectively deliver 1 to rat

Full text of DOI:10.1016/j.bmcl.2008.10.090

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6344–6347
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Orally bioavailable prodrugs of a BCS class 2 molecule, an inhibitor of HIV-1
reverse transcriptase
a
a
b
a
a
Todd R. Elworthy a, , James P. Dunn , J. Heather Hogg , Grace Lam , Y. David Saito , Tania M. P. C. Silva ,
*
Dimitrios Stefanidis c, Witold Woroniecki b, Eugenia Zhornisky c, Amy S. Zhou b, Klaus Klumpp d
a Department of Medicinal Chemistry, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304-1397, USA
b Department of Drug Metabolism and Pharmacokinetics, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304-1397, USA
c Department of Pharmaceutics, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304-1397, USA
d Department of Viral Biochemistry, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304-1397, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The N-2 position of pyridazinone 1, a potent HIV-1 NNRTI that has limited aqueous solubility, was deriv-
atized into a series of hydroxymethyl esters and carbonates as well as one phosphate. The derivatives
served as prodrugs to effectively deliver 1 to rat plasma upon oral treatment at 50 mpk. Increases of
4.3- to 8.6-fold in 24-hour exposure of 1 (over that of parent) were observed while the prodrugs and
the hydroxymethyl adduct 2 were undetectable.
Received 11 September 2008
Revised 16 October 2008
Accepted 20 October 2008
Available online 1 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Class 2 molecule
HIV-1 reverse transcriptase inhibitor
Prodrug
Pyridazinone
Oral bioavailability
Dissolution limiting pharmacokinetics
NNRTI
Hydroxymethyl derivatives
We recently described the discovery and SAR of a new class of
inhibitors of HIV-1 reverse transcriptase of which 1 is a represen-
tative.1 These pyridazinones display high potency and bind at the
allosteric site common to most nonnucleoside inhibitors (NNRTI).
Compound 1 has very good Caco2 permeability and stability in hu-
man liver microsomes as well as low iv clearance rates in rat and
dog, 16% and 14% of hepatic blood flow, respectively (see Fig. 1).
However, 1 displays poor aqueous solubility and a high melting
point, both of which are typical characteristics for the chemotype.
Compound 1 has some less than ideal pharmacokinetic properties
that are likely influenced by these poor physical properties.
Panel 1 illustrates blood plasma levels achieved in rat following
dosing of 1. Plasma exposure was less than dose-proportional after
oral administration with only a 3-fold increase in 24-hour AUC for
the 10-fold increase in dose. These data for 5 and 50 mpk suggest
that poor solubility and high crystallinity are playing significant
roles to limit oral exposure. Furthermore, levels of 1 at the
50 mpk dose did not reach target plasma levels desired to provide
a high antiviral inhibitory quotient of 1 that warranted its further
development as an antiviral candidate.2
Prodrugs have been utilized to overcome delivery problems of
drugs with limited solubility, for instance phenytoin.3 Phenytoin
is a molecule that displays poor solubility and acceptable perme-
ability thus designated a BCS Class 2 molecule.4 The prodrug fosph-
enytoin bears a solubilizing phosphate feature. The approach to
generate a stable prodrug was achieved by linking that feature
via a hydroxymethyl derivative. Phenytoin is a substituted hydan-
toin that is more acidic (calcd pKa 8) than an amide and this prop-
erty enables a rapid release of the parent upon cleavage of the
F
N
O
N
NH
Cl
O
Me
1
N
Caco2 perm: 19 x 10-6 cm/s
solubility at pH 6.5: 0.3 µg/mL
mp 230-232 o
measured pKa 10.5
C
* Corresponding author. Tel.: +1 650 852 3159.
E-mail address: todd.elworthy@Roche.com (T.R. Elworthy).
Figure 1. Structure and selected properties of 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.090

T. R. Elworthy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6344–6347
6345
10
1
erated. Isopropenyl chloroformate served as an excellent activating
agent (for acid substrates) as shown for Method A. The succinate
half ester 3 was generated and evaluated as the free acid, while
the nicotinate 6 was evaluated as the hydrochloride salt. The req-
uisite acid substrates for esters 7 and 8 were the N-Boc amino acids
of Gly and Pro, respectively. The esters 7 and 8 were obtained as
their HCl salts following the deprotection under acidic conditions.
