Carbamoyl pyridone HIV-1 integrase inhibitors. 2. Bi- and tricyclic derivatives result in superior antiviral and pharmacokinetic profiles

Article abstract of DOI:10.1021/jm301550c

This work is a continuation of our initial discovery of a potent monocyclic carbamoyl pyridone human immunodeficiency virus type-1 (HIV-1) integrase inhibitor that displayed favorable antiviral and pharmacokinetic properties. We report herein a series of

Full text of DOI:10.1021/jm301550c

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Article
Carbamoyl Pyridone HIV-1 Integrase Inhibitors 2. Bi- and Tri-cyclic
Derivatives Result in Superior Antiviral and Pharmacokinetic Profiles
takashi kawasuji, Brian A. Johns, Hiroshi Yoshida, Jason G. Weatherhead, Toshiyuki
Akiyama, Teruhiko Taishi, Yoshiyuki Taoda, Minako Mikamiyama, Hitoshi Murai,
Ryuichi Kiyama, Masahiro Fuji, Norihiko Tanimoto, Tomokazu Yoshinaga, Takahiro
Seki, Masanori Kobayashi, Akihiko Sato, Edward P. Garvey, and Tamio Fujiwara
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301550c • Publication Date (Web): 14 Jan 2013
Downloaded from http://pubs.acs.org on January 16, 2013
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Carbamoyl Pyridone HIVꢀ1 Integrase Inhibitors 2.
Biꢀ and Triꢀcyclic Derivatives Result in Superior
Antiviral and Pharmacokinetic Profiles
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Takashi Kawasuji,*,† Brian A. Johns,‡ Hiroshi Yoshida,† Jason G. Weatherhead,‡ Toshiyuki
Akiyama,† Teruhiko Taishi,† Yoshiyuki Taoda,† Minako Mikamiyama-Iwata,† Hitoshi Murai,†
Ryuichi Kiyama,† Masahiro Fuji,† Norihiko Tanimoto,† Tomokazu Yoshinaga,† Takahiro Seki,†
Masanori Kobayashi,† Akihiko Sato,† Edward P. Garvey,‡ Tamio Fujiwara,†
† Shionogi Pharmaceutical Research Center, Shionogi & Co., Ltd., 3ꢀ1ꢀ1, Futabaꢀcho, Toyonakaꢀ
shi, Osaka 561ꢀ0825, Japan
‡ GlaxoSmithKline Research & Development, Infectious Diseases Therapeutic Area Unit, Five
Moore Drive, Research Triangle Park, North Carolina 27709, U. S. A.
Abstract
This work is a continuation of our initial discovery of a potent monocyclic carbamoyl pyridone
human immunodeficiency virus typeꢀ1 (HIVꢀ1) integrase inhibitor that displayed favorable
antiviral and pharmacokinetic properties. We report herein a series of bicyclic carbamoyl
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pyridone analogs to address conformational issues from our initial SAR studies. This
modification of the core unit succeeded to deliver low nM potency in standard antiviral assays.
An additional hydroxyl substituent on the bicyclic scaffold provides remarkable improvement of
antiviral efficacies against clinically relevant resistant viruses. These findings led to additional
cyclic tethering of the naked hydroxyl group resulting tricyclic carbamoyl pyridone inhibitors to
address remaining issues and deliver potential clinical candidates. The tricyclic carbamoyl
pyridone derivatives described herein served as the immediate lead in molecules to the next
generation integrase inhibitor dolutegravir which is currently in late stage clinical evaluation.
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KEYWORDS
HIVꢀ1 integrase, twoꢀmetal binding pharmacophore, molecular design, carbamoyl pyridone
inhibitor, dolutegravir, DTG, S/GSK1349572, S/GSK572
1. Introduction
HIVꢀ1 integrase (IN) is a virally encoded enzyme essential for replication that catalyzes the
integration of viral DNA into the host chromatin. As a result of this unique retroviral step, IN has
become an attractive target for drug discovery.1 In 2007, raltegravir2 (RAL) (figure 1) became
the first approved HIVꢀ1 IN inhibitor (INI) thus opening up a new class of antiretroviral agents
and it has since become a preferred first line agent as part of the highly active antiretroviral
therapy (HAART) treatment guidelines since 2009.3 RAL requires a 400 mg twice daily dose and
has given rise to clear signature mutation pathways (N155H, Q148H/K/RꢀG140S and Y143C).4
Elvitegravir5 (EVG) (figure 1) is a second INI which was approved in 2012. EVG allows for a
once daily dose of 150 mg, however, this requires coꢀadministration of CYP3A inhibitor as a PK
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booster.6 Additionally, EVG has significant cross resistance with RAL in both the N155 and
Q148 mutation pathways.7 While both RAL and EVG are valued additions to the HAART
armamentarium, there remains significant room for differentiation and improvement. It was our
vision that a next generation INI would have to address attributes in the areas of overall dose
burden, dosing interval,8 potency against resistant mutations and genetic barrier to resistance.
Thus it was with these goals that we set out to design a novel scaffold that would deliver clear
value for patients. The work presented herein is the second in a series of manuscripts to
elaborate on the evolution of our scaffold design in pursuit of true next generation properties that
ultimately resulted in the discovery of dolutegravir (DTG) (figure 1) to deliver low dose, once
daily unboosted PK and exceptional resistance properties.9 We recently reported on the twoꢀ
metal pharmacophore10 driven design of a carbamoyl pyridone INI 1 shown in Figure 2a.11 This
compound showed remarkable antiviral potency against wildꢀtype HIVꢀ1 virus from just the
minimum pharmacophore elements consisting of C2 carboxylate and C3 hydroxy pyridone
moiety as a basic chelating unit and a benzyl carboxyamide unit to occupy the hydrophobic
region. Although the potency against some clinically relevant mutant viruses were comparable to
the prior generation INIs and insufficient for further development, this agent showed encouraging
PK properties with a T1/2 of 4.7 hours and robust plasma drug levels well in excess of the protein
adjusted IC50 (MT4PAIC50) at 24 hours after oral dosing in rat PK studies. The initial monocyclic
carbamoyl pyridone inhibitor 1 has an ester unit at the C2 position to coordinate with a metal
cofactor in the catalytic active site of the enzyme. We sought to explore further chemical space
adjacent to the requisite pharmacophore elements in hopes of addressing the potential risk of
chemical or metabolic stabilities of the ester unit as well as attempting to improve on the
remaining deficiencies around resistance profile. We initially tried amide derivatives including
primary, secondary and tertiary amides. Potentially the amide modification could provide an
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additional benefit in antiviral potency due to slightly higher basicity of carbonyl lone pair
favorable to coordinate with magnesium cofactors in the active site. However potencies were
remarkably reduced in both enzymatic and antiviral assays, presumably because the amide analog
causes an undesired intramolecular hydrogen bond between the central OH and amide NH moiety
(in the case of primary and secondary amide examples) or more generally simply disrupts the coꢀ
planarity of the metal chelation motif due to steric hindrance of the amide and the pyridone NH
as shown in Figure 2b. Eitherway, this distortion of the chelating unit critical for effective
binding to the adjoining two metal cofactors was not productive during the simple ester to amide
modification. An intramolecular tethering design was adopted as shown in Figure 2c as a logical
next step in the development of the carbamoyl pyridone SAR in order to fix the metal chelation
motif into a desired coꢀplanar orientation for effective metal coordination.
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Me
O
N
O
N
NH
F
Me
N
N
H
N
O
O
O-
K+
Raltegravir (RAL)
OH
MeO
N
O
Cl
F
O
OH
Elvitegravir (EVG)
F
O
H
N
O
N
N
H
F
O
O-
O
Na+
Me
Dolutegravir (DTG)
Figure 1. HIVꢀ1 integrase inhibitors.
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Explore Chemical Space
NH
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O
O
N
H
OMe
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F
OH
O
Compound 1
IC50 = 10 nM
(a)
O
O
O
H
O
H
N
N
H
N
H
N
R
N
O
R
O
O
N
OH
H
R
H
(b)
O
N
N
H
N
R
O
O
O
(c)
Figure 2. (a) The monocyclic carbamoyl pyridone lead inhibitor 1. (b) Unfavorable
intramolecular hydrogen bonding and steric hindrance potentially disrupt the coꢀplanarity of the
metal chelation motif by amide modifications. (c) A logical approach toward the cyclization
modification to fix the metal chelation motif into a coꢀplanar orientation.
2. Chemistry
All tested compounds were prepared through the key N1 acetaldehyde unit using one of the
strategies outlined in Figure 3. Series A and B were accessed through imine formation which
underwent enamine tautomerization and led to the unsaturated analogs (Series A) and upon
reduction the saturated counterparts (Series B). Condensation of the N1 acetaldehyde with a
pendent amide let to the hydroxyl containing Series C. Finally, access to the tricyclic Series D or
E was envisioned to result from condensation of the acetaldehyde functionalized core with an
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aminoalcohol or diamine which could then close to form a fused amide group and facilitate the
tricyclic ring formation.
