The design of 8-hydroxyquinoline tetracyclic lactams as HIV-1 integrase strand transfer inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.03.038

A novel series of HIV-1 integrase strand transfer inhibitors were designed using the venerable two-metal binding pharmacophore model and incorporating structural elements from two different literature scaffolds. This manuscript describes a number of 8-hyd

Full text of DOI:10.1016/j.ejmech.2016.03.038

Accepted Manuscript
The Design of 8-Hydroxyquinoline Tetracyclic Lactams as HIV-1 Integrase Strand
Transfer Inhibitors
Emile J. Velthuisen, Brian A. Johns, David P. Temelkoff, Kevin W. Brown, Susan C.
Danehower
PII:
S0223-5234(16)30212-4
10.1016/j.ejmech.2016.03.038
EJMECH 8463
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 January 2016
Revised Date: 14 March 2016
Accepted Date: 15 March 2016
Please cite this article as: E.J. Velthuisen, B.A. Johns, D.P. Temelkoff, K.W. Brown, S.C. Danehower,
The Design of 8-Hydroxyquinoline Tetracyclic Lactams as HIV-1 Integrase Strand Transfer Inhibitors,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.038.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
The Design of 8-Hydroxyquinoline Tetracyclic Lactams as HIV-1 Integrase Strand
Transfer Inhibitors
Emile J. Velthuisen*, Brian A. Johns, David P. Temelkoff, Kevin W. Brown, Susan C.
Danehower
GlaxoSmithKline Research & Development, Infectious Diseases Therapeutic Area Unit, Five
Moore Drive, Research Triangle Park, NC 27709, United States
*Corresponding author. E-mail address: emile.j.velthuisen@gsk.com
Abstract— A novel series of HIV-1 integrase strand transfer inhibitors were designed using the
venerable two-metal binding pharmacophore model and incorporating structural elements from
two different literature scaffolds. This manuscript describes a number of 8-hydroxyquinoline
tetracyclic lactams with exceptional antiviral activity against HIV-1 and little loss of potency
against the IN signature resistance mutations Q148K and G140S/Q148H.
Keywords: HIV, AIDS, integrase, integrase inhibitor, strand transfer inhibitor, quinoline
Abbreviations: ADME, absorption, distribution, metabolism, excretion; AIDS, acquired
immunodeficiency syndrome; Cl, clearance; CLND, chemi-luminescent nitrogen detection; CCD,
catalytic core domain; CTD, C-terminal domain; DIPEA, diisopropyl ethyl amine; DMPK, drug
metabolism and pharmacokinetics; DNAUC, dose-normalized area under the curve; dsDNA,
double-stranded deoxyribonucleic acid; EVG, elvitegravir; F, oral bioavailability; HAART, highly
active antiretroviral therapy; HIV, human immunodeficiency virus; HQT, hydroxyquinoline
tetracyclic; IN, Integrase; IN-ST, Integrase strand transfer; NTD, N-terminal domain or
naphthyridinone; RAL, raltegravir; Teoc, 2-(trimethylsilyl)ethyl carbamate; TMS, trimethylsilyl.
1. Introduction
HIV-1 integrase (IN) is one of the three essential enzymes required for viral replication and has
been the subject of intense research for two decades.[1,2,3,4,5] The enzyme is 32 kDa in size,
and consists of a 288 amino acid sequence that can be divided into three domains: N-terminal
(NTD), C-terminal (CTD), and the catalytic core (CCD). The catalytic core possesses a triad of
carboxylate residues that coordinate a pair of Mg2+ ions and is ultimately responsible for
catalyzing two biochemical steps required for incorporation of reverse transcribed viral DNA into
host chromatin.[6,7] In the first reaction, IN removes the two terminal nucleotides from the
respective 3’-ends of viral DNA. The enzyme then facilitates nicking of host chromosomal DNA
by the recessed 3’-hydroxyl of the viral DNA ends, resulting in strand transfer and effective
integration of the viral dsDNA.[8] Although there have been efforts to find inhibitors of both
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biochemical process, strand transfer inhibitors have proven to be much more amenable to
inhibition that is pharmacologically relevant.[9,10,11,12,13,14]
In general, strand transfer inhibitors contain three chelating heteroatoms in a rigidified co-planar
orientation linked to a flexible hydrophobic tail.[15,16,17] These molecules act as a bioisostere
of the substrate phosphodiester transition state by chelating two magnesium ions and
preventing progress of the strand transfer of viral DNA into the host’s chromatin. This generic
pharmacophore model [18] has proven highly successful in designing strand transfer inhibitors
and to date there have been three agents approved by the FDA for use in patients infected by
HIV-1.[2
]
In 2007, raltegravir (RAL) was approved as the first-in-class integrase strand transfer inhibitor
(Figure 1).[19] Although it requires a relatively high dose of 400 mg twice daily, it was well
tolerated, and provided a very rapid and exceptional viral load drop in patients.[20] However, a
noteworthy deficiency of this therapeutic was formation of three primary IN signature mutation
pathways: Y143, Q148, and N155.[21] These mutations can include additional mutations to
recover viral fitness (e.g. Q148H/G140S) and lead to decreased susceptibility and eventual
virological failure.[22] In 2012, a second strand transfer inhibitor was approved – elvitegravir
(EVG).[23] This agent was differentiated from RAL with the option of once daily dosing with the
co-administration of a CYP3A4 inhibitor such as ritonavir or cobicistat.[24] However, EVG was
also susceptible to formation of IN mutations albeit through a slightly different resistance
pathway and demonstrates significant cross-resistance with RAL.[25]
Figure 1
(insert figure 1)
With the advent of two marketed strand transfer inhibitors there was little justification for
introducing a third agent unless it was clearly differentiated in terms of the dosing regimen (i.e.
low dose, no PK booster) and/or resistance profile to existing IN resistance mutations.
Fortunately, GSK had several such molecules under clinical investigation with the tricyclic
carbamoylpyridone class of inhibitors.[18,26,27,28] These molecules represented a true second
generation profile in that they were not cross-resistance to RAL or EVG and the preclinical
pharmacokinetics were suggestive of unboosted once daily pharmacokinetics. The realization
that optimization of integrase inhibitors designs was feasible as evidenced by the
carbamoylpyridone scaffold coupled with early discovery uncertainties inherent to any drug
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discovery program led to an interest in identifying additional scaffolds with such attributes to
serve as backup to the clinical assets.[29]
2. Results and Discussion
2.1 HQT Scaffold Design
The carbamoylpyridone class of inhibitor is believed, in part, to have improved potency against
wild-type and key resistance mutations due to the additional rings built into the metal chelating
architecture.[28,30] Accordingly, we sought to combine this type of strategy with our earlier work
on naphthyridinone (NTD) inhibitors.[31] In combining the additional ring system “C” of the
carbamoylpyridone (CAB, 3)[26,27,28] with the 3-benzylpyridine portion of the naphthyridinone
4,[32,33,34] we would create a hydroxyquinoline tetracyclic scaffolding (HQT, 5) that has the
additional structural elements believed necessary for a superior resistance profile (Scheme 1).
Scheme 1
(insert Scheme 1)
2.2 HQT Chemistry
Our initial attempt to construct the hydroxyquinoline tetracyclic ring system 16 was through an
anionic Michael-Dieckmann condensation of pyridine 9 and cyclic hydrazide 14 (Scheme 2).
Synthesis of pyridine 9 started with Negishi coupling of (3-chloro-2-fluorobenzyl)zinc bromide
and commercially available methyl 5-bromo-2-chloronicotinate (6). The methyl ester was
reduced with sodium borohydride and the resulting 2-chloropyridine 8 carbonylated to afford
lactone 9. Synthesis of the coupling partner 14 commenced with alkylation of known tert-
butoxycarbonyl pyrazolidine 10[35] with allyl bromide. Following removal of the tert-
butoxycarbonyl protecting group under acidic conditions the pyrazolidine 12 was acylated with
acryloyl chloride and the olefins metathesized to provide cyclic hydrazide 14. The key Michael-
Dieckmann condensation was accomplished in a two step sequence. Lactone 9 was first
deprotonated with LiHMDS and treated with Michael acceptor 14. The intermediate alcohol 15
was then activated using methanesulfonyl chloride and subsequently eliminated to afford the
desired hydroxyquinoline tetracyclic ring system 16 in 24% yield. Although the key step provided
a low yield of desired product, optimization was delayed until the antiviral activity of HQT 16 was
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confirmed and could validate the additional structural complexity translating into an improved
profile.
Scheme 2
(insert Scheme 2)
Reagents and Conditions: (a) Pd(PPh3)4, (3-chloro-2-fluorobenzyl)zinc bromide, THF (72%); (b) NaBH4, THF (69%);
(c) Pd(OAc)2, Et3N, DPPP, DMF (22%); (d) Allyl bromide, K2CO3; (e) TFA; (f) acryloyl chloride (41% over 3-steps); (g)
Grubbs 2 (85%); (h) LiHMDS, THF; (i) MsCl, Et3N, CH2Cl2 (24% over 2-steps).
Fortunately, the initial HQT analog 16 was potent in a pseudotyped antiviral assay (pHIVIC50 = 16
nM) with an 9-fold shift in the presence of 40% human serum albumin (pHIVPAIC50 = 136 nM). In
addition, there was no loss of potency against theQ148K virus and only a 2.3-fold loss in
potency against G140S/Q148H. It is noteworthy that both raltegravir (1) and elvitegravir (2)
demonstrated a much greater loss of potency against these two integrase resistance mutations
(Figure 1). These results provided the necessary justification to reinvestigate the key Michael-
Dieckmann condensation and to further explore the SAR of this series.
Several attempts were made to optimize the Michael-Dieckmann cyclization, varying the
solvent, temperature, and base which had little impact on the outcome of the reaction. Instead,
we sought to fundamentally redesign the lactone coupling partner in order to decrease the pKa
of the benzylic position.[36,37] Synthesis of such a substrate commenced with activation of
benzylic alcohol 8 with methanesulfonyl chloride and subsequent displacement with sodium
cyanide (Scheme 3). The 2-chloropyridine 17 was then carbonylated in the presence of
methanol to afford methyl ester 18. In contrast to the previous Michael-Dieckmann cyclization of
lactone 9, deprotonation of pyridine 18 with LiHMDS and subsequent treatment with hydrazide
14 afforded the desired dihydroquinoline product 19 very efficiently. However, much to our
disappointment, the oxidation of the dihydroquinoline 19 to the corresponding HQT did not occur
under several conditions.
Scheme 3
(insert Scheme 3)
Reagents and Conditions: (a) NaCN (97%); (b) Pd(OAc)2, Et3N, DPPP, DMF (72%); (c) LiHMDS, THF (60%); (d)
DDQ, DCM or Pd/C, air, MeOH.
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As an alternative to the late stage oxidation of dihydroquinoline 19 we sought to increase the
oxidation state of the hydrazide coupling partner 14 by creating an ynoate. Synthesis of the new
Michael acceptor began with alkylation of tert-butoxycarbonyl pyrazolidine 10 with propynyl
methanesulfonate (Scheme 4). The alkyne 21 was deprotonated with n-butyl lithium and
quenched with methyl chloroformate to afford ynoate 22. Upon treatment of pyridine 18 with
LiHMDS and subsequent addition of ynoate 22, the desired Michael-Dieckmann cyclization
occurred. Following removal of the tert-butoxycarbonyl protecting group under acidic conditions,
the lactam formed spontaneously to provide HQT 20 in 89% yield.
Scheme 4
(insert Scheme 4)
Reagents and Conditions: (a) prop-2-yn-1-yl methanesulfonate, Cs2CO3, DMF (67%); (b) i) n-BuLi, ii) methyl
chloroformate (66%); (c) i) LiHMDS, ii) TFA (89%).
In a pseudotyped antiviral assay HQT 20 was very potent (pHIVIC50 = 2.2 nM) but demonstrated a
significant 188-fold shift in the presence of 40% human serum albumin (pHIVPAIC50 = 415 nM).
There was a 1.3-fold loss in potency against the Q148K virus and only a 8-fold loss against the
double mutant G140S/Q148H. With an improved HQT synthesis and an additional functional
handle to exploit, a variety of analogs were pursued using this strategy.
2.3 HQT Structure Activity Relationship
To follow up on the initial hit we were interested in constructing either proline or pipecolinic acid
derived hydroxyquinoline tetracyclic ring systems in order to investigate the effect of ring size