The phosphate ester 4 was generated as its di-sodium salt accord-
ing to Method B. The S-Val derived N-carboxyanhydride readily
consumed 2 at ambient temperature and ester 5 was obtained
upon acid treatment as indicated in Method C. Method D illustrates
the generation of carbonates by initial acylation of 2 with 4-nitro-
phenoxy chloroformate, followed by treatment with 2-(N,N-
dimethylamino)ethanol or 4-(2-hydroxyethyl)morpholine to give
9 or 10, respectively.7
i.v. 2.5 mpk
p.o. 5 mpk
p.o. 50 mpk
0.1
0.01
0.001
0
5
10
15
20
25
Time after Dosing (h)
Panel 1. Pharmacokinetic curves after dosing 1 to rat.
Table 1 summarizes relevant physical and rat pharmacokinetic
properties of 1 and prodrugs 3–10. All compounds were dosed at
50 mpk except 1, which was also dosed at 5 mpk (row 1). The pro-
drugs were dose adjusted to deliver 50 mpk of 1.
The stability of 2 was also evaluated and the estimated half-life
at either pH 2.0 or 6.5 was less than 60 s. Thus, it reverts to 1 rap-
idly in aqueous solutions.
One important characterization of the new derivatives was the
determination of thermodynamic solubility, which is typically
done by tumbling for 24 hours at pH 6.5. A prerequisite for this
determination is adequate stability of the compounds, however al-
most all had half-lives less than 5 hours under these conditions.
Therefore, exposing these prodrugs to the solubility assay would
have produced variable amounts of 1 and thus generating data
would have been uninterpretable.
All of the prodrugs displayed a mp less than the mp of the par-
ent 1. The crystallinity of these derivatives had been favorably al-
tered thus hinting that dissolution may be favorably impacted to
drive plasma exposure of 1.
phosphoryl bond of fosphenytoin. Inhibitor 1 displays acidity (pKa
10.5) similar to that of phenytoin thus a rapid in vivo release of
parent was anticipated upon application of this strategy.
We embarked on an investigation of prodrugs to address the
solubility and crystalline issues of 1. This letter describes a success-
ful approach to improve the blood plasma levels of 1. This was
achieved by introducing solubilizing features and perturbing the
crystallinity of 1 via the formaldehyde adduct 2 of which the pro-
drugs were produced. The opportunity to enhance exposure by ac-
tive transport5 was also examined.
Methods A (3, 6–8), B (4), C (5), and D (9, 10) enabled the gen-
eration of esters and carbonates and are highlighted in Scheme 1.
These prodrugs were designed as anionic or cationic species so as
to promote dissolution. Pyridazinone 16 cleanly gave adduct 2
upon heating with aqueous formaldehyde and the complete re-
moval of volatiles.7 The hydroxyl group required rapid conversion
in order to minimize reformation of 1 once a solution of 2 was gen-
The succinate 3 and nicotinate 6 esters improved 24-hour plas-
ma levels of 1. In addition, 3 showed acceptable stability at physi-
ological pH, whereas 6 hydrolyzed rapidly at pH 2. Phosphate 4
presented a clear improvement in stability as well as prodrug
effectiveness. Prodrug 4 gave rise to the highest exposure levels
with an 8.6-fold AUC increase of 1. Interestingly, the Tmax was de-
layed to 8 hours, likely a consequence of the higher stability of 4.
The action of nutrient processing enzymes such as phosphatases
would be necessary to cleave the phosphoryl bond of 4 and thus
the observation of continued absorption of 1 throughout the small
intestine. It is possible that this metabolism had been saturated
with this dose. However, this hypothesis was not fully evaluated
in rat. Surprisingly, carbonate 9 showed unacceptable aqueous sta-
bility at pH 6.5 as well as 9 and 10 gave no improvement in PK
properties.
Amino acid derivatives have previously been reported to in-
crease absorption by targeting oligopeptide transporters.5 Pro-
drugs 5, 7, and 8 were generated in this interest as well as the
opportunity of their salts to enhance dissolution. Valinate ester 5
displayed acceptable aqueous stability with t1/2 of 3.2 h at pH
6.5. However, 5 presented no benefit for the delivery of 1 within
the broader set of prodrugs. The glycinate 7 and prolinate 8 es-
ters showed 4- to 5-fold improvements in 24 h plasma levels but
were excluded from further consideration due to insufficient
stability.
F
N
O
O
N
N
OH
H
H
1
Cl
O
aq. MeOH
reflux
Me
2
N
Method A:
Method B:
Method C:
O
Method D:
O
i) i-Pr2NP(CH2CH2CN)2
ii) m-CPBA
iii) aq. NH3
NO2
NBoc
O
Cl
O
O
O
Cl
i) acid
iv) Na-salt exchange
on Dowex resin
O
substrate,
excess Et3N
i) cat. Et3N
ii) HCl, rt
i) cat.DMAP, Et 3N
ii) HOCH2CH2NX2
ii) HCl
F
N
O
N
N
O
Cl
O
Me
N
O
O
5
O
O
6
NH3 Cl
OH
P
4
ONa
ONa
NH Cl
The prodrugs and adduct 2 were not detected in rat plasma dur-
ing the 24-hour course of the pharmacokinetic evaluations.