7
8
CHO
O
9
N
N
H2N R1
+
H
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OMe
O
OBn O
N1-acetaldehyde unit
R1
O
O
N
O
N
N
H
N
N
H
N
R1
OMe
OH
O
O
Series A (unsaturated bridge)
Series B (saturated bridge)
OBn O
O
CHO
O
O
OH
N
N
N
H
N
H
H
N
N
O
R1
R1
OH
O
OBn O
Series C
O
X
N
NH
X
O
O
N
H
N
N
N
H
O
OMe
OH
Series D (X = O)
Series E (X = N-R2)
O
OBn O
Figure 3. Conceptual synthetic approach toward biꢀ and triꢀcyclic analogs.
The key synthetic divergence points were chosen as the benzyl protected intermediates 13, 14
(Scheme 1). These were chosen due to the potential for mild and selective modification of the
core and C2 carboxyl moiety to support our synthetic strategies outlines in Figure 3. In addition,
the benzyl protect central hydroxyl would be stably protected and able to be exposed
chemoselectively in a last step to produce the fully functionalized metal binding moiety. The
commercially available maltol 2 was protected as its benzyl ether followed by pyrone to pyridone
conversion via amination with ammonia to provide intermediate 4. C5 bromination was followed
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by palladium catalyzed carbonylation to give the methyl ester 6. After acetyl protection of 4ꢀ
hydroxyl, oxidation of the pyridine nitrogen with m-CPBA was performed followed by thermal
rearrangement of the resultant N-oxide in acetic anhydride to give the corresponding C2ꢀ
hydroxymethyl derivative 9. The acetoxy unit was selectively removed with anhydrous sodium
methoxide followed by amination with 4ꢀfluorobenzylamine on the C5 ester to provide 11. The
C2 alcohol was oxidized in a stepwise manner to produce the corresponding carboxylic acid 13.
Condensation with methanol was performed to conclude preparation of intermediate 14.
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Scheme 1. Synthesis of key intermediatesa
Br
MeO2C
O
a
c
e
b
d
O
O
NH
NH
NH
O
O
O
O
OH
OBn
OBn
OBn
OBn
2
3
4
5
6
MeO2C
O
MeO2C
MeO2C
AcO
O-
MeO2C
AcO
g
f
h
i
NH
N
N+
N
AcO
OBn OH
O
OBn
OBn
OBn OAc
O
10
7
8
9
O
O
O
p-F-Bn
p-F-Bn
p-F-Bn
p-F-Bn
j
k
l
N
NH
CO2Me
N
H
NH
N
H
NH
N
NH
CHO
H
H
O
O
CO2H
O
OBn
OBn
OBn OH
OBn
13
14
11
12
aReagents and conditions: (a) BnBr, K2CO3, DMF, 80 °C; (b) NH3, 2 M NaOH, ethanol, 90 °C;
(c) Nꢀbromosuccinimide, acetnitrile, rt; (d) Pd(OAc)2, 1,3ꢀbis(diphenylphosphino)propane, Et3N,
CO, methanol, DMF, 80 °C; (e) Ac2O, 130 °C; (f) mꢀCPBA, chloroform, rt; (g) Ac2O, 130 °C;
(h) MeONa, methanol, rt; (i) 4ꢀfluorobenzylamine, DMF, 120 °C; (j) MnO2, chloroform, 60 °C;
(k)
NaCl,
sulfamic
acid,
THFꢀwater,
rt;
(l)
HOBt,
1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide hydrochloride, Et3N, methanol, DMF, reflux.
The synthesis of series A and B are shown in Scheme 2. Nꢀallylation of 14 and oxidative
cleavage with osmium tetroxide and sodium metaperiodate gave the corresponding acetaldehyde
intermediate 16. After formation of an imine with a primary amine, intramolecular cyclization
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proceeded using microwave heating to provide 17. Acidic deprotection of 17 served to deprotect
the above mentioned benzyl ether and preserve the unsaturation to give the target series A
derivatives 18a-f. Subjecting 17 to hydrogenolysis conditions also resulted in removal of the
benzyl ether while concomitantly reducing the newly formed ring to give the partially saturated
compounds 19a-f (series B).
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Scheme 2. Synthesis of series A and Ba
O
p-F-Bn
a
b
N
H
N
14
O
CO2Me
O
OBn
15
O
p-F-Bn
p-F-Bn
c
N
H
N
CHO
N
H
N
N
O
CO2Me
O
R
OBn
OBn O
e
16
17
d
O
O
p-F-Bn
p-F-Bn
N
N
N
H
N
H
N
N
O
R
O
R
OH
O
OH
O
18
19
[17-19]a : R = 2-methoxyethyl
[17-19]b : R = 2-(N-morpholino)ethyl
[17-19]c : R = 2-dimethylcarbamoylmethyl
[17-19]d : R = 2-isopropoxyethyl
[17-19]e : R = 2-phenoxyethyl
[17-19]f : R = cyclohexyl
aReagents and conditions: (a) 3ꢀbromopropen, Cs2CO3, DMF, rt; (b) potassium osmate
dihydrate, sodium metaperiodate, 1,4ꢀdioxane, rt; (c) amine, dichloromethane, 140 °C
microwave; (d) TFA, rt; (e) H2, 10% PdꢀC, DMFꢀmethanol, rt.
Synthesis of series C is shown in Scheme 3. After amidation of the carboxylic acid 13 with a
primary amine, Nꢀallylation was performed to give intermediate 21. Oxidative cleavage of the
double bond resulted in formation of hemiꢀaminal derivatives 22, hydrogenolytic deprotection
concluded the sequence to produce compound 23.
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Scheme 3. Synthesis of series Ca
5
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7
8
9
10
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13
14
15
16
17
18
19
20
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aReagents and conditions: (a) HOBt, 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
hydrochloride, Et3N, DMF, reflux; (b) 3ꢀbromopropen, Cs2CO3, DMF, rt; (c) potassium osmate
dihydrate, sodium metaperiodate, 1,4ꢀdioxane, rt; (d) 10% PdꢀC, DMFꢀmethanol, rt.
Series D and E compounds were synthesized as shown in Scheme 4. The aldehyde 16 was
condensed with an aminoꢀalcohol (series D) or diamine (series E) to generate the corresponding
cyclic (hemi)ꢀaminal moiety, and direct heating under microwave irradiation promoted
intramolecular cyclization to provide the corresponding tricyclic compounds 24–36. Additional
modification on the secondary amine of 34 allowed introducing acetylꢀ or sulfonylꢀ functional
group. Subsequent deprotection completed preparation of compounds 37–48.
Scheme 4. Synthesis of series D and Ea
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e
O
O
O
O
p-F-Bn
O
24, 37 ; n = 0
25, 38 ; n = 1
26, 39 ; n = 2
N
H
N
a
16
N
N
n
n
OBn O
R
N
p-F-Bn
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N
H
N
b
OBn O
e
27, 40 ; n = 0, R = isopropyl
28, 41 ; n = 1, R = methyl
29, 42 ; n = 1, R = isopropyl
30, 43 ; n = 1, R = thiazol-2-ylmethyl
31, 44 ; n = 1, R = 2-methoxyethyl
32, 45 ; n = 2, R = methyl
33, 46 ; n = 2, R = isobutyl
34
; n = 1, R = H
c
d
35, 47 ; n = 1, R = acetyl
36, 48 ; n = 1, R = methanesulfonyl
aReagents and conditions: (a) aminoꢀalcohol, dichloromethane, 140 °C microwave; (b) diamine,
dichloromethane, 140 °C microwave; (c) Ac2O, cat. DMAP, pyridine, rt; (d) MsCl, DMAP,
pyridine, rt; (e) 10% PdꢀC, methanol, rt.
3. Results and Discussion
3.1. Antiviral profiles of bicyclic series A and B.
Inhibitory activities of series A and B are summarized in Table 1. Slightly improved antiviral
potencies against wild type HIVꢀ1 virus from the initial compound 1 were observed while the
uniformly high selectivity index over cytotoxicity to indicate a true antiviral effect was
maintained. Although variation on R did not have a significant influence on antiviral potency,
modifications toward hydrophilic substituents appeared to have a negative impact on potency as
seen in compounds 19b, 18c and 19c. Potency shifts in presence of human serum albumin (HSA)
resulted in a very acceptable protein adjustment to the IC50 (MT4PAIC50). Comparison of series A
(unsaturated) and series B (saturated) resulted in no significant difference in potency for the R
groups that were examined, however slightly larger protein shift was observed for the unsaturated
analogs. The saturated series B also had a slightly improved profile (vs. series A) against the
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Q148K site directed mutant virus across the R substitutents examined. An improvement of fold
change against the Q148K mutant compared to the original monocyclic compound 1 was
observed in compounds which have an ethereal R group (18a, 19a, 18d, 19d and 19e), however
the data still left room for improvement to achieve our original differentiation goals.