and chirality on antiviral potency, protein shift, and in vivo DMPK. Accordingly, a variety of chiral
ynoates were prepared from known aldehydes[38] as illustrated in Scheme 5. The sequence
started with homologation of a chiral aldehyde with Ohira-Bestmann reagent[39] followed by
formation of the propiolate from deprotonation of the terminal alkyne and quenching with methyl
chloroformate. In addition to the tert-butoxy carbonyl protected derivatives, it was also possible
to use a non-acid labile protecting group such as a trimethylsilylethyl carbamate (Teoc) by
performing a late stage protecting group swap.
Scheme 5
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(insert scheme 5)
Reagents and Conditions: (a) K2CO3, dimethyl (diazomethyl)phosphonate, K2CO3, MeOH; (b) i) n-BuLi, ii) methyl
chloroformate; (c) TFA, DCM; (d) Teoc-Cl, pyridine
The chiral ynoates described in Scheme 5 were used in Michael-Dieckmann cyclizations with
pyridine 18 to construct a variety of HQT analogs (Scheme 6). In general, the LiHMDS
promoted cyclization proceeded with good conversion to the desired substituted quinoline.
However, following deprotection of the tert-butoxy carbonyl protected proline or piperidine, the
nitrogen could either cyclize with the C7-ester to afford the desired lactam 40 or react with the
C5-nitrile to provide an undesired amidine ring system 41. Fortunately when attempting to
create a 6-membered ring, lactam 40 would form preferentially. However, when constructing a
5-membered ring, the acidic conditions required for removal of the tert-butoxy carbonyl
protecting group promoted formation of amidine 41. In order to circumvent this undesired
reaction pathway a non-acid labile nitrogen protecting group such as trimethylsilylethyl
carbamate (Teoc) was employed. Overall, the combined yields for the Michael-Dieckmann
cyclization and subsequent deprotection/lactam formation ranged from 12-78% (Table 1).
Scheme 6
(insert Scheme 6)
Reagents and Conditions: (a) LiHMDS, THF; (b) TFA, DCM; (c) NaOH, MeOH; (d) TBAF, THF or CsF, THF/DMF.
Table 1
(insert table 1)
1 Reported yield is over 3-steps for R = Boc (a, b, c) and 2-steps for R = Teoc (a, d).
2 Assay run in the presence of 40% human serum albumin.
In general, all the HQT analogs were potent in the pseudotyped antiviral assay (pHIVIC50 = 5 – 18
nM) although many demonstrated a significant shift in potency when the assay was run in the
presence of 40% human serum albumin (Table 1). Against the single mutant Q148K virus and
the double mutant G140S/Q148H virus, 6-membered lactams (42 and 43) exhibited a smaller
fold change in potency relative to the 5-membered lactams (44 – 47). Stereochemistry appeared
to be less of a predictor of antiviral potency against both wild type and mutant viruses although
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there was a significant difference in protein shift for the matched stereoisomers of both the 5-
and 6-memererd lactams (42/43, 44/45, and 46/47).
In order to further define the HQT structure activity relationship a variety of conditions were
explored to hydrolyze the C5-nitrile to the corresponding carboxylic acid. Unfortunately majority
of the standard conditions afforded decomposition products with the exception of an acidic
hydrolysis with acetic acid and sulfuric acid that provided a primary amide (Figure 2).
Interestingly these analogs proved to be exceptionally potent antiviral agents.
Figure 21
(insert figure 2 scheme)
(insert figure 2)
1 Reagents and Conditions: (a) AcOH/H2SO4, H2O.
2 Assay run in the presence of 40% human serum albumin.
The HQT C5-primary amides were very potent in the pseudotyped antiviral assay (pHIVIC50 = 0.6
– 8.0 nM) with reasonably small serum shifts that ranged from 2x to 5x (Figure 2). Most
encouraging was their potency against the IN resistance mutations. All four analogs were nearly
equipotent to, or more potent against the Q148K mutant. Against the double mutant
G140S/Q148H there was a slightly larger fold shift but this still translated into single digit
nanomolar potency which is a requirement for a true second generation integrase strand
transfer inhibitor.
2.4 HQT in vivo DMPK
In addition to an excellent virological profile it was also necessary to evaluate the in vivo
metabolic stability of the series (Table 2). In order to do so, HQT 52 was dosed both
intravenously and orally in Sprague-Dawley rats and Harlan beagle dogs. The compound had a
good t1/2, low i.v. clearance, but poor oral bioavailability in rats (7.2 h, 1.4 mL min-1 kg-1, 2 %)
and dogs (11.8 h, 2.8 mL min-1 kg-1, 14 %). Despite moderate permeability (PAPP = 60 nm/s),
poor intrinsic aqueous solubility (cLND = 25 µM) is likely limiting oral absorption for this
compound.
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Table 2
(insert table 2)
3.0 Conclusion
In conclusion, this manuscript describes the design and synthesis of novel IN strand transfer
inhibitors using the venerable two-metal binding pharmacophore model and incorporating
structural elements from two different literature scaffolds. Several examples of the 8-
hydroxyquinoline tetracyclic lactams have a virological profile consistent with other second
generation integrase stand transfer inhibitors and represent an excellent starting point for further
optimization wherein improvements in the pharmacokinetic profiles are needed.
4.0 Experimental Section
4.1 Chemistry
All commercially available reagents were used without further purification. Column
chromatography was carried out on silica gel (70-230 mesh). TLC was conducted on silica gel
250 micron, F254 plates. 1H NMR spectra were recorded at 300 or 400 MHz. Melting points are
uncorrected. Purities of test compounds were established by analytical HPLC (C-18 column,
5.0 micron, 0
→100% CH3CN (or MeOH)/water with 0.05-0.1% HCOOH (or TFA) and UV
detection with or without mass spectrometer detection. All test compounds showed >95% purity
(AUCs by UV detection).
4.1.1 Methyl 2-chloro-5-(3-chloro-2-fluorobenzyl)nicotinate (7). A suspension of zinc (5.0 g) in 4
N HCl (10 mL) was stirred at 30 ºC for 10 min and then filtered. The residue was crushed to
yield fine particles and then washed with water (5x) and acetone (2x). The activated zinc was
then dried in vacuo for 24 h.
A solution of 3-chloro-2-flurobenzyl bromide (1.12 g, 5 mmol) in THF (3 mL) was added
dropwise to suspension of activated zinc (0.98 g, 15 mmol), 1,2-dibromoethane (0.034 mL, 0.4
mmol), trimethylchlorosilane (0.038 mL, 0.3 mmol) and THF (1.5 mL) at 23 ºC. The reaction was
heated to 45 ºC and maintained at this temperature for 1.5 h. TLC indicated complete
consumption of 3-chloro-2-flurobenzyl bromide (sm: Rf = 0.4; 30% EtOAc-hexanes).
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A solution of (3-chloro-2-fluorobenzyl)zinc bromide in THF (7.98 ml, 7.98 mmol, 1.0 M) was
added dropwise over a 10 min period to a stirring mixture of 5-bromo-2-chloronicotinate (2.0 g,
7.98 mmol), tetrakis(triphenylphosphine)palladium (0.923 g, 0.798 mmol), and THF (26.6 ml) at
65 °C. The resulting mixture was stirred for 1 h an d then poured into 10% aq. NH4Cl and the
layers were separated. The aqueous phase was extracted with EtOAc and the combined
organics layers were dried (MgSO4), filtered and concentrated. The residue was purified by
silica gel chromatography (10-100% EtOAc-hexanes gradient) to yield the title compound as a
1
yellow oil. (1.8 g, 71.8 %): H NMR (400 MHz, CDCl3)
δ ppm 8.41 (d, J=2.38 Hz, 1 H), 7.97 (d,
J=2.38 Hz, 1 H), 7.37–7.29 (m, 1 H), 7.08–7.04 (m, 3 H), 4.04 (s, 2 H), 3.97–3.92 (m, 3 H); ES+
MS: 315 (M + 1).
4.1.2 {2-Chloro-5-[(3-chloro-2-fluorophenyl)methyl]-3-pyridinyl}methanol (8). To a –78 °C
solution of methyl 2-chloro-5-(3-chloro-2-fluorobenzyl)nicotinate (1.5 g, 4.77 mmol) in THF (24
mL) was added DiBAL-H (10.50 ml, 10.50 mmol, 1.0 M in CH2Cl2). After 30 min, the reaction
mixture was warmed to –30 °C and maintained at this temperature for 2 h. The reaction mixture
was poured into 1 N HCl and the layers were separated. The aqueous phase was extracted with
EtOAc and the combined organics layers were dried (MgSO4), filtered and concentrated. The
residue was purified by silica gel chromatography (10-60% EtOAc-hexanes gradient) to afford
the title compound as a white solid (1.1 g, 69% yield): 1H NMR (400 MHz, CDCl3)
δ ppm 8.22 (d,
J=2.20 Hz, 1 H), 7.70 (d, J=1.65 Hz, 1 H), 7.31 (m, 1 H), 7.10–7.03 (m, 2 H), 4.76 (d, J=5.86 Hz,
2 H), 4.02 (s, 2 H); ES+ MS: 287 (M + 1).
4.1.3 3-(3-chloro-2-fluorobenzyl)furo[3,4-b]pyridin-7(5H)-one (9). A solution of {2-Chloro-5-[(3-
chloro-2-fluorophenyl)methyl]-3-pyridinyl}methanol (0.6 g, 2.1 mmol), and triethylamine (1.45
mL, 10.5 mmol) in DMF (15 mL) was degassed with N2 for 10 min. The mixture was then
treated with palladium acetate (0.05 g, 0.21 mmol) and DPPP (0.09 g, 0.21 mmol) and
subsequently purged/backfilled with N2 3x and CO 3x. The reaction mixture was then placed
under an atmosphere of CO (50 psi) and heated to 90 °C. After 18 h, the reaction mixture was
cooled to ambient temperature and filtered through a pad of celite. The filtrate was partitioned
between EtOAc/water and the organics washed with water, brine, dried (MgSO4), filtered, and
concentrated in vacuo. The residue was purified by silica gel chromatography (10-50%
EtOAc/hexanes) to afford the title compound (0.13 g, 22% yield) as a yellow solid: 1H NMR (400
MHz, CDCl3)
δ ppm 8.82 (s, 1 H), 7.68 (s, 1 H), 7.36 (td, J=7.28, 2.11 Hz, 1 H), 7.16–7.04 (m, 2
H), 5.34 (s, 2 H), 4.18 (s, 2 H); ES+ MS: 278 (M + 1).
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4.1.4 1-Acryloyl-2-(2-propen-1-yl)pyrazolidine (13). A solution of 1,1-dimethylethyl 1-
pyrazolidinecarboxylate[40] (17.7 g, 103 mmol) in toluene (206 mL) was treated with potassium
carbonate (28.4 g, 206 mmol) and allyl bromide (13.34 ml, 154 mmol) and heated. After 4 h, the
reaction mixture was poured into water and the layers separated. The aqueous phase was
extracted with EtOAc and the combined organics layers were washed with brine, dried (MgSO4),
filtered and concentrated. The crude residue was dissolved in dichloromethane and cooled to 0
°C. Trifluoroacetic acid (103 ml, 1336 mmol) was ad ded and the reaction mixture was then
warmed to 23 °C. After 16 h, the reaction mixture w as concentrated in vacuo and azeotroped
with toluene (2x). The crude residue was dissolved in THF (500 mL) and triethyl amine (43.0
mL, 308 mmol) was added. The reaction mixture was then cooled to –78 °C and treated
dropwise with acryloyl chloride (9.2 mL, 113 mmol). The cooling bath was allowed to expire and
the reaction mixture was stirred for an additional 16 h. The reaction mixture was poured into sat.
aq. NH4Cl and the layers were separated. The aqueous phase was extracted with EtOAc and
the combined organics layers were washed with brine, dried (MgSO4), filtered and concentrated.
The residue was purified by Kugelrohr distillation (0.2 torr, 175 °C oven temperature) to afford
the title compound (7.0 g, 41%): 1H NMR (400 MHz, CDCl3)
δ ppm 6.98 (dd, J=17.3, 10.4 Hz, 1
H), 6.36 (dd, J=17.3, 2.1 Hz, 1 H), 5.97–5.79 (m, J=17.0, 10.3, 6.5, 6.5 Hz, 1 H), 5.64 (dd,
J=10.4, 2.1 Hz, 1 H), 5.26–5.14 (m, 2 H), 4.14–3.32 (m, 2 H), 3.25 (d, J=6.5 Hz, 2 H), 2.99 (dd,
J=0.8, 0.4 Hz, 2 H), 2.27–2.06 (m, 2 H).
4.1.5 2,3-dihydro-1H-pyrazolo[1,2-a]pyridazin-5(8H)-one (14). A solution 1-Acryloyl-2-(2-propen-
1-yl)pyrazolidine (2.5 g, 15.04 mmol) in dichloromethane (500 ml) was degassed with N2 for 15
min and shielded from ambient light. Benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-
imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium
(Grubbs
Catalyst,
2nd
Generation)(1.50 g, 0.11 mmol) was added and the reaction mixture was heated to reflux. After
15 h, ethyl vinyl ether (7.23 mL, 75 mmol) was added and stirring was continued for 15 min. The
reaction mixture was cooled to 23 °C and concentrat ed in vacuo. The crude residue was purified
by silica gel chromatography (0-100% ethyl acetate-dichloromethane) to afford the title
compound (1.5 g, 85%) as a black oil: 1H NMR (400 MHz, CDCl3)
δ ppm 6.56 (dt, J=9.7, 4.2 Hz,
1 H), 6.06–6.00 (m, 1 H), 3.77–3.69 (m, 2 H), 3.51 (dd, J=3.9, 1.4 Hz, 2 H), 3.05 (t, J=6.5 Hz, 2
H), 2.22–2.12 (m, 2 H).
4.1.6 3-[(3-chloro-2-fluorophenyl)methyl]-13-hydroxy-9,10-dihydro-8H-pyrazolo[1,2-b]pyrido[2,3-
g]phthalazin-12(6H)-one (16). A –78 °C solution of 3-[(3-chloro-2-fluorophenyl)me thyl]furo[3,4-
b]pyridin-7(5H)-one (0.2 g, 0.720 mmol) in dichloromethane (2.8 ml) was treated with lithium
10