Pyridazinone 1 was found to be an excellent substrate to deriv-
atize as hydroxymethyl linked prodrugs that in turn gave rapid
in vivo liberation of parent. Substantially improved plasma levels
3
O
O
O
8
O
O
O
NH3 Cl
NH2 Cl
NMe2
HCl
N
HCl
O
O
7
9
10
of 1 were observed (AUC24h > 50
lM h/mL) following the oral
Scheme 1. Preparation of hydroxymethyl derivatives of 1.
administration of prodrugs 3–10 to rat.

6346
T. R. Elworthy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6344–6347
Table 1
Physical and pharmacokinetic properties of prodrugs of 1.
Compound
Melting point (°C)
Method of prepn.
Stability t1/2 (h)a pH 2 pH 6.5
Rat PK of 1b
AUC, fold increased
Tmax (h)
Cmax
(l
M)
AUCc
(lM h/mL)
1e
1
3
4
5
6
7
8
9
230–232
230–232
167–171
194–197
177–178
173–174
162–163
163–165
75–90
—
—
A
B
—
—
462
>24f
1415
10
38
17
289
301
—
—
40
3.7
3.3
2.7
8.0
4.0
4.7
4.0
4.7
2.0
3.3
0.96
6.8
—
1
1.5
13.2
12.7
9.1
8.8
6.3
7.3
7.3
5.6
20.3
142
178
134
101
93
108
108
89
6.9
8.6
6.5
4.9
4.6
5.3
5.9
4.3
>24f
3.2
4.7
1.0
<0.3
0.9
3.5
C
A
A
A
D
D
10
79–101
a
Aqueous stability of prodrugs was assessed with a solution of 0.1 M HCl and 0.14 M KCl (pH 2) or a solution 25 mM of both NaH2PO4 and Na2HPO4 (pH 6.5).
Three male Hanover–Wister rats were orally dosed with the test molecule at 50 mpk or the equivalent of prodrug to deliver 50 mpk of 1. The values listed are the average
b
of the three. The quantification limit for 1 was P5 ng/mL.
c
Plasma was first sampled at 0.5 h while the animals were monitored for 24 h, at which time the last plasma sample was taken. At no time point was the prodrug or 2
detected and their quantification limits were 2–10 ng/mL.
d
24-hour exposure increase compared to same dose level of parent, 1.
PO dosed at 5 mpk.
No degradation was detected between 0 and 24 h at 40 °C.
e
f
Method A. A CH2Cl2 (150 mL) solution of 2 (3.13 g, 5.2 mmol) was immediately
added to a 0 °C solution of succinic acid (2.44 g, 20.6 mmol), triethylamine
(3.6 mL, 26 mmol), 4-dimethylaminopyridine (127 mg, 1.05 mmol), and
Phosphate prodrug 4 presented superior aqueous stability and
the most efficient delivery of 1. An 8.6-fold enhancement of the cir-
culating NNRTI 1 was observed during the 24-hour exposure.
In contrast to previous experience with nucleoside-amino ester
prodrugs, prodrugs of 1 bearing a lipophilic amino ester do not ap-
pear to be substrates for active transport, for example, 5, as they
did not enhance bioavailability relative to the other prodrugs.
Despite differences in aqueous stability, consistent and robust
increases in 24-hour exposure, from 4.3- to 6.9-fold were obtained
across the series of acyl prodrugs 3, 5–10 examined. Plasma con-
centrations of prodrugs were undetectable thus apparently consis-
tent with prodrug conversion by ubiquitous esterase activity.
Therefore, the proposal we forward is that the acyl cleavage of
the prodrugs is initially subject to enzymatic action during absorp-
tion that likely was not saturated under the study conditions.