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Table 1. Antiviral profiles of series A and B.a
O
N
N
18[a-f]; unsaturated bridge
19[a-f]; saturated bridge
H
N
F
O
R
OH
O
MT-4/MTTb
wild type IC50 (nM)
MT-4/MTTc
CC50 (nM)
MT4PAIC50
(nM)
e
Q148Kd
(FC)
potency
shiftf
compd
R
1
ꢀ
10 ± 2.9
19000 ± 850
>200000
200 ± 25
35 ± 6.8
18 ± 2.5
>250
120
27
12
17
18a
19a
18b
19b
18c
19c
18d
19d
18e
19e
18f
19f
1.6 ± 1.1
5.9 ± 0.044
6.6 ± 1.5
21 ± 5.5
OMe
52000 ± 8300
36000 ± 4100
18
3.1
3
20
N
160000 ± 25000
>200000
NT
34
1.6
4.1
2.4
4.4
3.1
11
O
O
35 ± 8.8
NT
144
62
NMe2
26 ± 7.9
>200000
NT
4.3 ± 1.4
4.2 ± 1.0
4.4 ± 1.6
6.8 ± 3.7
12 ± 3.1
49000 ± 19000
30000 ± 1900
2400 ± 710
10000 ± 1300
>200000
39 ± 14
9.9 ± 1.3
>250
19
O
13
48
OPh
17 ± 6.9
>250
65
9.5
13
156
29
6.5 ± 2.9
>240000
>250
4.4
aData represent the mean ± s.d. from independent three experiments. bAntiꢀHIV activities using
c
d
e
MTꢀ4 cells. Cytotoxicity using MTꢀ4 cells. Fold change against the resistant variant. Protein
adjusted antiviral potencies based on MTꢀ4/MTT IC50. fPotency shift in presence of human serum
albumin.
3.2. Antiviral profiles of hydroxyl series C and tricyclic series D and E.
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A remarkable potency improvement against the Q148K variant was observed from hydroxyl
compound 23 (3.8 nM IC50 vs. wild type with a 2.1 fold reduction against Q148K). This
potency profile combined with a very modest 3.7 fold shift in presence of HSA served as
encouragement for further exploration. However as is discussed in next section, the series C
compound did not show sufficient oral bioavailability. Moreover the hemiꢀaminal chiral center is
not configurationally stable and interconverts on a moderate time scale (minutes to hours)
resulting in lose of enantiomeric purity after chiral purification. Additional series C derivatives
wherein the R substituent was modified are not shown herein but delivered similar virological
and PK profiles and hence were not further improvements to justify continued pursuit. Although
we abandoned further exploration of series C, the hydroxyl substitution on the saturated bridge
provided impetus to address the perceived PK and configurational stability issues. In order to
address the immediate issue of configurational stability, two series of tricyclic analogs were
devised whereby the hemiꢀaminal was effectively ‘locked down’ into a third ring such that
equilibration through an open chain intermediate would not be feasible. The oxygen and
nitrogen containing third ring analogs, series D and E respectively, showed high antiviral
potencies against wildꢀtype virus and excellent selectivity over cytotoxicity as previously
observed (Table 2). The ring size (fiveꢀ, sixꢀ and sevenꢀmember) does not affect antiviral efficacy
for the wild type virus regardless of hemiꢀaminal or aminal analog, however the fiveꢀmembered
analogs 37 and 40 lost potency against the mutant. Although the series E aminal unit does not
appear to impact potency or resistance profile significantly, hydrophilic functional groups as seen
in 47 and 48 do begin to erode the antiviral activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Antiviral profiles of series C, D and Ea
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2
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4
5
6
O
O
R
Y
H
N
N
N
H
F
n
OH
O
7
8
9
MT-4/MTTb
MT-4/MTTc
CC50
e
Q148Kd
(FC)
MTTPAIC50
(nM)
potency
shiftf
compd
Y
n
R
wild type IC50
(nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(nM)
23
37
38
39
40
41
42
43
44
45
46
47
48
-
-
0
1
2
0
1
1
1
1
2
2
1
1
-
3.8 ± 0.53
2.7 ± 0.63
3.4 ± 1.1
2.9 ± 0.70
2.3 ± 0.55
3.8 ± 0.96
7.5 ± 0.42
3.3 ± 0.91
5.2 ± 1.3
6.4 ± 3.7
5.4 ± 0.92
29 ± 4.2
83000 ± 22000
72000 ± 24000
18000 ± 1600
26000 ± 7000
8000 ± 2500
20000 ± 3000
7200 ± 790
2800 ± 820
9800 ± 2200
>200000
2.1 ± 0.50
19 ± 5.1
2.8 ± 0.32
2.2 ± 1.2
39 ± 7.4
4.7 ± 1.0
9.1 ± 1.7
4.2 ± 1.6
5.7 ± 0.47
4.2 ± 1.0
3.2 ± 1.1
NT
14.1
140.4
26.9
22.0
7.8
3.7
52
O
O
O
N
N
N
N
N
N
N
N
N
ꢀ
ꢀ
7.9
7.6
3.4
4.3
4.3
3.9
3.9
4.9
17
ꢀ
isopropyl
methyl
16.3
32.3
12.9
20.3
31.4
91.8
58.0
120.4
isopropyl
thiazolꢀ2ꢀylmethyl
2ꢀmethoxyethyl
methyl
isobutyl
13000 ± 5000
>200000
acetyl
2
methanesulfonyl
43 ± 22
>220000
NT
2.8
aData represent the mean ± s.d. from independent three experiments. bAntiꢀHIV activities using
MTꢀ4 cells. Cytotoxicity using MTꢀ4 cells. Fold change against the resistant variant. Protein
adjusted antiviral potencies based on MTꢀ4/MTT IC50. fPotency shift in presence of human serum
albumin.
c
d
e
3.3. Efficacy against additional RAL signature pathways and rat PK profiles of 18a, 19a,
23 and 38.
Antiviral potency against clinically relevant RAL resistant mutants (Q148K, N155H and
Y143R) and rat PK profiles are summarized in Table 3. The bicyclic analogs 18a, 19a, hydroxyl
examples 23, and tricyclic analog 38 showed a low fold change against the N155H and Y143R
site directed mutants. As was discussed above, the tricyclic derivatives furthermore succeeded in
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improving efficacy against Q148K which was the primary remaining issue left unaddressed by
monocyclic lead analog 1. Low dose rat PK profiles resulted in low CLt (1 mL/min/kg) and
moderate to long T1/2 estimates (3ꢀ17 hours) in every compound in Table 3. Although the
moderate to low oral bioavailability of the hydroxyl derivative 23 raised concerns, the tricyclic
analog 38 delivered a yet further improved T1/2 accompanied with an oral bioavailability of 50%.
The rat 24 hour plasma concentration (C24) of 7127 ng/mL from a 5 mg/kg oral dose robustly
covered the MT4PAIC50 of 10 ng/mL. No remarkable CYP inhibition nor metabolic concerns were
identified during in vitro studies.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Although these virological advances and promising PK profile of the tricyclic series D were
significant toward our goal, this data set was from racemate. As an initial approach to understand
contributions of the individual enantiomers toward the antiviral profile, the isomers were isolated
after chiral chromatographic separation of the Oꢀbenzyl protected precursor 25 to give the
enantiopure benzyl ethers 25R and 25S. Subsequent deprotection via hydrogenolysis provided
the pure individual enantiomers 38R and 38S as shown in Figure 4. We were initially concerned
that the hemiꢀaminal stability would be an issue but no interconversion or chemical stability
issues were observed with the purified isomers of 38. Notably the isomers had almost identical
antiviral activity against wild type virus however significantly differed in their loss of activity
against a Q148K mutation. The S derivative displayed only a 2 fold potency change while the R
analog had a 10 fold shift in potency. The isomers also showed different protein binding shifts
with the S isomer showing a near 50 fold shift while the R isomer had a very modest 5 fold loss
of potency in the presence of HSA. These differences in protein binding on potency also suggest
a different PK profile might be expected to arise as well. These preliminary data on the purified
isomers demonstrated excellent potency and previous PK data from the racemate 38 was
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consistent with once daily PK potential with a low risk for CYP450 interaction and the need for a
boosting agent.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Resistance profile against RAL resistant mutants and PK parameters in rat.a
C24 /
Q148K
(FC)
N155H
(FC)
Y143R
(FC)
CLt
(mL/min/kg)
T1/2
(hours)
Vdss
(mL/Kg)
Cmpd.