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bis(trimethylsilylamide) (0.72 mL, 0.72 mmol, 1.0 M THF) dropwise. After 12 min, a solution of
2,3-dihydro-1H-pyrazolo[1,2-a]pyridazin-5(8H)-one (0.104 g, 0.756 mmol) in THF (2.8 mL) was
added dropwise over a 5 min period. After an additional 30 min, the reaction mixture was
treated with lithium bis(trimethylsilylamide) (0.72 mL, 0.72 mmol, 1.0 M THF) and stirring
continued for 20 min. The reaction mixture was poured into sat. aq. NH4Cl and the layers
separated. The aqueous phase was extracted with ethyl acetate and the combined organic
layers were dried (MgSO4), filtered and concentrated. The crude residue was dissolved in THF
(3.0 mL), cooled to 0 °C and treated sequentially w ith triethyl amine (0.60 mL, 4.32 mmol) and
methane sulfonyl chloride (0.23 mL, 2.88 mmol). After 30 min, the reaction mixture was poured
into sat. aq. NH4Cl and the layers separated. The aqueous phase was extracted with ethyl
acetate, dried (MgSO4), filtered and concentrated. The residue was purified by silica gel
chromatography (0-10% methanol-dichloromethane) to afford the title compound (63 mg, 24%)
as a brown solid: 1H NMR (400 MHz, CDCl3)
δ ppm 8.85 (d, J=1.83 Hz, 1 H), 7.78 (s, 1 H), 7.32
(td, J=7.32, 1.65 Hz, 1 H), 7.17–6.98 (m, 2 H), 6.94 (s, 1 H), 4.20 (s, 2 H), 4.13 (s, 3 H), 3.95–
3.87 (m, 2 H), 3.22 (t, J=6.41 Hz, 2 H), 2.35–2.22 (m, 2 H); ES+ MS: 398 (M + 1).
4.1.7 {2-Chloro-5-[(3-chloro-2-fluorophenyl)methyl]-3-pyridinyl}acetonitrile (17). A 0 °C solution
of {2-chloro-5-[(3-chloro-2-fluorophenyl)methyl]-3-pyridinyl}methanol (1.6 g, 5.59 mmol) and
Et3N (3.12 ml, 22.37 mmol) in dichloromethane (50 mL) was treated with methanesulfonyl
chloride (0.865 ml, 11.18 mmol). After 20 min the reaction mixture was poured into sat. sat.
NH4Cl and the layers were separated. The aqueous phase was extracted with dichloromethane
and the combined organics layers were dried (MgSO4), filtered and concentrated. The crude
residue was dissolved in N,N–dimethylformamide (50 mL) and sodium cyanide (0.301 g, 6.15
mmol) was added in one portion. After stirring overnight, the reaction mixture was poured into a
water/diethyl ether mixture and the layers were separated. The aqueous phase was extracted
with diethyl ether and the combined organics layers were washed with water, brine, dried
(MgSO4), filtered and concentrated. The crude residue was used without further purification (1.6
1
g, 97%): H NMR (400 MHz, CDCl3)
δ ppm 8.29 (d, J=1.83 Hz, 1 H), 7.70 (d, J=1.46 Hz, 1 H),
7.33 (m, 1 H), 7.11–7.03 (m, 2 H), 4.04 (s, 2 H), 3.83 (s, 3 H); ES+ MS: 296 (M + 1).
4.1.8 Methyl 5-[(3-chloro-2-fluorophenyl)methyl]-3-(cyanomethyl)-2-pyridinecarboxylate (18). A
pressure vessel was charged with methyl 5-[(3-chloro-2-fluorophenyl)methyl]-3-(cyanomethyl)-
2-pyridinecarboxylate (1.60 g, 5.42 mmol), triethylamine (3.76 ml, 27.1 mmol), N,N–
11

ACCEPTED MANUSCRIPT
dimethylformamide (27 ml), and methanol (27 ml). After degassing with N2 for 10 min, Pd(OAc)2
(0.122 g, 0.542 mmol) and DPPP (0.224 g, 0.542 mmol) were added. The reaction mixture was
then purged/backfilled with N2 (3x), followed by CO (3x) and finally pressurized with CO at 50
psi and heated to 90 °C. After stirring 18 h, the r eaction mixture was cooled to 23 °C and filtered
through a pad of celite and rinsed with diethyl ether. The filtrate was poured into water and the
layers were separated. The aqueous phase was extracted with diethyl ether and the combined
organics layers were dried (MgSO4), filtered and concentrated. The residue was purified by
silica gel chromatography (10-100% EtOAc-hexanes gradient) to afford the title compound as
an off-white solid (1.25 g, 72%): 1H NMR (400 MHz, CDCl3)
δ ppm 8.62 (s, 1 H), 7.79 (s, 1 H),
7.34 (td, J=6.96, 2.75 Hz, 1 H), 7.12–7.04 (m, 2 H), 4.27 (s, 2 H), 4.13 (s, 2 H), 4.01 (s, 3 H);
ES+ MS: 319 (M+1).
4.1.9 3-(3-chloro-2-fluorobenzyl)-12,13-dioxo-5a,6,8,9,10,12,12a,13-octahydro-5H-pyrazolo[1,2-
b]pyrido[2,3-g]phthalazine-5-carbonitrile (19). A -78 °C solution of Methyl 5-[(3-chloro-2-
fluorophenyl)methyl]-3-(cyanomethyl)-2-pyridinecarboxylate (30 mg, 0.094 mmol) in THF (1 mL)
was treated with LiHMDS (104 µL, 0.104 mmol). After 20 min, the reaction mixture was treated
with a solution of 2,3-dihydro-1H-pyrazolo[1,2-a]pyridazin-5(8H)-one (15.6 mg, 0.113 mmol) in
THF (0.2 mL). After 20 min the reaction mixture was warmed to ambient temperature. After an
additional 30 min, the reaction mixture was quenched with 10% NH4Cl and diluted with EtOAc.
The layers were partitioned and the organic phase was washed with brine, dried (MgSO4),
filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (0-
10% MeOH/DCM) to afford the title compound (24 mg, 60% yield).
4.1.10 1,1-dimethylethyl 2-(2-propyn-1-yl)-1-pyrazolidinecarboxylate (21). A 0 °C solution of
propargyl alcohol (2.90 ml, 49.8 mmol) in dichloromethane (100 mL) was treated with
triethylamine (13.89 ml, 100 mmol) and methanesulfonyl chloride (5.81 ml, 74.7 mmol). After 20
min, the reaction mixture was poured into sat. aq. NH4Cl and the layers separated. The aqueous
phase was extracted with dichloromethane and the combined organic extracts were dried
(MgSO4), filtered and concentrated in vacuo.
A solution of 1,1-dimethylethyl 1-pyrazolidinecarboxylate40 (4.29 g, 24.91 mmol) and DMF (80
mL) was treated with cesium carbonate (12.2 g, 37.4 mmol) and a solution of 2-propyn-1-yl
methanesulfonate (6.67 g, 49.8 mmol) and heated to 50 °C. After 16 h, the reaction mixture was
poured into water and diethyl ether and the layers separated. The aqueous phase was extracted
with diethyl ether and the combined organic extracts were washed with water, brine, dried
12