isopropenyl chloroformate (0.80 mL, 7.3 mmol) in CH2Cl2 (110 mL). The
bubbling solution was stirred cold for 1.5 h and poured into 30 mL of 10% aq
HOAc. The mixture was extracted with EtOAc (4Â 50 mL) and stored over
sodium sulfate. After removal of the volatiles, the residue was loaded onto a pad
of silica and washed with 2:1 EtOAc/hexane. Acid 3 (1.36 g, 2.6 mmol, 50%) was
eluted with 0.5% HOAc in 3:1 EtOAc/hexane and recrystallized from warm EtOAc
and hexane giving the following characteristics: mp 167–171 °C; 1H NMR (d6-
DMSO, 300 MHz) d 12.2 (br s, 1H), 8.22 (t, J = 1.2 Hz, 1H), 7.95 (d, J = 1.2 Hz, 2H),
7.51 (dd, J = 1.5, 8.4 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.30 (apparent d, J = 1.2 Hz,
1H), 5.89 (s, 2H), 3.99 (s, 2H), 2.50 (t, J = 1.5 Hz, 4H), 2.07 (d, J = 1.2 Hz, 3H); 13C
NMR (75 MHz) d 171.9, 169.9, 158.7, 155.6, 152 (d), 144.0, 139.2, 135.6, 135.4,
129.75, 129.6, 127.8, 127.7, 124.9, 124.8, 124.7, 122.1, 115.1, 113.1, 71.6, 31.8,
14.7; ESMS m/z 525 (M+1)+; Anal. calcd for C25H18FClN4O6: C, 57.20; H, 3.46; N,
10.68. Found: C, 57.26; H, 3.61; N, 10.52.
Method B. 1-H tetrazole (225 mg, 3.2 mmol) and 2 (90% purity, 682 mg, 1.4 mmol)
were suspended in CH3CN (10 mL) immediately following by dropwise addition of
bis(2-cyanoethyl)-N,N-di-i-Pr phosphoramidite (Spectrum Chemicals, 871 mg,
3.2 mmol) over 3 min. The mixture was stirred for 16 h at rt, filtered, and
concentrated to yield a clear oil. The desired phosphite was purified (SiO2, 60–90%
ethyl acetate/hexanes) to yield (866 mg, quantitative) a clear oil and dissolved in
CH2Cl2 (14 mL). Immediately, the solution was cooled to 0 °C and to this was
added m-chloroperbenzoic acid in one portion (Aldrich Co. 77% purity, 326 mg,
1.45 mmol). The reaction was stirred for 4 h at 0 °C, treated with 1 M Na2S2O4 and
extracted twicewith CH2Cl2. Theorganic layerswere combined, driedoverMgSO4,
filtered and concentrated. The desired material was purified (SiO2, 0–2%
methanol/EtOAc) to yield (620 mg, 69%) a white foam. The phosphate triester
(612 mg, 1.0 mmol) was suspended in CH3CN (2.5 mL) then treated with
concentrated aq NH3 (10 mL) and allowed to stir at rt. After 48 h, another
portion of aq NH3 (5 mL) was added and stirred for an additional 24 h. The
suspension was then filtered, washed with CH3CN to obtain the desired product as
a white ammonium salt (456 mg, 86%). A portion of this was passed through a pad
of Dowex 50 Â 2 (Na+) to obtain the 4 as an off-white sodium salt: mp 194–197 °C;
Anal. calcd for C21H13FClN4O6PÁ(H2O)2: C, 43.10; H, 2.93; N, 9.58. Found: C, 43.12;
H, 2.67; N, 9.23.
Method C. A DMF (25 mL) solution of 2 (4.5 g, 10.6 mmol) was generated at rt and
without delay treated with triethylamine (0.3 mL, 2.1 mmol) and a toluene
(15 mL) solution of N-tert-butoxycarbonyl (S)-valine N-carboxyanhydride (3.1 g,
12.7 mmol). The resulting solution was stirred for 2 h and poured into water
(120 mL) and extracted with 2:1 hexane/EtOAc (4 Â 100 mL). The combined
organic extracts was washed brine, stored over anhydrous sodium sulfate, and
loaded onto a pad of silica gel (eluant gradient: 1:1 to 3:1 EtOAc/hexane). The
desired N-Boc amino ester was obtained (5.6 g, 8.9 mmol, 84%) as a foam and was
dissolved in EtOAc at rt and treated with hydrochloric acid (4 mL, 4 M solution in
1,4-dioxane). The solution deposited a white solid and stirring was continued for
14 h. Et2O (25 mL) was added and the suspension was stored at 0 °C. The crude
solid was collected and dissolved in warm iso-propanol and EtOAc and then stored
at rt for 18 h. Amine HCl salt of 5 (1.3 g, 2.3 mmol, 22%): mp 176.7–178.3 °C; Anal.
calcd for C26H23FClN5O4HCl: C, 55.72; H, 4.32; N, 12.50. Found: C, 55.71; H, 4.28; O,
12.35.
Acknowledgments
We appreciate the early input provided by Professor Ronald T.