%F
MT4PAIC50
83 ± 6.6b
>1700b
8.4 ± 1.8b
25 ± 7.8b
21 ± 8.9
16 ± 3.9b
1.8 ± 0.16b
1.5 ± 0.59
1.6 ± 0.40
1.5 ± 0.12
1.5 ± 0.28
1.6 ± 0.85
RAL
EVG
1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
200 ± 25
35 ± 6.8
18 ± 2.5
2.1 ± 0.50
2.8 ± 0.32
1.2 ± 0.41
0.02 ± 0.00
0.51 ± 0.04
0.24 ± 0.02
0.10 ± 0.03
4.7 ± 0.15
11 ± 2.6
3.4 ± 0.28
7.6 ± 0.2
17 ± 6.7
200 ± 38
15 ± 1.1
120 ± 6
140 ± 13
130 ± 11
53 ± 20
40 ± 7.1
41 ± 2.7
14 ± 6.3
51 ± 8.5
3.4
2600
27
18a
19a
23
3.7 ± 1.0
1.5 ± 0.16
1.5 ± 0.054
1.6 ± 0.75
117
685
38
aData represent the mean ± s.d. FC; fold change. bData reported in reference 9.
25
chiral separation
H
N
H
N
O
O
N
N
O
O
OBn O
OBn O
25S
25R
deprotection
38R
38S
OH
OH
Figure 4. Chiral separation of the compound 38.
5. Conclusions
The carbamoyl pyridone scaffold was optimized through series’ of bicyclic and tricyclic
analogs for the inhibition of HIVꢀ1 IN utilizing a divergent synthetic strategy from a key Nꢀ
pyridonylꢀacetaldehyde intermediate. We have demonstrated the addition of a second and third
ring have added significant attributes to the carbamoyl pyridone core scaffold in terms of
antiviral activity against both wildꢀtype and key resistant mutants. Furthermore, protein binding
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effects as well as in vitro and in vivo PK profiles were taken into consideration in resulting in the
optimization to the tricyclic analogs 38. As such the tricyclic series D was identified as a true
next generation INI series which appeared to deliver on our original differentiation goals centered
on resistance and once daily PK. Further optimization of the individual enantiomers of
compounds such as 38 will be the subject of future publications.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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35
36
37
38
39
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6. Experimental section
6.1. Chemistry
Reactions were carried out under a nitrogen atmosphere with anhydrous solvents. Melting
points were collected up to 300 °C with a Stanford Research Systems Optimelt for every
crystalized sample. Purity of samples tested were assessed using combustion analyses or
LC/MS/UV/ELSD, and the analytical data confirmed the purities to be ≥95%. Combustion
analyses were performed with a Yanaco CORDER MTꢀ6 and a DIONEX ICSꢀ2000, LC/MS/UV
were with Waters Acquity SQD System. Microwave irradiation was performed with Biotage
1
Initiator. H NMR spectra were measured on a Varian MERCURY or Gemini 300MHz
spectrometer in a solution of either CDCl3 or DMSOꢀd6, using tetramethylsilane as the internal
standard. Chemical shifts are expressed as δ (ppm) values for protons relative to the internal
standard.
3-Benzyloxy-2-methylpyran-4-one (3). Maltol 2 (189 g, 1.5 mol) was dissolved in DMF
(1890 mL), and benzyl bromide (184 mL, 1.5 mol) was added. After stirring the solution at 80 °C
for 15 minutes, potassium carbonate (228 g, 1.65 mol) was added, and the mixture was stirred for
1 hour. After the reaction solution was cooled to room temperature, inorganic salt was filtered
off, and the filtrate was evaporated in vacuo. To the precipitated inorganic salt was added THF
(1000 mL), and the salt was filtered off. The filtrate was again evaporated in vacuo to obtain the
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product (329 g, quant.) as colorless solid. H NMR (CDCl3) δ 2.09 (s, 3H), 5.15 (s, 2H), 6.36 (d,
5
6
J = 5.6 Hz, 1H), 7.29–7.41 (m, 5H), 7.60 (d, J = 5.6 Hz, 1H).
7
8
9
3-Benzyloxy-2-methyl-1H-pyridine-4-one (4). The compound 3 (162 g, 750 mmol) was
dissolved in ethanol (487 mL), and aqueous ammonia (28%, 974 mL) and a 6 M aqueous sodium
hydroxide solution (150 mL, 900 mmol) was added. After strring the reaction solution at 90 °C
for 1 hour, the solution was cooled to under iceꢀcooling, and ammonium chloride (58 g, 1.08
mol) was added. The solution was extracted with chloroform, and the organic layer was washed
with an aqueous saturated sodium bicarbonate solution, and dried with anhydrous sodium sulfate.
The solvent was evaporated in vacuo, then isopropylalcohol and diethyl ether were added to the
residue. Precipitated solid was filtered to obtain the product (69.1 g, 43% yield) as pale yellow
solid. 1H NMR (DMSOꢀd6) δ 2.05 (s, 3H), 5.04 (s, 2H), 6.14 (d, J = 7.2 Hz, 1H), 7.31–7.42 (m,
5H), 7.46 (d, J = 7.2 Hz, 1H), 11.29 (brs, 1H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-Benzyloxy-5-bromo-2-methyl-1H-pyridine-4-one (5). The compound 4 (129 g, 599 mmol)
was suspended in acetonitrile (1300 mL), and Nꢀbromosuccinimide (117 g, 659 mmol) was
added. After stirring at room temperature for 1.5 hours precipitated crystals were filtered and
washed with acetonitrile and diethyl ether to obtain the product (154 g, 88% yield) as colorless
solid. 1H NMR (DMSOꢀd6) δ 2.06 (s, 3H), 5.04 (s, 2H), 7.32–7.42 (m, 5H), 8.03 (d, J = 5.5 Hz,
1H), 11.82 (brs, 1H).
Methyl 5-benzyloxy-6-methyl-4-oxo-1,4-dihydropyridine-3-carboxylate (6). To a solution
of the compound 5 (88.0 g, 300 mmol), Pd(OAc)2 (13.4 g, 60.0 mmol) and 1,3ꢀ
bis(diphenylphosphino)propane (30.8 g, 516 mmol) in DMF (660 mL) were added methanol (264
mL) and triethylamine (210 mL, 1.50 mol) at room temperature. The interior of a reaction vessel
was replaced with CO, and the solution was stirred at room temperature for 30 minutes, then
stirred at 80 °C for 18 hours. To a mixture of ethyl acetate (1500 mL), an aqueous saturated
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ammonium chloride solution (1500 mL) and water (1500 mL) stirred under iceꢀcooling the
reaction solution was added. Precipitates were filtered and washed with water (300 mL), ethyl
acetate (300 mL) and diethyl ether (300 mL) to obtain the product (44.9 g, 55% yield) as
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
colorless solid. H NMR (DMSOꢀd6) δ 2.06 (s, 3H), 3.72 (s, 3H), 5.02 (s, 2H), 7.33–7.42 (m,
5H), 8.07 (s, 1H).
Methyl 4-acetoxy-5-benzyloxy-6-methylnicotinate (7). After a solution of the compound 6
(19.1 g, 70.0 mmol) in acetic anhydride (134 mL) was stirred at 130 °C for 40 minutes, the acetic
anhydride was evaporated in vacuo to obtain the product (19.9 g, 90% yield) as flesh colored
1
solid. H NMR (CDCl3) δ 2.29 (s, 3H), 2.52 (s, 3H), 3.89 (s, 3H), 4.98 (s, 2H), 7.36–7.41 (m,
5H), 8.85 (s, 1H).
Methyl 4-acetoxy-5-benzyloxy-6-methyl-1-oxynicotinate (8). To a solution of the compound
7 (46.2 g, 147 mmol) in chloroform (370 mL) was added 65% mꢀCPBA (42.8 g, 161 mmol) in
portions under iceꢀcooling, and the solution was stirred at room temperature for 90 minutes. To
the reaction solution was added a 10% aqueous potassium carbonate solution, and the mixture
was stirred for 10 minutes. The solution was extracted with chloroform, and the organic layer
was washed with successively with a 10% aqueous potassium carbonate solution, an aqueous
saturated ammonium chloride solution, and an aqueous saturated sodium chloride solution. The
organic layer was dried with anhydrous sodium sulfate, and the solvent was evaporated in vacuo.
The residual precipitate was washed with diisopropyl ether to obtain the product (42.6 g, 87%
yield) as colorless solid. 1H NMR (CDCl3) δ 2.30 (s, 3H), 2.41 (s, 3H), 3.90 (s, 3H), 5.02 (s, 2H),
7.37–7.39 (m, 5H), 8.70 (s, 1H).
Methyl 4-acetoxy-6-acetoxymethyl-5-benzyloxynicotinate (9). To acetic anhydride (500 mL)
which had been heated at 130 °C was added the compound 8 (42.6 g, 129 mmol) over 2 minutes,
and the mixture was stirred for 20 minutes. The acetic anhydride was evaporated in vacuo to
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obtain the product (49.6 g, quant.) as black oil. H NMR (CDCl3) δ 2.10 (s, 3H), 2.28 (s, 3H),
5
6
3.91 (s, 3H), 5.07 (s, 2H), 5.20 (s, 2H), 7.35–7.41 (m, 5H), 8.94 (s, 1H).