ACCEPTED MANUSCRIPT
(MgSO4), filtered and concentrated. The residue was purified by silica gel chromatography (0-
1
50% ethyl acetate-hexanes) to afford the title compound (3.5 g, 67%) as an orange solid: H
NMR (400 MHz, CDCl3)
δ ppm 3.64–3.53 (m, 4 H), 3.23 (t, J=6.87 Hz, 2 H), 2.24–2.13 (m, 2 H),
2.05 (s, 1 H), 1.50 (s, 9 H).
4.1.11
3-[(3-chloro-2-fluorophenyl)methyl]-13-hydroxy-12-oxo-6,9,10,12-tetrahydro-8H-
pyrazolo[1,2-b]pyrido[2,3-g]phthalazine-5-carbonitrile (20). A –78 °C solution of methyl 5-[(3-
chloro-2-fluorophenyl)methyl]-3-(cyanomethyl)-2-pyridinecarboxylate (300 mg, 0.941 mmol) in
THF (1.3 mL) was treated with lithium bis(trimethylsilyl amide) (1035 µl, 1.035 mmol) dropwise.
After stirring for 20 min, a solution of 1-dimethylethyl 2-[4-(methyloxy)-4-oxo-2-butyn-1-yl]-1-
pyrazolidinecarboxylate (303 mg, 1.129 mmol) in THF (0.9 mL) was added and stirred at this
temperature for 20 min. The reaction mixture was then warmed to 0 °C for 30 min and poured
into 0.1 M HCl and the layers separated. The aqueous phase was extracted with ethyl acetate
and the combined organic layers were dried (MgSO4), filtered and concentrated. The crude
residue was dissolved in 1,2-dichloroethane (1.3 mL) and treated with trifluoroacetic acid. After
stirring at 23 °C for 1 h, the reaction mixture was heated to 90 °C. After 20 min, the reaction
mixture was cooled to ambient temperature and concentrated in vacuo to afford the title
compound (355 mg, 89%): 1H NMR (400 MHz, CDCl3)
δ ppm 8.93 (s, 1 H), 8.19 (s, 1 H), 7.34 (t,
J=6.87 Hz, 1 H), 7.17–7.03 (m, 2 H), 4.39 (s, 2 H), 4.27 (s, 2 H), 3.91 (t, J=7.42 Hz, 2 H), 3.29
(t, J=6.32 Hz, 2 H), 2.43–2.25 (m, 2 H); ES+ MS: 423 (M+1).
4.1.12 1,1-dimethylethyl 2-[4-(methyloxy)-4-oxo-2-butyn-1-yl]-1-pyrazolidinecarboxylate (22). A
–78 °C solution of 1,1-dimethylethyl 1-pyrazolidine carboxylate (1.61 g, 7.66 mmol) in THF (30.6
mL) was added n-BuLi (5.74 ml, 9.19 mmol, 1.6 M hexanes) dropwise. After stirring for 30 min,
the reaction mixture was cannulated into a –78 °C s olution of methyl chloroformate (0.652 ml,
8.42 mmol) in THF (30 mL). The reaction mixture was warmed to 23 °C and after 16 h, poured
into sat. aq. NH4Cl and the layers were separated. The aqueous phase was extracted with ethyl
acetate and the combined organic extracts were dried (MgSO4), filtered and concentrated. The
residue was purified by silica gel chromatography (0-30% ethyl acetate-hexanes) to afford the
1
title compound (1.35 g, 66% yield): H NMR (400 MHz, CDCl3)
δ ppm 3.78 (s, 3 H), 3.76 (s, 2
H), 3.60 (t, J=7.23 Hz, 2 H), 3.22 (t, J=6.96 Hz, 2 H), 2.20 (quin, J=7.10 Hz, 2 H), 1.50 (s, 9 H).
13

ACCEPTED MANUSCRIPT
4.1.13 1,1-dimethylethyl (2S)-2-[4-(methyloxy)-4-oxo-2-butyn-1-yl]-1-pyrrolidinecarboxylate (25).
A suspension of potassium carbonate (3.20 g, 23.16 mmol) in methanol (20 mL) was cooled to
0 °C and diethyl 1-diazo-2-oxopropylphosphonate (2. 83 mL, 17.37 mmol) was added. 1,1-
dimethylethyl (S)-tert-butyl 2-(2-oxoethyl)pyrrolidine-1-carboxylate[41] (2.34 g, 10.97 mmol) was
added, the ice bath was removed and the mixture was stirred for 2 hours after which time the
yellow solution took on a green hue. Saturated NH4Cl was added and the mixture was
extracted with ether. The ethereal extracts were washed with brine, dried over Na2SO4, filtered
and concentrated to give the crude, 1,1-dimethylethyl (2S)-2-[4-(methyloxy)-4-oxo-2-butyn-1-yl]-
1-pyrrolidinecarboxylate (2.61 g, quantitative), as pale yellow oil. TLC (ethyl acetate/hexanes
1:4, Rf = 0.53).
To a -78 °C solution of 1,1-dimethylethy l (2S)-2-(2-propyn-1-yl)-1-
pyrrolidinecarboxylate (2.611 g, 12.48 mmol) in THF (25 mL) was slowly added n-butyllithium
(8.32 mL, 13.32 mmol, 1.6 M in hexanes) to give a dark mixture. After 30 minutes at -78 °C the
dark solution was added to an awaiting -78 °C solut ion of methyl chloroformate (0.987 mL,
12.74 mmol) in THF (10 mL) via cannula. After the addition the dark mixture was stirred for 5
minutes at -78 °C and then the bath was removed. A fter 5 minutes TLC was taken and showed
the reaction to be complete. Saturated NH4Cl was added and the mixture was diluted with ethyl
acetate. After extraction with ethyl acetate, the extracts were washed with brine, dried over
Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (0-
15-30% ethyl acetate,hexanes) to give 1,1-dimethylethyl (2S)-2-[4-(methyloxy)-4-oxo-2-butyn-1-
1
yl]-1-pyrrolidinecarboxylate (2.30 g, 78% over 3 steps) as a pale yellow oil. H NMR (400 MHz,
CDCl3)
δ ppm 4.03 – 3.88 (m, 1 H), 3.77 (s, 3 H), 3.48 – 3.32 (m, 2 H), 2.87 – 2.42 (m, 2 H),
2.16 – 1.77 (m, 4 H), 1.47 (s, 9 H); TLC (ethyl acetate/hexanes 15:85, Rf = 0.30).
4.1.14 1,1-dimethylethyl (2R)-2-[4-(methyloxy)-4-oxo-2-butyn-1-yl]-1-pyrrolidinecarboxylate (26).
A suspension of potassium carbonate (0.49 g, 3.56 mmol) in methanol (3 mL) was cooled to 0
°C and diethyl 1-diazo-2-oxopropylphosphonate (0.58 mL, 2.67 mmol) was added. (R)-tert-butyl
2-(2-oxoethyl)pyrrolidine-1-carboxylate[42] (0.38 g, 1.78 mmol) was added, the ice bath was
removed and the mixture was stirred for 2 hours after which time the yellow solution took on a
green hue. Saturated NH4Cl was added and the mixture was extracted with ether. The ethereal
extracts were washed with brine, dried over Na2SO4, filtered and concentrated to give the crude,
1,1-dimethylethyl (2R)-2-[4-(methyloxy)-4-oxo-2-butyn-1-yl]-1-pyrrolidinecarboxylate (0.25 g,
quantitative), as pale yellow oil. To a -78 °C solu tion of 1,1-dimethylethyl (2R)-2-(2-propyn-1-yl)-
1-pyrrolidinecarboxylate (0.25 g, 1.19 mmol) in THF (6 mL) was slowly added n-butyllithium
14

ACCEPTED MANUSCRIPT
(0.72 mL, 1.79 mmol, 2.5M solution in hexanes) to give a dark mixture. After 30 minutes at -78
°C the dark solution was added to an awaiting -78 ° C solution of methyl chloroformate (0.138
mL, 12.74 mmol) in THF (3 mL) via cannula. After the addition the dark mixture was stirred for 5
minutes at -78 °C and then the bath was removed. A fter 5 minutes TLC was taken and showed
the reaction to be complete. Saturated NH4Cl was added and the mixture was diluted with ethyl
acetate. After extraction with ethyl acetate, the extracts were washed with brine, dried over
Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (0-
15-30% ethyl acetate,hexanes) to give 1,1-dimethylethyl (2R)-2-[4-(methyloxy)-4-oxo-2-butyn-1-
1
yl]-1-pyrrolidinecarboxylate (0.1 g, 31% yield) as a pale yellow oil. H NMR (400 MHz, CDCl3)
δ
ppm 4.03 – 3.88 (m, 1 H), 3.77 (s, 3 H), 3.48 – 3.32 (m, 2 H), 2.87 – 2.42 (m, 2 H), 2.16 – 1.77
(m, 4 H), 1.47 (s, 9 H).
4.1.15 1,1-dimethylethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-pyrrolidinecarboxylate
(29). (S)-tert-butyl 2-formylpyrrolidine-1-carboxylate[43] (21 g, 0.11 mol) and diethyl 1-diazo-2-
oxopropylphosphonate (30.4 g, 0.158 mol) were dissolved in methanol (200 mL), cooled to 0 °C
and potassium carbonate (30.4 g, 0.158 mol) was added. The mixture was stirred at ice bath
temp for 30 minutes. After 18h, the mixture was quenched with saturated NH4Cl and extracted
with ether. The extracts were washed with brine, dried over MgSO4, filtered and concentrated to
a yellow oil (14 g) that was used in the next step without further purification. A solution of the
crude alkyne in THF (280 mL) was cooled to -78 °C a nd treated slowly with a n-butyllithium
(31.7 mL, 7.314 mmol, 2.5 M in hexanes). After 30 min, the reaction mixture was transferred via
cannula into a -78 °C solution of methyl chloroform ate (5.92 mL, 76 mmol) in THF (60 mL). After
30 min, the reaction mixture was allowed to warm to ambient temperature and quenched with
NH4Cl and extracted with ethyl acetate. The extracts were washed with brine, dried, filtered and
concentrated. The crude material was purified by silica gel chromatography (0-30% ethyl
acetate/hexanes) to give 1,1-dimethylethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-
pyrrolidinecarboxylate (7.7 g, 46% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3)
δ ppm
4.69 – 4.48 (m, 1 H), 3.78 (br. s., 3 H), 3.54 – 3.26 (m, 2 H), 2.24 – 1.86 (m, 4 H), 1.49 (s, 9 H).
4.1.16 1,1-dimethylethyl (2R)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-pyrrolidinecarboxylate
(30). (R)-tert-butyl 2-formylpyrrolidine-1-carboxylate[44] (1.22 g, 6.14 mmol) and diethyl 1-
diazo-2-oxopropylphosphonate (1.499 mL, 9.21 mmol) were dissolved in methanol (20 mL),
cooled to 0 °C and potassium carbonate (1.697 g, 12 .28 mmol) was added. The mixture was
15