Borchardt. This prodrug campaign would not have been possible
if it were not for the efforts of the following excellent process re-
search chemists, namely Michael Martin, Justin Vitale, Felicia Thai,
Gary F. Cooper, Harlan Reese, Anton Constaninescu, and Jiang Zhu.
References and notes
1. Sweeney, Z. K.; Dunn, J. P.; Li, Y.; Heilek, G.; Dunten, P.; Elworthy, T. R.; Han, X.;
Harris, S. F.; Hirschfeld, D. R.; Hogg, J. H.; Huber, W.; Kaiser, A. C.; Kertesz, D. J.;
Kim, W.; Mirzadegan, T.; Roepel, M. G.; Saito, Y. D.; Silva, T. M. P. C.; Swallow, S.;
Tracy, J. L.; Villasenor, A.; Vora, H.; Zhou, A.; Klumpp, K. Bioorg. Med. Chem. Lett.
2008, 18, 4352.
2. Sweeney, Z. K.; Klumpp, K. Curr. Opin. Drug Discov. Dev. 2008, 11, 458.
3. Varia, S. A.; Schuller, S.; Sloan, K. B.; Stella, V. J. J. Pharm. Sci. 1984, 73, 1068.
4. Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. Pharm. Res. 1997, 12, 413.
5. Li, F.; Maag, H.; Alfredson, T. J. Pharm. Sci. 2008, 97, 1109.
6. Kertesz, D. J.; Martin, M.; Palmer, W. S. U.S. Pat. Appl. Publ. 2005, US
2005234236 A1 20051020. CAN 143:405914.
7. A suspension of pyridazinone 1 (3.3 g, 8.4 mmol) in methanol (55 mL) and 37%
aqueous formaldehyde (15 mL, 185 mmol) was heated to reflux for 2.5 h during
which a solution formed. The reaction mixture was removed from heating and
allowed to stand at rt for 1 h during which solids formed. The mixture was
diluted with 20 mL of ice water and filtered. The solid was stored at 50 °C for
16 h in vacuo. The desired adduct 2 (3.3 g, 7.7 mmol, 92%) was obtained: mp
172–179 °C; 1H NMR (d6-DMSO, 300 MHz) d 8.22 (t, J = 1.2 Hz, 1H), 7.93 (d,
J = 1.2 Hz, 2H), 7.50 (dd, J = 1.5, 8.4 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.25
(apparent d, J = 1.2 Hz, 1H), 6.60 (t, J = 7.5 Hz, 1H), 5.27 (d, J = 7.5 Hz, 2H), 3.98
(s, 2H), 2.06 (d, J = 1.2 Hz, 3H); 13C NMR (75 MHz) d 160.3, 157.3, 154 (d), 144.4,
140.7, 137.3 (d), 131.4, 130.7, 129.5, 127.0 (d), 126.4, 123.7, 116.8, 114.8, 73.8,
33.5, 16.5; ESMS m/z 425 (M+1)+; Anal. calcd for C21H14FClN4O3: C, 59.37; H,
3.32; N, 13.19. Found: C, 59.08; H, 3.18; 13.14.
Method D. A CH2Cl2 (10 mL) solution of 2 (600 mg, 1.4 mmol) was cooled to 0 °C
and immediately treated with triethylamine (0.6 mL, 4.2 mmol), 4-
dimethylaminopyridine
(85 mg,
0.71 mmol),
and
para-nitrophenoxy
chloroformate (571 mg, 2.8 mmol). The yellow solution was stirred at 0 °C for
2 h and poured into 30 mL of aq NaHCO3. The mixture was extracted with EtOAc
(4 Â 50 mL) and stored over sodium sulfate. The residue was loaded onto a pad of

T. R. Elworthy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6344–6347
6347
silica and the intermediate carbonate (170 mg, 0.3 mmol) was eluted with 1:2
EtOAc/hexane and subsequently dissolved in CH3CN (12 mL) and CH2Cl2
(10 mL) at rt and treated with 2-(N,N-dimethylamino)ethanol (0.1 mL,
1.1 mmol). After stirring for 16 h, the mixture was poured into aq NaHCO3,
extracted with EtOAc (3 Â 25 mL) and stored over anhydrous sodium sulfate.
The crude (85 mg, 11%) was dissolved in CH2Cl2 and treated with HCl (0.5 mL,
1 M Et2O) and allowed to stand for 18 h at rt. Amine HCl salt of 9 was filtered
and the volatiles were removed in vacuo: mp 75–90 °C; Anal. calcd for
C26H23FClN5O5HCl.(CH2Cl2)0.65: C, 50.68; H, 4.04; N, 11.09. Found: C, 50.68; H,
3.95; O, 10.91.