7
8
9
Methyl 5-benzyloxy-6-hydroxymethyl-4-oxo-1,4-dihydropyridine-3-carboxylate (10). To a
solution of the compound 9 (9.50 g, 25.4 mmol) in methanol (50 mL) was added a 28 wt%
sodium methoxide methanol solution (7.35 mL, 38.1 mmol) under iceꢀcooling. The solution was
stirred at room temperature for 30 minutes. To an iceꢀcooled sat. ammonium chloride aqueous
solution the reaction solution were added and stirred for 30 minutes. Precipitated crystals were
filtered, and washed with water and diethyl ether to obtain the product (4.68 g, 64% yield) as
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
colorless solid. H NMR (DMSOꢀd6) δ 3.72 (s, 3H), 4.37 (s, 2H), 5.06 (s, 2H), 5.72 (s, 1H),
7.34–7.40 (m, 5H), 8.06 (s, 1H), 11.47 (brs, 1H).
5-Benzyloxy-N-(4-fluorobenzyl)-6-hydroxymethyl-4-oxo-1,4-dihydropyridine-3-
carboxamide (11). To a solution of the compound 10 (289 mg, 1.0 mmol) in DMF (3 mL) was
added 4ꢀfluorobenzylamine (126 ꢁL, 1.10 mmol). The mixture was stirred at 120 °C for 1.5
hours. After the reaction solution was cooled to room temperature, 2 M aq HCl (6 mL) was
added. The solution was extracted with ethyl acetate, and the organic layer was washed with
water and an aqueous saturated sodium chloride solution. The organic layer was dried with
anhydrous sodium sulfate, and the solvent was evaporated in vacuo to obtain the product (198
1
mg, 52% yield) as colorless solid. H NMR (DMSOꢀd6) δ 4.45 (d, J = 5.0 Hz, 2H), 4.52 (d, J =
6.0 Hz, 2H), 5.09 (s, 2H), 5.84 (t, J = 5.0 Hz, 1H), 7.14–7.20 (m, 2H), 7.35–7.40 (m, 7H), 8.30 (s,
1H), 10.72 (t, J = 6.0 Hz, 1H), 11.97 (s, 1H).
5-Benzyloxy-N-(4-fluorobenzyl)-6-formyl-4-oxo-1,4-dihydropyridine-3-carboxamide (12).
To a suspension of the compound 11 (9.80 g, 25.6 mmol) in chloroform (490 mL) manganese
dioxide (49.0 g) was added. The mixture was stirred at room temperature for 1 hour, then stirred
at 60 °C for 20 minutes. The manganese dioxide was filtrated off under celite filtration, and
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washed with chloroform heated at 50 °C. The filtrate was evaporated in vacuo to obtain the
product (8.20 g, 84% yield) as pale yellow solid. 1H NMR (DMSOꢀd6) δ 4.53 (d, J = 5.8 Hz, 2H),
5.38 (s, 2H), 7.15–7.21 (m, 2H), 7.35–7.46 (m, 7H), 8.33 (s, 1H), 9.90 (s, 1H), 10.35 (t, J = 5.8
Hz, 1H), 12.49 (s, 1H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
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50
51
52
53
54
55
56
57
58
59
60
3-Benzyloxy-5-(4-fluorobenzylcarbamoyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid
(13). To an aqueous solution (105 mL) of sodium chlorite (7.13 g, 78.8 mmol) and sulfamic acid
(7.65 g, 78.8 mmol) was added a solution of the compound 12 (15.0 g, 39.4 mmol) in THF (630
mL) under iceꢀcooling. The mixture was stirred at room temperature for 1 hour, and water (2500
L) was added to the reaction solution. The precipitated crystals were filtered, and washed with
1
diethyl ether to obtain the product (14.0 g, 90% yield) as colorless solid. H NMR (DMSOꢀd6) δ
4.52 (d, J = 5.8 Hz, 2H), 5.13 (s, 2H), 7.14–7.19 (m, 2H), 7.31–7.40 (m, 5H), 7.47–7.49 (m, 2H),
8.31 (d, J = 4.5 Hz, 1H), 10.44 (t, J = 5.8 Hz, 1H), 12.47 (brs, 1H).
Methyl
3-benzyloxy-5-(4-fluorobenzylcarbamoyl)-4-oxo-1,4-dihydropyridine-2-
carboxylate (14). The compound 13 (12.0 g, 30.3 mmol), 1ꢀ(3ꢀdimethylaminopropyl)ꢀ3ꢀ
ethylcarbodiimide hydrochloride (6.96 g, 36.3 mmol) and 1ꢀhydroxybenzotriazole (4.91 g, 36.3
mmol) in DMF (180 mL) was stirred at room temperature for 1.5 hours. To the mixture methanol
(180 mL) and triethylamine (9.29 mL, 66.7 mmol) were added, and the mixture was heated to
reflux for 1.5 hours. The reaction solution was diluted with ethyl acetate, then washed with an
aqueous saturated sodium bicarbonate solution, a 10% aqueous citric acid solution, and an
aqueous saturated sodium chloride solution. The organic layer was dried with anhydrous sodium
sulfate, and the solvent was evaporated in vacuo. The residual precipitate was washed with
diethyl ether to obtain the product (8.74 g, 70% yield) as colorless crystals. 1H NMR (DMSOꢀd6)
δ 3.85 (s, 3H), 4.52 (d, J = 6.0 Hz, 2H), 5.15 (s, 2H), 7.13–7.21 (m, 2H), 7.31–7.47 (m, 7H), 8.33
(s, 1H), 10.41 (t, J = 6.0 Hz, 1H), 12.59 (s, 1H).
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1-allyl-3-benzyloxy-5-(4-fluorobenzylcarbamoyl)-4-oxo-1,4-dihydropyridine-2-
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carboxylate (15). To a solution of the compound 14 (6.79 g, 16.5 mmol) 3ꢀbromopropene (2.15
mL, 24.8 mmol) and Cs2CO3 (8.09 g, 24.8 mmol) in DMF (54 mL) was added, and the mixture
was stirred at room temperature for 4.5 hours. To the reaction solution was added an aq.
ammonium chloride solution, and the mixture was extracted with ethyl acetate. The organic layer
was washed with water and an aq. saturated sodium chloride solution. The organic layer was
dried with anhydrous sodium sulfate, and the solvent was evaporated in vacuo. The residual
precipitate was washed with diethyl ether to obtain the product (6.15 g, 83% yield) as colorless
solid. 1H NMR (CDCl3) δ 3.76 (s, 3H), 4.54 (d, J = 6.0 Hz, 2H), 4.60 (d, J = 6.0 Hz, 2H), 5.20–
5.37 (m, 2H), 5.25 (s, 2H), 5.80–5.93 (m, 1H), 6.98–7.04 (m, 2H), 7.31–7.35 (m, 7H), 8.45 (s,
1H), 10.41 (m, 1H).
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Methyl
3-benzyloxy-5-(4-fluorobenzylcarbamoyl)-4-oxo-1-(2-oxoethyl)-1,4-
dihydropyridine-2-carboxylate (16). To a solution of the compound 15 (7.6 g, 16.9 mmol) in
1,4ꢀdioxane (228 mL) was added an aqueous solution (38 mL) of potassium osmate dihydrate
(372 mg, 1.01 mmol), and sodium metaperiodate (14.5 g, 67.6 mmol) was further added. After
stirring at room temperature for 2 hours the reaction solution was added to a vessel to which
ethyl acetate (300 mL) and water (300 mL) had been added, while stirring. The organic layer was
washed with water, a 5% aq. sodium hydrogen sulfite solution and an aq. saturated sodium
chloride solution. The organic layer was dried with anhydrous sodium sulfate, and the solvent
was evaporated in vacuo, and the residual precipitate was washed with diethyl ether to obtain the
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product (5.39 g, 71% yield) as colorless solid. H NMR (CDCl3) δ 3.74 (s, 3H), 4.60 (d, J = 5.9
Hz, 2H), 4.87 (s, 2H), 5.27 (s, 2H), 6.98–7.04 (m, 2H), 7.30–7.40 (m, 7H), 8.39 (s, 1H), 9.58 (s,
1H), 10.38 (s, 1H).
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9-Benzyloxy-2-(2-methoxyethyl)-1,8-dioxo-1,8-dihydro-2H-pyrid[1,2-a]pyrazine-7-
carboxylic acid 4-fluorobenzylamide (17a). To a solution of the compound 16 (400 mg, 0.884
mmol) in dichloromethane (12 mL) were added 2ꢀmethoxyethylamine (77.0 ꢁL, 0.884 mmol)
and acetic acid (18 ꢁL). After stirring at room temperature for 5 minutes, the reaction was
performed at 140 °C for 30 minutes in a microwave reaction apparatus. The solvent was
evaporated in vacuo, and the residue was subjected to silica gel column chromatography.