ACCEPTED MANUSCRIPT
stirred at ice bath temp for 30 minutes. After 18h, the mixture was quenched with saturated
NH4Cl and extracted with ether. The extracts were washed with brine, dried over MgSO4, filtered
and concentrated to a yellow oil (1.24 g) that was used in the next step without further
purification. A solution of the crude alkyne in THF (12 mL) was cooled to -78 °C and treated
slowly with a n-butyllithium (1.6 M in hexanes) (4.57 mL, 7.31 mmol). The resultant was stirred
for 30 minutes at -78 °C and transferred by cannula to a -78 °C solution of methyl chloroformate
(0.54 mL, 7.0 mmol). After 30 min, the cooling bath was removed and the mixture was allowed
to warm to rt. The mixture was quenched with NH4Cl and extracted with ethyl acetate. The
extracts were washed with brine, dried, filtered and concentrated. The crude material was
purified by silica gel chromatography (0-30% ethyl acetate/hexanes) to give 1,1-dimethylethyl
(2R)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-pyrrolidinecarboxylate (0.780 g, 50% over two
1
steps) as a colorless oil. H NMR (400 MHz, CDCl3)
δ ppm 4.69 – 4.48 (m, 1 H), 3.78 (br. s., 3
H), 3.54 – 3.26 (m, 2 H), 2.24 – 1.86 (m, 4 H), 1.49 (s, 9 H).
4.1.17 2-(trimethylsilyl)ethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-pyrrolidinecarboxylate
(31).
A
solution of 1,1-dimethylethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-
pyrrolidinecarboxylate (7.7 g, 30.4 mmol) in dichloromethane (30 mL) was treated with
trifluoroacetic acid (9.75 mL) to give a tan colored solution. The mixture was stirred for 15
minutes and concentrated in vacuo, and azeotroped twice with toluene and then placed under
vacuum overnight to give methyl 3-[(2S)-2-pyrrolidinyl]-2-propynoate trifluoroacetate as a pale
yellow oil. The crude trifluoroacetate salt was dissolved in dichloromethane (162 mL) and then
cooled to 0 °C. Pyridine (12.3 mL, 152 mmol) was a dded followed by slow addition of 2-
(trimethylsilyl)ethyl chloridocarbonate (6.05 g, 33.5 mmol). The mixture was stirred at 0 °C for
20 minutes then room temperature for 20 minutes. Water was added and the mixture was
diluted with dichloromethane. The layers were separated and the organic layer was washed
with saturated NaHCO3 (x2) and brine (x2), dried over MgSO4, filtered and concentrated. The
crude material was purified by silica gel chromatography (0-15-50% EtOAc/hexanes gradient) to
give 2-(trimethylsilyl)ethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-pyrrolidinecarboxylate
1
(6.0 g, 67% yield) as a light yellow oil. H NMR (400 MHz, CDCl3)
δ ppm 4.73 – 4.55 (m, 1 H),
4.26 – 4.16 (m, 2 H), 3.76 (s, 3 H), 3.59 – 3.30 (m, 2 H), 2.22 – 1.90 (m, 4 H), 1.10 – 0.97 (m, 2
H), 0.05 (s, 9 H).
16

ACCEPTED MANUSCRIPT
4.1.18 2-(trimethylsilyl)ethyl (2R)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-pyrrolidinecarboxylate
(32).
A
solution of 1,1-dimethylethyl (2R)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-
pyrrolidinecarboxylate (0.489 g, 1.931 mmol) in dichloromethane (2 mL) was treated with
trifluoroacetic acid (0.4 mL) to give a tan colored solution. After stirring for 1 hour, additional
trifluoroacetic acid (0.2 mL) was added. The mixture was stirred for 15 minutes and judged
complete by TLC. The mixture was concentrated, azeotroped twice with toluene and then
placed under vacuum overnight to give methyl 3-[(2R)-2-pyrrolidinyl]-2-propynoate
1
trifluoroacetate as a pale yellow oil. H NMR (400 MHz, CHLOROFORM-d)
δ ppm 10.40 – 9.30
(m, 1 H), 8.33 – 7.78 (m, 1 H), 4.58 (t, J=6.87 Hz, 1 H), 3.80 (s, 3 H), 3.60 – 3.43 (m, 2 H), 2.50
– 2.40 (m, 1 H), 2.32 – 2.09 (m, 3 H). The crude trifluoroacetate salt was dissolved in
dichloromethane (10 mL) and then cooled to 0 °C. P yridine (0.781 mL, 9.65 mmol) was added
followed by slow addition of 2-(trimethylsilyl)ethyl chloridocarbonate (0.384 mL, 2.124 mmol).
The mixture was stirred at 0 °C for 20 minutes then room temperature for 20 minutes. Water
was added and the mixture was diluted with dichloromethane. The layers were separated and
the organic layer was washed with saturated NaHCO3 (x2) and brine (x2), dried over MgSO4,
filtered and concentrated. The crude material was purified by silica gel chromatography (0-15-
50% EtOAc/hexanes gradient) to give 2-(trimethylsilyl)ethyl (2R)-2-[3-(methyloxy)-3-oxo-1-
propyn-1-yl]-1-pyrrolidinecarboxylate (0.481, 84% over 2 steps) as a light yellow oil. 1H NMR
(400 MHz, CDCl3)
δ ppm 4.73 – 4.55 (m, 1 H), 4.26 – 4.16 (m, 2 H), 3.76 (s, 3 H), 3.59 – 3.30
(m, 2 H), 2.22 – 1.90 (m, 4 H), 1.10 – 0.97 (m, 2 H), 0.05 (s, 9 H); TLC (ethyl acetate/hexanes
15:85, Rf = 0.29).
4.1.19 1,1-Dimethylethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-piperidinecarboxylate
(35). To an ice cold suspension of potassium carbonate (38.6 g, 0.28 mol) and diethyl 1-diazo-
2-oxopropylphosphonate (41 g, 0.21 mmol) was added (S)-tert-butyl 2-formylpiperidine-1-
carboxylate[45] (30 g, 0.14 mol) via pipet. The mixture was stirred for 1 hour at which time the
reaction was judged complete by TLC (15:85 EtOAc/hexanes). The mixture had turned a pale
green in color. The reaction was quenched by the addition of saturated NH4Cl and extracted
with ether. The extracts were washed with brine, dried over Na2SO4, filtered and concentrated
in vacuo. The residue was dissolved in THF (400 mL), cooled to -78 °C and treated dropwise
with n-BuLi (47.2 mL, 118 mmol, 2.5M in THF). After 30 minutes at -78 °C, the dark solution was
cannulated into a -78 °C solution of methyl chlorof ormate (11.2 g, 118 mmol) in THF (50 mL).
The mixture was stirred for 5 minutes at -78 °C the n warmed to ambient temperature. Saturated
17

ACCEPTED MANUSCRIPT
NH4Cl was added the mixture was diluted with ethyl acetate. The organic layer was washed with
brine, dried over Na2SO4, filtered and concentrated. The crude material was purified by silica
gel chromatography (0-30% ethyl acetate/hexanes) to give 1,1-dimethylethyl (2S)-2-[3-
(methyloxy)-3-oxo-1-propyn-1-yl]-1-piperidinecarboxylate (13.5 g, 59%) as a clear oil. 1H NMR
(400 MHz, CDCl3)
δ ppm 5.39 – 5.12 (m, 1 H), 3.91 – 4.07 (m, 1 H), 3.78 (s, 3 H), 3.08 – 2.91
(m, 1 H), 1.90 – 1.32 (m, 15 H).
4.1.20 1,1-Dimethylethyl (2R)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-piperidinecarboxylate
(36). To an ice cold suspension of potassium carbonate (4.51 g, 32.64 mmol) and diethyl 1-
diazo-2-oxopropylphosphonate (4.20 g, 24.48 mmol) was added 1,1-dimethylethyl (R)-tert-butyl
2-formylpiperidine-1-carboxylate[46] (3.48 g, 16.32 mmol) via pipet. The mixture was stirred for
1 h at which time the reaction was judged complete by TLC (15:85 EtOAc/hexanes). The
mixture had turned a pale lime green in color. The reaction was quenched by the addition of
saturated NH4Cl and extracted with ether. The extracts were washed with brine, dried over
Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in THF (25 mL), cooled
to -78 °C and treated dropwise with n-BuLi (13.89 m L, 22.23 mmol, 1.6M in THF). After 30
minutes at -78 °C, the dark solution was cannulated into a -78 °C solution of methyl
chloroformate (1.579 mL, 20.38 mmol) in THF (10 mL). The mixture was stirred for 5 minutes at
-78 °C then warmed to room temp. TLC right before dry ice bath was removed indicated the
reaction was nearly complete by formation of new more polar spot. Saturated NH4Cl was added
the mixture was diluted with ethyl acetate. The organic layer was washed with brine, dried over
Na2SO4, filtered and concentrated.
The crude material was purified by silica gel
chromatography (0-15-30% ethyl acetate/hexanes) to give 1,1-dimethylethyl (2R)-2-[3-
(methyloxy)-3-oxo-1-propyn-1-yl]-1-piperidinecarboxylate (2.86 g, 58%) as a clear oil. 1H NMR
(400 MHz, CDCl3)
δ ppm 5.39 – 5.12 (m, 1 H), 3.91 – 4.07 (m, 1 H), 3.78 (s, 3 H), 3.08 – 2.91
(m, 1 H), 1.90 – 1.32 (m, 15 H); TLC (ethyl acetate/hexanes 15:85, Rf = 0.49).
4.1.21 2-(trimethylsilyl)ethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-piperidinecarboxylate
(37).
A
solution of 1,1-dimethylethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-
piperidinecarboxylate (1.0 g, 3.74 mmol) in dichloromethane (4 mL) was treated with
trifluoroacetic acid (1.2 mL). After 30 minutes, the reaction mixture was concentrated and then
azeotroped with toluene. The crude amine was dissolved in dichloromethane (20 mL), cooled to
18

ACCEPTED MANUSCRIPT
0 °C and then treated with pyridine (1.5 mL, 18.7 m mol) followed by slow addition of Teoc-Cl
(0.74 g, 4.11 mmol). The mixture was stirred for 5 minutes at 0 °C and then warmed to room
temperature for 30 minutes. The mixture was diluted with dichloromethane. The organic phase
was washed with saturated NaHCO3 (x2) and then brine, dried with MgSO4, filtered and
concentrated.
EtOAc/hexanes) to give 2-(trimethylsilyl)ethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-
piperidinecarboxylate (0.35 g, 30%) as a clear oil. 1H NMR (400 MHz, CDCl3)
ppm 5.38 – 5.17
The crude material was purified by silica gel chromatography (0-30%
δ
(m, 1 H), 4.20 (t, J=8.42 Hz, 2 H), 4.10 – 3.95 (m, 1 H), 3.78 (s, 3 H), 3.11 – 2.98 (m, 1 H), 1.92
– 1.63 (m, 5 H), 1.48 – 1.35 (m, 1 H), 1.05 – 0.98 (m, 2 H), 0.05 (s, 9 H).
4.1.22 2-(trimethylsilyl)ethyl (2R)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-piperidinecarboxylate
(38).
A
solution of 1,1-dimethylethyl (2R)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-1-
piperidinecarboxylate (1.119 g, 4.19 mmol) in dichloromethane (12 mL) was treated with
trifluoroacetic acid (4 mL). After 30 minutes, TLC indicated complete consumption of starting
material and only baseline material (15:85 ethyl acetate/hexanes).
The mixture was
concentrated and then azeotroped with toluene. The crude amine was dissolved in
dichloromethane (15 mL), cooled to 0 °C and then tr eated with pyridine (1.016 mL, 12.56 mmol)
followed by slow addition of Teoc-Cl (0.840 mL, 4.60 mmol). The mixture was stirred for 5
minutes at 0 °C and then warmed to room temperature for 30 minutes. The mixture was diluted
with dichloromethane. The organic phase was washed with saturated NaHCO3 (x2) and then
brine, dried with MgSO4, filtered and concentrated. The crude material was purified by silica gel
chromatography (0-15-30% EtOAc/hexanes) to give 2-(trimethylsilyl)ethyl (2R)-2-[3-(methyloxy)-
1
3-oxo-1-propyn-1-yl]-1-piperidinecarboxylate (1.092 g, 84%) as a clear oil. H NMR (400 MHz,
CDCl3)
δ ppm 5.38 – 5.17 (m, 1 H), 4.20 (t, J=8.42 Hz, 2 H), 4.10 – 3.95 (m, 1 H), 3.78 (s, 3 H),
3.11 – 2.98 (m, 1 H), 1.92 – 1.63 (m, 5 H), 1.48 – 1.35 (m, 1 H), 1.05 – 0.98 (m, 2 H), 0.05 (s, 9
H).
4.1.23
(R)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-oxo-6,6a,7,8,9,11-
hexahydroindolizino[7,6-g]quinoline-5-carbonitrile (42). A -78 °C solution of methyl 5-[(3-chloro-
2-fluorophenyl)methyl]-3-(cyanomethyl)-2-pyridinecarboxylate (0.200 g, 0.63 mmol) in THF (3.0
mL) was treated with lithium bis(trimethylsilyl)amide (0.75 mL, 0.75 mmol, 1.0 M in THF). After 1
h, the deep red solution was treated with a solution of (R)-tert-butyl 2-(4-methoxy-4-oxobut-2-yn-
1-yl)pyrrolidine-1-carboxylate (0.168 g, 0.75 mmol) in THF (1.0 mL). After 20 min, the reaction
19