Fractions eluting with tolueneꢀacetone were collected and evaporated in vacuo to obtain the
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product (226 mg, 54% yield) as a yellow solid. H NMR (CDCl3) δ 3.35 (s, 3H), 3.65 (t, J = 5.2
Hz, 2H), 3.98 (t, J = 5.2 Hz, 2H), 4.62 (d, J = 6.0 Hz, 2H), 5.28 (s, 2H), 6.57 (s, 2H), 6.98–7.04
(m, 2H), 7.30–7.38 (m, 5H), 7.637.67 (m, 2H), 8.59 (s, 1H), 10.60–10.64 (m, 1H).
9-Benzyloxy-2-[2-(morpholin-4-yl)ethyl]-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-
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a]pyrazine-7-carboxylic acid 4-fluorobenzylamide (17b). H NMR (CDCl3) δ 2.59 (s, 4H),
2.74 (s, 2H), 3.73 (s, 4H), 3.95 (s, 2H), 4.62 (d, J = 6.0 Hz, 2H), 5.28 (s, 2H), 6.53 (d, J = 6.0 Hz,
1H), 6.63 (d, J = 6.0 Hz, 1H), 7.01 (t, J = 8.7 Hz, 2H), 7.26–7.38 (m, 5H), 7.64 (d, J = 6.9 Hz,
2H), 8.61 (s, 1H), 10.61 (t, J = 5.4 Hz, 1H).
9-Benzyloxy-2-dimethylcarbamoylmethyl-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-
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a]pyrazine-7-carboxylic acid 4-fluorobenzylamide (17c). H NMR (CDCl3) δ 3.01 (s, 3H),
3.13 (s, 3H), 4.59 (s, 2H), 4.63 (d, J = 6.0 Hz, 2H), 5.26 (s, 2H), 6.42 (d, J = 6.0 Hz, 1H), 6.64 (d,
J = 6.0 Hz, 1H), 6.98–7.05 (m, 2H), 7.16–7.36 (m, 5H), 7.60–7.66 (m, 2H), 8.60 (s, 1H), 10.59
(brt, J = 6.0 Hz, 1H).
9-Benzyloxy-2-(2-isopropoxyethyl)-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-
carboxylic acid 4-fluorobenzylamide (17d). 1H NMR (CDCl3) δ 1.12 (d, J = 6.0 Hz, 6H), 3.51–
3.59 (m, 1H), 3.68 (t, J = 4.8 Hz, 2H), 3.96 (t, J = 4.8 Hz, 2H), 4.62 (d, J = 6.0 Hz, 2H), 5.28 (s,
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2H), 6.58–6.64 (m, 2H), 6.98–7.04 (m, 2H), 7.30–7.39 (m, 5H), 7.64–7.66 (m, 2H), 8.59 (brs,
1H), 10.63 (s, 1H).
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carboxylic acid 4-fluorobenzylamide (17e). H NMR (CDCl3) δ 4.17–4.20 (m, 2H), 4.25–4.28
(m, 2H), 4.62 (d, J = 5.6 Hz, 2H), 5.28 (s, 2H), 6.60–6.66 (m, 2H), 6.86 (d, J = 8.0 Hz, 2H),
6.95–7.04 (m, 2H), 7.28–7.37 (m, 8H), 7.64 (d, J = 7.0 Hz, 2H), 8.59 (s, 1H), 10.60 (brs, 1H).
9-Benzyloxy-2-cyclohexyl-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic
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acid 4-fluorobenzylamide (17f). H NMR (CDCl3) δ 1.15–1.92 (m, 10H), 4.62 (d, J = 6.1 Hz,
2H), 4.70–4.78 (m, 1H), 5.27 (s, 2H), 6.43 (d, J = 6.4 Hz, 1H), 6.69 (d, J = 6.3 Hz, 1H), 7.01–
7.16 (m, 2H), 7.18–7.37 (m, 5H), 7.66–7.68 (m, 2H), 8.63 (s, 1H), 10.67 (t, J = 5.5 Hz, 1H).
9-Hydroxy-2-(2-methoxyethyl)-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-
carboxylic acid 4-fluorobenzylamide (18a). To the compound 17a (140 mg, 0.293 mmol) was
added trifluoroacetic acid (1.4 mL) under iceꢀcooling. The mixture was stirred at 0 °C for 5
minutes and at room temperature for 1.5 hours. The solvent was evaporated in vacuo and the
residue was diluted with chloroform and ice water. The organic layer was washed with an aq.
saturated sodium bicarbonate solution, a 10% aq. citric acid solution and water. The organic layer
was dried with anhydrous sodium sulfate, and the solvent was evaporated in vacuo. The residual
crystals were recrystallized with dichloromethaneꢀethanol to obtain the product (89.0 mg, 79%
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yield) as yellow crystals. mp 223–224 °C; H NMR (DMSOꢀd6) δ 3.25 (s, 3H), 3.58 (t, J = 5.4
Hz, 2H), 3.92 (t, J = 5.1 Hz, 2H), 4.53 (d, J = 5.7 Hz, 2H), 6.87 (d, J = 6.3 Hz, 1H), 7.14 (t, J =
9.0 Hz, 2H), 7.33–7.38 (m, 2H), 7.47 (d, J = 6.0 Hz, 1H), 8.77 (s, 1H), 10.56 (t, J = 6.0 Hz, 1H),
12.00 (brs, 1H). Anal. Calcd for C19H18FN3O5: C, 58.91; H, 4.68; F, 4.90; N, 10.85. Found: C,
58.68; H, 4.63; F, 4.73; N, 10.80.
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9-Hydroxy-2-[2-(morpholin-4-yl)ethyl]-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-
7-carboxylic acid 4-fluorobenzylamide (18b). mp 212–215 °C; 1H NMR (DMSOꢀd6) δ 2.51 (s,
4H), 2.38 (s, 2H), 3.55 (s, 4H), 3.90 (s, 2H), 4.55 (d, J = 6.0 Hz, 2H), 6.95 (d, J = 6.3 Hz, 2H),
7.17 (t, J = 8.7 Hz, 2H), 7.35–7.40 (m, 2H), 7.50 (d, J = 6.3 Hz, 1H), 8.78 (s, 1H), 10.58 (t, J =
6.3 Hz, 1H), 12.10 (s, 1H). LC/MS: m/z 443 [M + H]+.
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2-Dimethylcarbamoylmethyl-9-hydroxy-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-
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7-carboxylic acid 4-fluorobenzylamide (18c). mp >300 °C; H NMR (DMSOꢀd6) δ 2.87 (s,
3H), 3.03 (s, 3H), 4.55 (d, J = 6.0 Hz, 2H), 4.71 (s, 2H), 6.80 (d, J = 6.3 Hz, 1H), 7.16 (m, 2H),
7.38 (m, 2H), 7.48 (d, J = 6.3 Hz, 1H), 8.82 (s, 1H), 10.54 (brt, J = 6.0 Hz, 1H), 11.83 (s, 1H).
Anal. Calcd for C20H19FN4O5(H2O)0.4: C, 56.98; H, 4.73; F, 4.51; N, 13.29. Found: C, 57.03; H,
4.44; F, 4.50; N, 13.13.
9-Hydroxy-2-(2-isopropoxyethyl)-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-
carboxylic acid 4-fluorobenzylamide (18d). mp 209ꢀ210 °C; 1H NMR (DMSOꢀd6) δ 1.06 (d, J
= 6.3 Hz, 6H), 3.54–3.64 (m, 3H), 3.90 (t, J = 5.4 Hz, 2H), 4.62 (d, J = 6.0 Hz, 2H), 6.89 (d, J =
6.3 Hz, 1H), 7.13–7.19 (m, 2H), 7.35–7.39 (m, 2H), 7.47 (d, J = 6.3 Hz, 1H), 8.77 (s, 1H), 10.58
(t, J = 5.7 Hz, 1H), 12.04 (brs, 1H). Anal. Calcd. for C21H22FN3O5: C, 60.72; H, 5.34; F, 4.57; N,
10.12. Found: C, 60.78; H, 5.29; F, 4.34; N, 10.11.
9-Hydroxy-1,8-dioxo-2-(2-phenoxyethyl)-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-
carboxylic acid 4-fluorobenzylamide (18e). mp 237–239 °C; 1H NMR (CDCl3) δ 4.18–4.21 (m,
2H), 4.26–4.29 (m, 2H), 4.62 (d, J = 5.7 Hz, 2H), 6.57 (d, J = 6.3 Hz, 1H), 6.71 (d, J = 6.3 Hz,
1H), 6.86 (d, J = 8.1 Hz, 2H), 6.97–7.02 (m, 3H), 7.29–7.35 (m, 4H), 8.56 (s, 1H), 10.58 (t, J =
5.7 Hz, 1H), 11.84 (brs, 1H). Anal. Calcd for C24H20FN3O5: C, 64.14; H, 4.49; F, 4.23; N, 9.35.
Found: C, 64.46; H, 4.50; F, 4.01; N, 9.12.