ACCEPTED MANUSCRIPT
mixture was warmed to 0 °C. After an additional 30 min, the reaction mixture was quenched with
0.1 N HCl and extracted with EtOAc. The combined extracts were washed with brine, dried
(Na2SO4), filtered and concentrated in vacuo. The residue was dissolved in DCM (8.0 mL) and
treated with TFA (2.0 mL). After 3h, the reaction mixture was concentrated in vacuo. The
residue was dissolved in MeOH (5 mL), and treated with 1N NaOH (2.5 mL). A precipitate
formed and the reaction mixture was then acidified to pH 3 with 1N HCl and extracted with
EtOAc. The organic later was dried over Na2SO4, filtered and concentrated in vacuo. The
residue was suspended in MeOH and filtered to afford the title compound (50 mg, 20% yield).
1H NMR (400MHz, DMSO-d6)
δ = 14.95 (s, 1H), 8.92 (s, 1H), 8.16 (s, 1H), 7.56 - 7.38 (m, 2H),
7.23 (t, J=7.9 Hz, 1H), 4.38 (s, 2H), 3.75 - 3.65 (m, 1H), 3.58 - 3.46 (m, 2H), 3.16 - 3.04 (m, 1H),
2.39 - 2.29 (m, 1H), 2.13 - 2.01 (m, 1H). ES+ MS: 422 (M + 1).
4.1.24 (S)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-oxo-6,6a,7,8,9,11-hexahydroindolizino[7,6-
g]quinoline-5-carbonitrile (43). A -78 °C solution of methyl 5-[(3-chloro-2-fluoroph enyl)methyl]-3-
(cyanomethyl)-2-pyridinecarboxylate (0.300 g, 0.94 mmol) in THF (5.0 mL) was treated with
lithium bis(trimethylsilyl)amide (1.13 mL, 1.13 mmol, 1.0 M in THF). After 1 h, the deep red
solution was treated with a solution of (S)-tert-butyl 2-(4-methoxy-4-oxobut-2-yn-1-yl)pyrrolidine-
1-carboxylate (0.168 g, 0.75 mmol) in THF (1.0 mL). After 20 min, the reaction mixture was
warmed to 0 °C. After an additional 30 min, the rea ction mixture was quenched with 0.1 N HCl
and extracted with EtOAc. The combined extracts were washed with brine, dried (Na2SO4),
filtered and concentrated in vacuo. The residue was dissolved in DCM (4.0 mL) and treated with
TFA (1.0 mL). After 2h, the reaction mixture was concentrated in vacuo. The residue was
dissolved in MeOH (5 mL), and treated with 1N NaOH (2.5 mL). A precipitate formed and the
reaction mixture was then acidified to pH 3 with 1N HCl and extracted with EtOAc. The organic
later was dried over Na2SO4, filtered and concentrated in vacuo. The residue was suspended in
1
MeOH and filtered to afford the title compound (189 mg, 48% yield) as a tan solid. H NMR
(400MHz, DMSO-d6)
δ = 14.95 (s, 1H), 8.92 (d, J=1.8 Hz, 1H), 8.16 (s, 1H), 7.55 - 7.38 (m, 2H),
7.27 - 7.19 (m, 1H), 4.38 (s, 2H), 4.00 - 3.89 (m, 1H), 3.74 - 3.64 (m, 1H), 3.58 - 3.45 (m, 2H),
3.09 (dd, J=13.3, 15.8 Hz, 1H), 2.40 - 2.01 (m, 2H), 1.93 - 1.69 (m, 2H). ES+ MS: 422 (M + 1).
4.1.25
(R)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-oxo-5b,6,7,8,9,11-
hexahydroindolizino[1,2-g]quinoline-5-carbonitrile (44). A -78 °C solution of methyl 5-[(3-chloro-
20

ACCEPTED MANUSCRIPT
2-fluorophenyl)methyl]-3-(cyanomethyl)-2-pyridinecarboxylate (0.255 g, 0.800 mmol) in THF
(4.0 mL) was treated with lithium bis(trimethylsilyl)amide (0.960 mL, 0.960 mmol, 1.0 M in THF).
After 1 h, the deep red solution was treated with a solution of 2-(trimethylsilyl)ethyl (2R)-2-[3-
(methyloxy)-3-oxo-1-propyn-1-yl]-1-piperidinecarboxylate (0.299 g, 0.960 mmol) in THF (1.0
mL). After 20 min, the reaction mixture was warmed to 0 °C. After an additional 20 min, the
reaction mixture was quenched with 0.1 N HCl and extracted with EtOAc. The combined
extracts were washed with brine, dried (Na2SO4), filtered and concentrated in vacuo. The
residue was dissolved in THF (7.0 mL) and treated with TBAF (2.4 mL, 2.4 mmol, 1.0 M in THF)
and heated to 70 °C. After 3h, the reaction mixture was treated with 1.0N HCl and extracted with
EtOAc. The extracts were washed with brine, dried over Na2SO4, filtered and concentrated.
Methanol an diethyl ether were added and a precipitate formed. The solvents were removed
and the solid was washed repeatedly with minimal amounts of methanol. The solid was dried
under vacuum to give the title compound (0.264 g, 50%) as an orange solid. 1H NMR (400
MHz, CDCl3)
δ ppm 8.92 (d, J=1.81 Hz, 1 H), 8.31 (d, J=1.20 Hz, 1 H), 7.37 – 7.32 (m, 1 H),
7.17 – 7.12 (m, 1 H), 7.11 – 7.05 (m, 1 H), 4.67 (dd, J=11.84, 3.81 Hz, 1 H), 4.41 - 4.55 (m, 1
H), 4.29 (s, 2 H), 3.10 – 3.00 (m, 1 H), 2.90 – 2.82 (m, 1 H), 2.14 – 2.06 (m, 1 H), 1.94 – 1.86
(m, 1 H), 1.83 – 1.69 (m, 1 H), 1.54 – 1.41 (m, 1 H), 1.32 - 1.19 (m, 1 H); ES+ MS: 422 (M + 1).
4.1.26 (S)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-oxo-5b,6,7,8,9,11-hexahydroindolizino[1,2-
g]quinoline-5-carbonitrile (45). A -78 °C solution of methyl 5-[(3-chloro-2-fluoroph enyl)methyl]-3-
(cyanomethyl)-2-pyridinecarboxylate (0.502 g, 1.45 mmol) in THF (9.0 mL) was treated with
lithium bis(trimethylsilyl)amide (2.5 mL, 2.5 mmol, 1.0 M in THF). After 1 h, the deep red solution
was treated with a solution of 2-(trimethylsilyl)ethyl (2S)-2-[3-(methyloxy)-3-oxo-1-propyn-1-yl]-
1-piperidinecarboxylate (0.54 g, 1.73 mmol) in THF (3.0 mL). After 20 min, the reaction mixture
was warmed to 0 °C. After an additional 20 min, the reaction mixture was quenched with 0.1 N
HCl and extracted with EtOAc. The combined extracts were washed with brine, dried (Na2SO4),
filtered and concentrated in vacuo. The residue was dissolved in THF (7.0 mL) and treated with
TBAF (5.8 mL, 5.8 mmol, 1.0 M in THF) and heated to 70 °C. After 3h, the reaction mixture was
treated with 1.0N HCl and extracted with EtOAc. The extracts were washed with brine, dried
over Na2SO4, filtered and concentrated. Methanol an diethyl ether were added and a precipitate
formed. The solvents were removed and the solid was washed repeatedly with minimal
amounts of methanol. The solid was dried under vacuum to give the title compound (0.07 g,
12%) as an orange solid. 1H NMR (400MHz, DMSO-d6)
δ
= 8.96 (s, 1H), 8.25 (s, 1H), 7.55 -
21

ACCEPTED MANUSCRIPT
7.38 (m, 2H), 7.28 - 7.17 (m, 1H), 4.74 (dd, J=3.5, 11.5 Hz, 1H), 4.40 (s, 2H), 4.21 (dd, J=3.6,
13.0 Hz, 1H), 3.00 - 2.89 (m, 1H), 2.58 (d, J=10.2 Hz, 1H), 2.02 - 1.84 (m, 1H), 1.81 - 1.63 (m,
2H), 1.37 - 1.03 (m, 2H); ES+ MS: 422 (M + 1).
4.1.27
(R)-3-(3-chloro-2-fluorobenzyl)-11-hydroxy-10-oxo-6,7,8,10-tetrahydro-5bH-
pyrrolizino[1,2-g]quinoline-5-carbonitrile (46). A -78 ºC solution of methyl 5-[(3-chloro-2-
fluorophenyl)methyl]-3-(cyanomethyl)-2-pyridinecarboxylate (0.237 g, 0.744 mmol) in THF (4.0
mL) was treated with lithium bis(trimethylsilyl)amide (0.892 mL, 0.892 mmol, 1.0M in THF).
The red mixture was stirred at -78 ºC for 20 min before adding a solution of (R)-2-
(trimethylsilyl)ethyl 2-(3-methoxy-3-oxoprop-1-yn-1-yl)pyrrolidine-1-carboxylate (0.265 g, 0.892
mmol) in THF (1 mL). The mixture was stirred at -78 °C for 20 minutes and then warmed to 0
°C. After 15 minutes the reaction was judged compl ete by LCMS. The mixture was quenched
with 0.1 N HCl and extracted with ethyl acetate. The extracts were washed with brine, dried
over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in THF/DMF (1:1 -
5.0 mL) and treated with cesium fluoride (0.282 g, 1.859 mmol) and heated to 90 °C. After 7
hours, the reaction mixture was cooled to ambient temperature and 1.0 N HCl was added. The
mixture was extracted with ethyl acetate. The extracts were washed with brine, dried over
Na2SO4, filtered and concentrated in vacuo. The material was by silica get chromatography (0-
100% ethyl acetate /hexanes gradient) to afford the title compound (60 mg, 20%) as an orange
1
solid. H NMR (400 MHz, CDCl3)
δ ppm 8.90 (m, 1 H), 8.29 (m, 1 H), 7.38 – 7.32 (m, 1 H), 7.18
– 7.06 (m, 2 H), 5.0 (dd, J=10.63, 6.02 Hz, 1 H), 4.30 (s, 2 H), 3.85 – 3.77 (m, 1 H), 3.52 – 3.44
(m, 1 H), 2.73 – 2.64 (m, 1 H), 2.50 – 2.34 (m, 2 H), 1.48 – 1.37 (m, 1 H); ES+ MS: 408 (M + 1).
4.1.28
(S)-3-(3-chloro-2-fluorobenzyl)-11-hydroxy-10-oxo-6,7,8,10-tetrahydro-5bH-
pyrrolizino[1,2-g]quinoline-5-carbonitrile (47). A -78 ºC solution of methyl 5-[(3-chloro-2-
fluorophenyl)methyl]-3-(cyanomethyl)-2-pyridinecarboxylate (0.153 g, 0.480 mmol) in THF (3.0
mL) was treated with lithium bis(trimethylsilyl)amide (0.576 mL, 0.576 mmol, 1.0M in THF).
The red mixture was stirred at -78 ºC for 20 min before adding a solution of (R)-2-
(trimethylsilyl)ethyl 2-(3-methoxy-3-oxoprop-1-yn-1-yl)pyrrolidine-1-carboxylate (0.171 g, 0.576
mmol) in THF (0.6 mL). The mixture was stirred at -78 °C for 20 minutes and then warmed to 0
°C. After 25 minutes the reaction was judged compl ete by LCMS. The mixture was quenched
with 0.1 N HCl and extracted with ethyl acetate. The extracts were washed with brine, dried
22