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acid 4-fluorobenzylamide (18f). mp >300 °C; H NMR (DMSOꢀd6) δ 1.15–1.84 (m, 10H),
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4.43–4.49 (m, 1H), 4.53 (d, J = 5.8 Hz, 2H), 7.05 (d, J = 6.4 Hz, 1H), 7.13–7.19 (m, 2H), 7.34–
7.39 (m, 2H), 7.53 (d, J = 6.4 Hz, 1H), 8.79 (s, 1H), 10.61 (t, J = 5.8 Hz, 1H), 12.23 (brs, 1H).
LC/MS: m/z 412 [M + H]+.
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9-Hydroxy-2-(2-methoxyethyl)-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-
carboxylic acid 4-fluorobenzylamide (19a). The compound 17a (157 mg, 0.329 mmol) was
dissolved in DMF (18 mL) and methanol (1 mL), 10% palladiumꢀcarbon powder (31 mg) was
added, and the mixture was stirred at room temperature for 20 hours under hydrogen atmosphere.
The reaction solution was filtered with celite, and the filtrate was evaporated in vacuo. The
residue was dissolved in chloroform, and insoluble residue was filtered with celite again, and the
filtrate was evaporated in vacuo. The residual crystals were recrystallized from dichloromethaneꢀ
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methanol to obtain the product (66 mg, 52% yield) as brown crystals. mp 197–199 °C; H NMR
(DMSOꢀd6) δ 3.27 (s, 3H), 3.55 (t, J = 5.1 Hz, 2H), 3.68 (t, J = 5.1 Hz, 2H), 3.79 (s, 2H), 4.36 (s,
2H), 4.51 (d, J = 5.7 Hz, 2H), 7.15 (t, J = 8.7 Hz, 2H), 7.32–7.37 (m, 2H), 8.38 (s, 1H), 10.46 (t,
J = 5.4 Hz, 1H), 12.41 (s, 1H). LC/MS: m/z 390 [M + H]+.
9-Hydroxy-2-[2-(morpholin-4-yl)ethyl]-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-
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a]pyrazine-7-carboxylic acid 4-fluorobenzylamide (19b). mp 205–207 °C; H NMR (DMSOꢀ
d6) δ 2.43 (s, 2H), 2.50 (s, 4H), 3.54 (s, 4H), 3.63 (s, 2H), 3.81 (s, 2H), 4.40 (s, 2H), 4.52 (d, J =
6.0 Hz, 2H), 7.16 (t, J = 9.0 Hz, 2H), 7.33–7.37 (m, 2H), 8.43 (s, 1H), 10.45 (t, J = 5.7 Hz, 1H),
12.48 (s, 1H). LC/MS: m/z 445 [M + H]+.
2-Dimethylcarbamoylmethyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-
a]pyrazine-7-carboxylic acid 4-fluorobenzylamide (19c). mp 245 °C; 1H NMR (CDCl3) δ 3.00
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(s, 3H), 3.08 (s, 3H), 3.83–3.87 (m, 2H), 4.37–4.41 (m, 2H), 4.42 (s, 2H), 4.60 (s, 2H), 6.98–7.04
(m, 2H); 7.30–7.34 (m, 2H), 8.33 (s, 1H). LC/MS: m/z 417 [M + H]+.
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9-Hydroxy-2-(2-isopropoxyethyl)-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-
7-carboxylic acid 4-fluorobenzylamide (19d). mp 210–212 °C; 1H NMR (DMSOꢀd6) δ 1.08 (d,
J = 6.0 Hz, 6H), 3.54–3.66 (m, 5H), 3.79–3.83 (m, 2H), 4.35–4.39 (m, 2H), 4.52 (d, J = 6.0 Hz,
2H), 7.12–7.18 (m, 2H), 7.32–7.37 (m, 2H), 8.40 (s, 1H), 10.44 (t, J = 6.0 Hz, 1H), 12.42 (brs,
1H). Anal. Calcd for C21H24FN3O5(H2O)0.3: C, 59.65; H, 5.86; F, 4.49; N, 9.94. Found: C, 59.58;
H, 5.63; F, 4.40; N, 9.96.
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9-Hydroxy-1,8-dioxo-2-(2-phenoxyethyl)-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-
carboxylic acid 4-fluorobenzylamide (19e). mp 200–201 °C; 1H NMR (CDCl3) δ 3.96–4.02 (m,
4H), 4.20–4.28 (m, 4H), 4.60 (d, J = 6.0 Hz, 2H), 6.86–6.89 (m, 2H), 6.96–7.02 (m, 3H), 7.28–
7.34 (m, 4H), 8.31 (s, 1H), 10.43 (brs, 1H), 12.15 (brs, 1H). Anal. Calcd for C24H20FN3O5: C,
63.85; H, 4.91; F, 4.21; N, 9.31. Found: C, 63.72; H, 4.93; F, 4.07; N, 9.29.
2-Cyclohexyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-
carboxylic acid 4-fluorobenzylamide (19f). mp >300 °C; 1H NMR (DMSOꢀd6) δ 1.03–1.81 (m,
10H), 3.69–3.72 (m, 2H), 4.29–4.36 (m, 3H), 4.52 (d, J = 6.1 Hz, 2H), 7.13–7.19 (m, 2H), 7.33–
7.37 (m, 2H), 8.43 (s, 1H), 10.47 (t, J = 5.8 Hz, 1H), 12.59 (brs, 1H). LC/MS: m/z 414 [M + H]+.
3-Benzyloxy-N5-(4-fluorobenzyl)-N2-(2-methoxyethyl)-4-oxo-1,4-dihydropyridine-2,5-
dicarboxamide (20). Using the compound 13, compound 20 was synthesized according to the
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method of synthesizing the compound 14. H NMR (CDCl3) δ 3.27 (s, 3H), 3.76–3.40 (m, 2H),
3.47–3.52 (m, 2H), 4.63 (d, J = 6.1 Hz, 2H), 5.48 (s, 2H), 6.99–7.05 (m, 2H), 7.33–7.45 (m, 7H),
8.54–8.58 (m, 1H), 8.62 (d, J = 6.7 Hz, 1H), 10.44–10.48 (m, 1H).
1-Allyl-3-benzyloxy-N5-(4-fluorobenzyl)-N2-(2-methoxyethyl)-4-oxo-1,4-dihydropyridine-
2,5-dicarboxamide (21). Using the compound 20, compound 21 was synthesized according to
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the method of synthesizing a compound 15. 1H NMR (CDCl3) δ 3.21 (s, 3H), 3.32–3.35 (m, 2H),
3.38–3.45 (m, 2H), 4.58–4.61 (m, 4H), 5.14–5.30 (m, 4H), 5.82–5.93 (m, 1H), 6.72 (brs, 1H),
6.98–7.04 (m, 2H), 7.30–7.39 (m, 7H), 8.41 (s, 1H), 10.43–10.47 (m, 1H).
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9-Benzyloxy-N-(4-fluorobenzyl)-3-hydroxy-2-(2-methoxyethyl)-1,8-dioxo-2,3,4,8-
tetrahydro-1H-pyrido[1,2-a]pyrazine-7-carboxamide (22). Using the compound 21,
compound 22 was synthesized according to the method of synthesizing a compound 16. 1H NMR
(CDCl3) δ 3.07–3.17 (m, 1H), 3.42 (s, 3H), 3.44–3.49 (m, 1H), 3.61–3.69 (m, 1H), 4.11–4.17 (m,
4H), 4.26–4.31 (m, 1H), 4.43–4.49 (m, 1H), 4.59–4.62 (m, 2H), 5.00–5.08 (m, 2H), 5.20 (d, J =
9.9 Hz, 1H), 5.34 (d, J = 9.9 Hz, 1H), 6.98–7.04 (m, 2H), 7.30–7.35 (m, 5H), 7.60–7.63 (m, 2H),
8.38 (s, 1H), 10.45–10.49 (m, 1H).
3,9-Dihydroxy-2-(2-methoxyethyl)-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrid[1,-2-
a]pyrazine-7-carboxylic acid 4-fluorobenzylamide (23). According to the method of
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synthesizing 19, compound 23 was synthesized from 22. mp 163–164 °C; H NMR (CDCl3) δ
3.24–3.32 (m, 1H), 3.46 (s, 3H), 3.52–3.57 (m, 1H), 3.72–3.80 (m, 1H), 4.17–4.39 (m, 3H), 4.62
(d, J = 5.8 Hz, 2H), 5.12 (s, 1H), 5.80 (brs, 1H), 6.97–7.03 (m, 2H), 7.30–7.35 (m, 2H), 8.32 (s,
1H), 10.36–10.38 (m, 1H), 12.02 (brs, 1H). Anal. Calcd for C19H20FN3O6: C, 56.29; H, 4.97; F,
4.69; N, 10.37. Found: C, 56.13; H, 4.99; F, 4.55; N, 10.34.