ACCEPTED MANUSCRIPT
over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in THF (5.0 mL)
and treated with TBAF (0.96 mL, 0.96 mmol) and heated to 70 °C. After 8h, the reaction mixture
was treated with 1.0N HCl and extracted with EtOAc. The extracts were washed with brine,
dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel
chromatography (0-100% EtOAc/hexanes) to afford the title compound (16 mg, 22%) as an
orange solid. 1H NMR (400MHz, CDCl3)
δ = 8.87 (s, 1H), 8.27 (s, 1H), 7.33 (s, 1H), 7.17 - 7.02
(m, 2H), 4.97 (dd, J=6.0, 10.6 Hz, 1H), 4.27 (s, 2H), 3.79 (d, J=11.5 Hz, 1H), 3.51 - 3.40 (m,
1H), 2.71 - 2.60 (m, 1H), 2.49 - 2.31 (m, 2H), 1.40 (dd, J=8.5, 11.1 Hz, 1H). ES+ MS: 408 (M +
1).
4.1.29
(R)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-oxo-6,6a,7,8,9,11-
hexahydroindolizino[7,6-g]quinoline-5-carboxamide (50). A suspension of (R)-3-(3-chloro-2-
fluorobenzyl)-12-hydroxy-11-oxo-6,6a,7,8,9,11-hexahydroindolizino[7,6-g]quinoline-5-
carbonitrile (0.034 g, 0.081 mmol) in acetic acid (0.6 mL) and water (0.6 mL) was treated with
sulfuric acid (0.6 mL) to affect dissolution and heated to 120 ºC. After 8 h, the reaction mixture
was concentrated in vacuo and the crude product recrystallized from MeOH to afford the title
compound (24 mg, 67%) as a yellow solid. 1H NMR (400MHz, DMSO-d6)
δ = 14.15 (s, 1H), 8.82
(d, J=1.8 Hz, 1H), 7.96 (s, 2H), 7.82 (br. s., 1H), 7.49 (t, J=6.9 Hz, 1H), 7.42 - 7.35 (m, 1H), 7.25
- 7.17 (m, 1H), 4.31 - 4.23 (m, 2H), 3.87 - 3.63 (m, 2H), 3.57 - 3.46 (m, 1H), 3.31 - 3.22 (m, 2H),
2.86 - 2.72 (m, 1H), 2.31 (dd, J=5.5, 11.3 Hz, 1H), 2.11 - 1.98 (m, 1H), 1.92 - 1.65 (m, 2H) ES+
MS: 440 (M + 1).
4.1.30 (S)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-oxo-6,6a,7,8,9,11-hexahydroindolizino[7,6-
g]quinoline-5-carboxamide (51). A suspension of (S)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-
oxo-6,6a,7,8,9,11-hexahydroindolizino[7,6-g]quinoline-5-carbonitrile (0.082 g, 0.194 mmol) in
acetic acid (0.7 mL) and water (0.7 mL) was treated with sulfuric acid (0.7 mL) to affect
dissolution. The mixture was heated at 125 ºC for 8 hours after which time the reaction was
judged complete by LCMS. The mixture was purified by reverse phase chromatography to give
the title compound (74 mg, 87%) as a yellow solid. 1H NMR (400MHz, CDCl3)
δ ppm 8.77 (s,
1H), 8.14 (s, 1H), 7.35 - 7.30 (m, 1H), 7.14 - 7.01 (m, 2H), 6.32 (br. s., 1H), 4.14 (s, 2H), 3.81
(br. s., 1H), 3.70 (t, J=10.5 Hz, 1H), 3.52 - 3.39 (m, 2H), 2.81 - 2.67 (m, 1H), 2.41 - 2.30 (m, 1H),
2.18 - 2.06 (m, 1H), 1.97 - 1.83 (m, 1H), 1.78 - 1.63 (m, 1H). ES+ MS: 440 (M + 1).
23

ACCEPTED MANUSCRIPT
4.1.31
(R)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-oxo-5b,6,7,8,9,11-
hexahydroindolizino[1,2-g]quinoline-5-carboxamide (52). A suspension of (R)-3-(3-chloro-2-
fluorobenzyl)-12-hydroxy-11-oxo-5b,6,7,8,9,11-hexahydroindolizino[1,2-g]quinoline-5-
carbonitrile (0.092 g, 0.218 mmol) in acetic acid (0.7 mL) and water (0.7 mL) was added sulfuric
acid (0.5 ml) to affect dissolution. The solution was stirred at room temperature for 15 minutes
and then heated to 90 °C for 1 hour. Heating was i ncreased to 125 °C for 4 hours and then the
mixture was cooled and allowed to stir overnight at room temperature. LCMS indicated
conversion to the primary amide and decarboxylation products (1:1 ratio). The mixture was
purified by reverse phase chromatography twice to give the title compound (25 mg, 26%) as a
1
grey-tinged solid. H NMR (400 MHz, METHANOL-d4)
δ ppm 8.87 (m, 1 H), 8.48 (m, 1 H), 7.43
– 7.29 (m, 2 H), 7.19 – 7.12 (m, 1 H), 4.81 – 4.74 (m, 1 H), 4.43 – 4.32 (m, 3 H), 3.10 – 3.00 (m,
1 H), 2.58 – 2.51 (m, 1 H), 2.05 – 1.95 (m, 1 H), 1.89 – 1.67 (m, 2 H), 1.48 – 1.32 (m, 1 H), 1.23
– 1.09 (m, 1 H); ES+ MS: 440 (M + 1).
4.1.32 (S)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-oxo-5b,6,7,8,9,11-hexahydroindolizino[1,2-
g]quinoline-5-carboxamide (53). A suspension of (S)-3-(3-chloro-2-fluorobenzyl)-12-hydroxy-11-
oxo-5b,6,7,8,9,11-hexahydroindolizino[1,2-g]quinoline-5-carbonitrile (0.050 g, 0.12 mmol) in
acetic acid (0.5 mL) and water (0.5 mL) was added sulfuric acid (0.5 ml) to affect dissolution and
then heated to 95 °C. After 6h, the reaction mixtur e was cooled to ambient temperature and
poured into water and DCM/MeOH, The layers were partitioned and the organic phase washed
with brine, dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by
reverse phase chromatography twice to give the title compound (10 mg, 20%). 1H NMR (300
MHz, DMSO-d6)
δ ppm 8.87 (m, 1 H), 8.48 (m, 1 H), 7.43 – 7.29 (m, 2 H), 7.19 – 7.12 (m, 1 H),
4.81 – 4.74 (m, 1 H), 4.43 – 4.32 (m, 3 H), 3.10 – 3.00 (m, 1 H), 2.58 – 2.51 (m, 1 H), 2.05 –
1.95 (m, 1 H), 1.89 – 1.67 (m, 2 H), 1.48 – 1.32 (m, 1 H), 1.23 – 1.09 (m, 1 H); ES+ MS: 440 (M
+ 1).
4.2 Virology
Reagents. Reverse transcriptase and integrase were prepared as previously described.[47]
Deoxynucleotide triphosphates (dNTPs) and protease-free Bovine Serum Albumin (BSA;
24

ACCEPTED MANUSCRIPT
Fraction V) were acquired from Roche Applied Science (Indianapolis, IN).
[8-3H(N)]-
deoxyguanosine 5'-triphosphate (dGTP) and streptavidin-coated polystyrene SPA imaging
beads were acquired from PerkinElmer (Waltham, MA). 1,4-dithiothreitol (DTT), CHAPS and
dimethly sulfoxide (DMSO) were acquired from Sigma-Aldrich (St. Louis, MO). NP-40 was
acquired from Thermo Scientific (Rockford, IL). Nuclease-free dH2O, Tris pH 8.0, NaCl, KCl,
MgCl, and EDTA were acquired from Life Technologies (Grand Island, NY). All compounds
were prepared as 10 mM stocks in DMSO and stored at -20 °C.
The following oligonucleotides were used and were acquired from Midland Certified Reagent
Company (Midland, TX) and Integrated DNA Technologies (Coralville, IA).
Oligo 1, 5’-AGGUG AGUGA GAUGA UAACA AAUUU GCGAG CCCCA GAUGC-3’
Oligo 2, 5’-Biotin-GCATC TGGGG CTCGC AAATT TG-3’
Oligo 3, 5’-CCCCC CCCCC CCCCC AGGTG AGTGA GATGA TAAC-cordycepin-3’
All lyophilized oligonucleotides were reconstituted to 200 µM in 10 mM Tris pH 8.0, 10 mM NaCl
and stored at -20°C in small aliquots prior to use.
Substrate A was prepared by equilibrating 20 µM of Oligo 1 and 20 µM of Oligo 2 in 10 mM Tris
pH 8.0, 10 mM NaCl at 95°C, followed by a gradual d ecrease to 80°C (0.1°C decrease /6
seconds) and then to 4°C (0.1C/20 seconds) with a D yad DNA Engine thermal cycler (BioRad;
Hercules, CA).
Pseudo-HIV assay. The antiviral activities of compounds were measured in a single-round
assay using a self-inactivating PHIV lentiviral vector. CIP4 cells (2 _ 104/well) infected with an
amount of PHIV sufficient to produce approximately 50,000 relative light units were added to 96-
well black, clear-bottom plates and were incubated for 2 days with S/GSK1349572 at varying
concentrations.Infected cells were measured as a function of luciferase activity in a luminometer
using the Steady-Glo reagent (Promega Corporation).29
4.3 In vivo DMPK
Male non-fasted Sprague Dawley (CD) rats (n=2 per study arm) received test article at doses of
1 mg/kg i.v. (1 mL/kg) or 5 mg/kg p.o. (5 mL/kg), formulated in a 10% DMSO/10% Solutol/80%
0.05 M N-methylglucamine dosing vehicle. For all animals, water was provided ad libitum.
Blood samples were withdrawn from a surgically-implanted venous cannula at timed intervals
25