7-Benzyloxy-N-(4-fluorobenzyl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-[1,3]oxazino[3,2-
d]pyrido[1,2-a]pyrazine-9-carboxamide (25). To the solution of the compound 16 (200 mg,
0.442 mmol) in dichloromethane (5 mL) 3ꢀaminopropaneꢀ1ꢀol (35.0 ꢂL, 0.511 mmol) and acetic
acid (10.0 mg, 0.167 mmol) were added. The reaction was performed with a microwave reaction
apparatus at 140 °C for 30 minutes. After cooled to room temperature, solvent was evaporated in
vacuo, the residue was subjected to silica gel column chromatography, and fractions eluting with
chloroformꢀmethanol were collected and concentrated to obtain the product (173 mg, 74% yield)
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as colorless solid. 1H NMR (CDCl3) δ 1.62–1.67 (m, 1H), 1.91–ꢀ2.07 (m, 1H), 3.07 (dt, J = 12.9,
3.3 Hz, 1H), 3.84 (dt, J = 11.7, 2.4 Hz, 1H), 4.11–4.20 (m, 2H), 4.32 (dd, J = 13.8, 3.3 Hz, 1H),
4.63 (d, J = 6.0 Hz, 2H), 4.71–4.78 (m, 1H), 5.01 (t, J = 4.2 Hz, 1H), 5.26 (d, J = 9.9 Hz, 1H),
5.31 (d, J = 9.9 Hz, 1H), 7.00–7.07 (m, 2H), 7.32–7.42 (m, 5H), 7.64–7.66 (m, 2H), 8.39 (s, 1H),
10.45 (brs, 1H).
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6-Benzyloxy-N-(4-fluorobenzyl)-5,7-dioxo-2,3,5,7,11,11a-hexahydrooxazolo[3,2-
d]pyrido[1,2-a]pyrazine-8-carboxamide (24). 1H NMR (DMSOꢀd6) δ 3.52–3.62 (m, 1H), 3.78–
3.91 (m, 1H), 4.01–4.12 (m, 2H), 4.24–4.31 (m, 1H), 4.50–4.62 (m, 2H), 4.88 (dd, J = 3.3, 12.0
Hz, 1H), 5.07 (d, J = 10.2 Hz, 1H), 5.21 (d, J = 10.2 Hz, 1H), 5.31 (dd, J = 9.9, 3.3 Hz, 1H), 7.19
(t, J = 8.7 Hz, 2H), 7.32–7.42 (m, 5H), 7.58 (d, J = 6.6 Hz, 2H), 8.64 (s, 1H), 10.49–10.54 (t, J =
6.3 Hz, 1H).
1-Benzyloxy-2,11-dioxo-2,5,5a,7,8,9,10,11-octahydro-6-oxa-4a,10a-
diazacyclohepta[b]naphthalene-3-carboxylic acid 4-fluorobenzylamide (26). 1H NMR
(CDCl3) δ 1.55–1.60 (m, 1H), 1.77–1.91 (m, 2H), 2.03–2.13 (m, 1H), 3.13–3.22 (m, 1H), 3.64–
3.81 (m, 2H), 4.15–4.21 (m, 1H), 4.31–4.37 (m, 1H), 4.39–4.48 (m, 1H), 4.63 (t, J = 6.0 Hz, 2H),
4.99 (t, J = 2.4 Hz, 1H), 5.26 (d, J = 10.2 Hz, 1H), 5.41 (d, J = 10.2 Hz, 1H), 7.03 (d, J = 9.0 Hz,
2H), 7.20–7.40 (m, 5H), 7.63–7.65 (m, 2H), 8.40 (s, 1H), 10.49–10.54 (m, 1H).
6-Benzyloxy-N-(4-fluorobenzyl)-1-isopropyl-5,7-dioxo-2,3,5,7,11,11a-hexahydro-1H-
imidazo[1,2-d]pyrido[1,2-a]pyrazine-8-carboxamide (27). 1H NMR (DMSOꢀd6) δ 1.06 (d, J =
6.3 Hz, 3H), 1.16 (d, J = 6.3 Hz, 3H), 2.77–2.85 (m, 1H), 2.98–3.07 (m, 1H), 3.15–3.22 (m, 1H),
3.54–3.58 (m, 2H), 3.87 (t, J = 11.4 Hz, 1H), 4.48–4.57 (m, 3H), 4.81–4.85 (m, 1H), 5.06 (d, J =
10.2 Hz, 1H), 5.20 (d, J = 10.2 Hz, 1H), 7.19 (t, J = 9.0 Hz, 2H), 7.31–7.42 (m, 5H), 7.57 (d, J =
7.8 Hz, 2H), 8.62 (s, 1H), 10.49 (t, J = 6.0 Hz, 1H).
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5-Benzyloxy-1-methyl-6,10-dioxo-1,2,3,4,6,9,9a,10-octahydro-1,4a,8a-triazaanthracene-7-
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carboxylic acid 4-fluorobenzylamide (28). H NMR (CDCl3) δ 1.61–1.68 (m, 2H), 2.31 (s,
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3H), 2.85–2.93 (m, 1H), 3.06–3.11 (m, 1H), 4.32–4.39 (m, 2H), 4.58–4.66 (m, 3H), 4.69–4.79
(m, 2H), 5.32 (s, 2H), 7.02–7.08 (m, 2H), 7.33–7.42 (m, 5H), 7.64–7.66 (m, 2H), 8.41 (s, 1H),
10.50 (brs, 1H).
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5-Benzyloxy-1-isopropyl-6,10-dioxo-1,2,3,4,6,9,9a,10-octahydro-1,4a,8a-triazaanthracene-
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7-carboxylic acid 4-fluorobenzylamide (29). H NMR (DMSOꢀd6) δ 0.95–0.99 (m, 6H), 1.52–
1.56 (m, 1H), 1.64–1.69 (m, 1H), 2.49–2.56 (m, 1H), 2.89–2.98 (m, 2H), 2.32–2.41 (m, 1H),
2.40–2.55 (m, 1H), 3.68–3.70 (m, 2H), 4.36 (brs, 1H), 4.54–4.59 (m, 1H), 4.88–4.93 (m, 1H),
5.07 (d, J = 10.2 Hz, 1H), 5.13 (d, J = 10.2 Hz, 1H), 7.18–7.24 (m, 2H), 7.35–7.45 (m, 5H),
7.58–7.62 (m, 2H), 8.78 (s, 1H), 10.52 (t, J = 6.0 Hz, 1H).
5-Benzyloxy-6,10-dioxo-1-thiazol-2-ylmethyl-1,2,3,4,6,9,9a,10-octahydro-1,4a,8a-
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triazaanthracene-7-carboxylic acid 4-fluorobenzylamide (30). H NMR (CDCl3) δ 1.67–1.71
(m, 1H), 2.00–2.13 (m, 1H), 2.84–2.97 (m, 2H), 3.19–3.23 (m, 1H), 4.13 (s, 2H), 4.35–4.38 (m,
2H), 4.56–4.67 (m, 3H), 4.71–4.80 (m, 1H), 5.29 (d, J = 9.9 Hz, 1H), 5.36 (d, J = 9.9 Hz, 1H),
7.03–7.08 (m, 2H), 7.24 (d, J = 3.3 Hz, 1H), 7.34–7.42 (m, 5H), 7.66–7.69 (m, 2H), 7.79 (d, J =
3.3 Hz, 1H), 8.05 (brs, 1H), 10.49 (t, J = 6.0 Hz, 1H).
5-Benzyloxy-1-(2-methoxyethyl)-6,10-dioxo-1,2,3,4,6,9,9a,10-octahydro-1,4a,8a-
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triazaanthracene-7-carboxylic acid 4-fluorobenzylamide (31). H NMR (CDCl3) δ 1.56ꢀ1.65
(m, 1H), 1.82–1.95 (m, 1H), 2.62–2.70 (m, 1H), 2.80–2.92(m, 3H), 2.98–3.05 (m, 1H), 3.28–
3.34 (m, 1H), 3.34 (s, 3H), 3.58–3.65 (m, 1H), 4.28 (dd, J = 13.8 Hz, 3.6 Hz, 1H), 4.38 (brs, 1H),
4.53–4.59 (m, 1H), 4.63 (d, J = 6.0 Hz, 2H), 4.69–4.76 (m, 1H), 5.29 (s, 2H), 7.00–7.05 (m, 2H),
7.30–7.38 (m, 5H), 7.63–7.66 (m, 2H), 8.38 (s, 1H), 10.53 (t, J = 6.0 Hz, 1H).
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