ACCEPTED MANUSCRIPT
for 24 h after dose administration, treated with EDTA, and centrifuged to harvest plasma for
LC/MS/MS analysis. Plasma concentration-time data for individual rats were analyzed using
non compartmental analysis (WinNonlin v. 4.1a; Pharsight, Mountain View CA) to generate
pharmacokinetic parameter estimates.
All studies were conducted in accordance with the GSK Policy on the Care, Welfare and
Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use
Committee either at GSK or by the ethical review process at the institution where the work was
performed.
[1] A. D. Frankel, J. A. Young, Annu. Rev. Biochem. 67 (1998) 1-25.
[2] B. Johns, HIV-1 Integrase. In Burger’s Medicinal Chemistry and Drug Discovery, 7th Edition;
D. Abraham, D. Rotella, Eds.; Wiley: New York, 2010.
[3] D. Hauzuda, Curr. Opin. HIV AIDS 7 (2012) 383-389.
[4] O. Sari, V. Roy, M. Métifiot, C. Marchand, Y. Pommier, S. Bourg, P. Bonnet, R. Schinazi, L. Agrofoglio, Eur. J.
Med. Chem. 104 (2015) 127-138.
[5] K. Gupta, T. Brady, B. Dyer, N. Malani, Y. Hwang, F. Male, R. Notle, L. Wang, E. Velthuisen, J. Jeffrey, G. Van
Duyne, F. Bushman, J. Biological Chem. 289 (2014) 26430-26440.
[6] P. Rice, R. Craigie, D. R. Davies, Curr Opin Struct Biol. 6 (1996) 76-83.
[7] F. Dyda, A. B. Hickman, T. M. Jenkins, A. Engelman, R. Craigie, D. R. Davies, Science, 266 (1994) 1981.
[8] J. A. Grobler, K. Stillmock, B. Hu, M. Witmer, P. Felock, A. S. Espeseth, A. Wolfe, M. Egbertson, M. Bourgeois, J.
Melamed, J. S. Wai, S. Young, J. Vacca, D. J. Hazuda, Proc. Natl. Acad. Sci. USA. 66 (2002) 6661-6666.
[9] S. Hare, G. Maertens, P. Cherepanov, EMBO J. 31 (2012) 3020-3028.
[10] C. Yoakim, M. Amad, M. Bailey, R. Bethell, M. Bös, P. Bonneau, M. Cordingley, R. Coulombe, J. Duan, P.
Edwards, L. Fader, A. Faucher, M. Garneau, A. Jakalian, S. Kyeaawai, L. Lamorte, S. LaPlante, L. Luo, S. Mason, M.
Poupart, N. Rioux, B. Simoneau, Y. Tsantrizos, M. Witvrouw, C. Fenwick, 51st Interscience Conference on
Antimicrobial Agents and Chemotherapy (ICAAC). September 17-20, 2011, F1-1369.
[11] E. Deprez, S. Barbe, M. Kolaski, H. Leh, F. Zouhiri, C. Auclair, J.-C. Brochon, M. Le Bret, J.-F. Mouscadet, Mol.
Pharmacol. 65 (2004) 85-98.
[12] M. Billamboz, V. Suchaud, F. Bailly, C. Lion, J. Demeulemeester, C. Calmels, M.-L. Andreola, F. Christ, Z.
Debyser, P. Cotelle, ACS Med. Chem. Lett. 4 (2013) 606-611.
[13] C. Auclair, B. Giethlen, M. Michaut, C. Monneret, E. Soma, L. Thibault, G. Wermuth, WO 2010/010147.
[14] E. Velthuisen, B. Johns, P. Gerondelis, Y. Chen, M. Li, K. Mou, W. Zhang, J. Seal, K. Hightower, S. Miranda, K.
Brown, L. Leesnitzer, Eur. J. Med. Chem. 83 (2014) 609-616.
[15] L. Krishnan, X. Li, H. L. Naraharisetty, S. Hare, P. Cherepanov, A. Engelman, Proc. Natl. Acad. Sci. USA, 107
(2010) 15910-15915.
[16] S. Hare, S. S. Gupta, E. Valkov, A. Engelman, P. Cherepanov, Nature, 464 (2010) 232-236.
[17] S. Hare, A. M. Vos, R. F. Clayton, J. W. Thuring, M. D. Cummings, P. Cherepanov, Proc. Natl. Acad. Sci. USA,
107 (2010) 20057-20062.
[18] T. Kawasuji, B. A. Johns, H. Yoshida, T. Taishi, Y. Taoda, H. Murai, R. Kiyama, M. Fuji, T. Yoshinaga, T. Seki, M.
Kobayashi, A. Sato, T. Fujiwara, J. Med. Chem. 55 (2012) 8735-8744.
[19] V. Summa, A. Petrocchi, F. Bonelli, B. Crescenzi, M. Donghi, M. Ferrara, F. Fiore, C. Gardelli, O. Paz, D. Hazuda,
P. Jones, O. Kinzel, R. Laufer, E. Monteagudo, E. Muraglia, E. Nizi, F. Orvieto, P. Pace, G. Pescatore, R. Scarpelli, K.
Stillmock, M. Witmer, M. Rowley, J. Med. Chem. 51 (2008), 5843-5855.
26

ACCEPTED MANUSCRIPT
[20] R. Steigbigel, D. Cooper, H. Teppler, J. Enron, J. Gatell, P. Kumar, J. Rockstroh, M. Schecter, C. Katlama, M.
Markowitz, P. Yeni, M. Loutfy, A. Lazzarin, J. Lennox, B. Clotet, J. Zhao, H. Wan, R. Rhodes, K. Strohmaier, R.
Barnard, R. Isaacs, B. Nguyen, Clin. Infect. Dis. 50 (2010) 605-612.
[21] M. Métifiot, K. Maddali, A. Naumova, X. Zhang, C. Marchand, Y. Pommier, Biochemistry 49 (2010) 3715-3722.
[22] A. Marcelin, F. Ceccherini, C. Perno, V. Calvez, Curr. Opin. HIV AIDS 4 (2009) 531-537
[23] M. Sato, H. Kawakami, T. Motomura, H. Aeamaki, T. Matsuda, M. Yamashita, Y. Ito, Y. Matsuzaki, K. Yamataka,
S. Ikeda, H. Shinkai, J. Med. Chem. 52 (2009) 4869-4882.
[24] J. Molina, A. LaMarca, J. Andrade-Villanueva, B. Clotet, N. Clumeck, Y. Liu, L. Zhong, N. Margot, A. Cheng, S.
Chuck, Lancet Infect. Dis. 12 (2012) 27-35.
[25] O. Goethals, R. Clayton, M. Van Ginderen, I. Vereycken, E. Wagemans, P. Geluykens, K. Dockx, R. Strijbos, V.
Smits, A. Vos, G. Meersseman, D. Jocjmans, K. Vermeire, D. Schols, S. Hallenberger, K. Hertogs, J. Virology, 82
(2008) 10366-10374.
[26] B. Johns, T. Kawasuji, E. Velthuisen, “HIV Integrase Inhibitors” in Success Strategies in Antiviral Drug Discovery;
M. Desai, N. Meanwell, Ed.; RSC Publishing: Cambridge, 2013; Vol. 32; p 149.
[27] B. Johns, T. Kawasuji, J. Weatherhead, T. Taishi, D. Temelkoff, H. Yoshida, T. Akiyama, Y. Taoda, H. Murai, R.
Kiyama, M. Fuji, N. Tanimoto, J. Jeffrey, S. Foster, T. Yoshinaga, T. Seki, M. Kobayahi, A. Sato, M. Johnson, E.
Garvey, T. Fujiwara, J. Med. Chem. 56 (2013), 5901-5916
[28] T. Kawasuji, B. Johns, H. Yoshida, J. Weatherhead, T. Akiyama, T. Taishi, Y. Taoda, M. Mikamiyama-Iwata, H.
Murai, R. Kiyama, M. Fuji, N. Tanimoto, T. Yoshinaga, T. Seki, M. Kobayashi, A. Sato, E. Garvey, T. Fujiwara, J. Med.
Chem. 56 (2013) 1124-1135.
[29] M. Kobayashi M, T. Yoshinaga, T. Seki, C. Wakasa-Morimoto, K. Brown, R. Ferris, S. Foster, R. Hazen, S. Miki, A.
Suyama-Kagitan, S. Kawauchi-Miki, T. Taishi, T. Kawasuji, B. Johns, M. Underwood, E. Garvey, A. Sato, T. Fujiwara,
Antimicrob. Agents and Chemother. 55 (2011) 813-821.
[30] F. DeAnda, K. Hightower, R. Notle, K. Hattori, T. Yoshinaga, T. Kawasuji, M. Underwood, PLOS One (2013)
e77448.
[31] Garvey, E. P.; Johns, B. A., Gartland, M.; Foster, S. A.; Miller, W. H.; Ferris, R. G; Hazen, R. J.; Underwood, M. R.;
Boros, E. E.; Thompson, J. B.; Weatherhead, J. G.; Koble, C. S.; Allen, S. H.; Schaller, L. T.; Sherrill, R. G.; Yoshinaga,
T.; Kobayashi, M.; Wakasa-Morimoto, C.; Miki, S.; Nakahara, K.; Noshi, T.; Sato, A.; Fujiwara, T. Antimicrob. Agents
and Chemother. 2008, 52, 901-908.
[32] B. Johns, T. Kawasuji, J. Weatherhead, E. Boros, J. Thompson, C. Koble, E. Garvey, S. Foster, J. Jeffrey, T.
Fujiwara, Bioorg. Med. Chem.Lett. 24 (2014) 3104-3107.
[33] B. Johns, T. Kawasuji, J. Weatherhead, E. Boros, J. Thompson, C. Koble, E. Garvey, S. Foster, J. Jeffrey, T.
Fujiwara, Bioorg. Med.l Chem.Lett. 23 (2013) 422-425.
[34] E. Garvey, B. Johns, M. Gartland, S. Foster, W. Miller, R. Ferris, R. Hazen, M. Underwood, E. Boros, J.
Thompson, Antimicrob. Agents and Chemother. 52 (2008) 901-908.
[35] R. Melensez, W. Lubell, J. Am. Chem. Soc. 126 (2004) 6759-6764.
[36] W. Eisenmuth, H. Renfroe, H. Schmid, Helv. Chim. Acta. 48 (1965) 375-379.
[37] F. Hauser, R. Rhee, J. Org. Chem. 43 (1978) 178–180.
[38] References for known aldehydes provided in Section 4.1.
[39] S. Müller, B. Liepold, G. Roth, H. Bestmann. Synlett 6 (1996) 521-522.
[40] R. Melensez, W. Lubell, J. Am. Chem. Soc. 126 (2004) 6759-6764.
[41] Y. Jagadeesh, K. Reddy, B. Rao, Tetrahedron: Asymmetry 22 (2011) 1485-1489.
[42] A. Fürstner, J. Kennedy, Chem. Eur. J. 12 (2006) 7398-7410.
[43] R. Beckett, S. Davies, A. Mortlock, Tetrahedron: Asymmetry 3 (1992) 123-136.
[44] E. Trybulski, R. Kramss, R. Mangano, A. Rusinko, J. Med. Chem. 33 (1990) 3190-3198.
[45] T. Wilkinson, N. Stehle, P. Beak, Org. Lett. 2 (2000) 155-158.
[46] G. Balboni, M. Marastoni, S. Merighi, P. Borea, R. Tomatis, Euro. J. Med. Chem. 35 (2000) 979-988.
[47] E. P. Garvey, B. Schwartz, M. J. Gartland, S. Lang, W. Halsey, G. Sathe, H. L. Carter III, and K. L. Weaver,
Biochemistry 48 (2009) 1644-1653.
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Figure 1. Representative integrase strand transfer inhibitors
Scheme 1. Design of the 8-hydroxyquinoline tetracyclic lactam scaffold
Scheme 2. Initial synthesis of the HQT scaffold
Scheme 3. Unsuccessful strategy to synthesizing the HQT scaffold
Scheme 4. Successful Michael-Dieckmann cyclization with an ynoate coupling partner
Scheme 5. Synthesis of ynoate coupling partners
Scheme 6. Synthesis of HQT analogs using a Michael-Dieckmann cyclization
Figure 2. Hydrolysis of the C5-nitrile to afford a primary amide
Table 2. In vivo DMPK profile of HQT analog 52
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