Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led to a Preclinical Candidate for the Treatment of Visceral Leishmaniasis

Article abstract of DOI:10.1021/acs.jmedchem.1c00047

There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.

Full text of DOI:10.1021/acs.jmedchem.1c00047

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Article
Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led
to a Preclinical Candidate for the Treatment of Visceral
Leishmaniasis
Michael Thomas,§ Stephen Brand,§ Manu De Rycker, Fabio Zuccotto, Iva Lukac, Peter G. Dodd,
Eun-Jung Ko, Sujatha Manthri, Kate McGonagle, Maria Osuna-Cabello, Jennifer Riley, Caterina Pont,
Frederick Simeons, Laste Stojanovski, John Thomas, Stephen Thompson, Elisabet Viayna,
Jose M. Fiandor, Julio Martin, Paul G. Wyatt, Timothy J. Miles, Kevin D. Read, Maria Marco,*
and Ian H. Gilbert*
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ABSTRACT: There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily
large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as
a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a
phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity.
The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving
medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The
compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure
and the impact this has on our understanding of the series’ structure−activity relationships.
infection causes irregular bouts of fever, substantial weight
loss, swelling of the spleen and liver, and anemia. Current
available treatments are not adequate due to high toxicity,
resistance issues, treatment cost and duration, or the need for
hospitalization to administer the treatment, a particularly
relevant aspect given the settings where the disease is endemic.
Indeed, the only effective oral treatment for VL is miltefosine,
although it requires 28 days of dosing, and contraception is
necessary for women both during and after treatment due to its
INTRODUCTION
■
Bites from infected sandflies are known to transmit Leishmania
parasites, a protozoan parasite. There are more than 20
different species of Leishmania, which cause a group of diseases
that manifest in three main clinical forms: cutaneous,
mucocutaneous, and visceral leishmaniasis (VL, also referred
to as kala-azar).1−3 There is also the complication of VL, called
post kala-azar dermal leishmaniasis. While the other three
forms cause major health problems such as nonhealing ulcers
and scarring, VL is the most severe and becomes lethal if not
treated. Recurrent epidemics of VL in East Africa (Ethiopia,
Kenya, South Sudan, and Sudan) have caused high morbidity
and mortality in those countries and together with Brazil,
India, and Somalia accounted for more than 90% of the new
cases reported in 2018.4 VL is usually due to infection with
either Leishmania donovani or Leishmania infantum. The
Received: January 11, 2021
Published: April 27, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.jmedchem.1c00047
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Figure 1. Compounds 1 (DDD01305143/GSK3494245), 2 (LXE408), 3 (initial hit), and 4 (early lead).
teratogenicity. For reasons that are poorly understood, clinical
outcomes for these drugs seem to vary from region to region of
the world, with much reduced cure rates being observed in
East Africa and South America. Given these factors, the need
for safer, oral, short-course, and low-cost medicines for the
treatment of VL is urgent.
the structure−activity relationships (SARs) around the 2-
amino group, is described in detail herein. Although the series
was developed phenotypically, it was subsequently found to act
principally through the inhibition of the β5-subunit of the
proteasome, specifically at the “chymotrypsin” site, and the
relationship between the antiparasitic SAR and the identified
target is also discussed in detail.10
There is some cause for optimism, as for the first time, there
are a number of compounds in late preclinical or early clinical
development, as indicated on the Drugs for Neglected Diseases
Initiative (DNDi) website.5 We recently reported a leishmanial
CRK-12 inhibitor as a preclinical candidate;6,7 Novartis
(LXE408, compound 2)8,9 and ourselves (GSK3494245/
DDD01305143, compound 1)10 have both reported protea-
some inhibitors (Figure 1); DNDi have other compounds in
clinical development, notably an oxaborole and a nitro-
imidazole, although the mechanism of action (MoA) of these
has not been reported.5 In all these cases, the optimization was
run phenotypically, with the MoA only being elucidated during
or after the medicinal chemistry program. It must be noted10
that there is a lack of validated drug targets for Leishmania.
As we reported recently, compound 1 was the product of a
program which started from substituted 2-phenyloxazolo[4,5-
b]pyridine 3 which was identified through phenotypic
screening against the related pathogen Trypanosoma cruzi,
the causative agent of Chagas’ disease (Figure 1). Sub-
sequently, compound 3 was screened against the physiologi-
cally relevant intramacrophage form of L. donovani, where it
showed weak activity. In parallel to our efforts, N-(4-chloro-3-
(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-carboxamide (the
4-chloro phenyl analogue of 3) had also been identified by
the groups of Buckner, Gelb, Baell, and Guy as a compound
with antikinetoplastid activity, with their optimization being
subsequently reported.8,11−15
As previously described, an initial scaffold hop from 3 led to
a 2-phenylimidazo[1,2-a]pyrimidine and optimization of the 6-
position of the bi-cycle led to a 6-morpholino substituent
which increased the potency and metabolic stability. Alongside
this, the fluorination of the phenyl ring increased potency and
replacement of the furanyl amide with a pyrrolidinyl urea
improved solubility (similar changes had also been observed
on related scaffolds by other groups13,15). This led to
compound 4, which showed efficacy in a mouse model of
VL.10 The key to the success of this compound series was a
further set of scaffold hops from 2-phenylimidazo[1,2-
a]pyrimidine, and this strategy, along with the discussion of
RESULTS
■
Compounds were initially screened against L. donovani in an
intracellular assay (L. donovani cultured in differentiated THP1
cells, referred to as the “INMAC assay”16), where we targeted
pEC50 values >5.8, alongside stability in mouse liver micro-
somes of <5.0 mL/min/g (Cli) and kinetic aqueous solubility
>100 μM (measured by chemiluminescent nitrogen detection,
CLND). We had successfully used these criteria in the hit to
lead stage to identify compounds for in vivo proof-of-concept
studies in another program, which subsequently led to the
identification of preclinical candidate DDD853651/
GSK3186899 active against CRK12.7 As reported previously10
and shown in Table 1, in vitro profiling of 4 shows that it met
these targets, with an INMAC pEC50 value of 6.2, Cli of <0.5
mL/min/g in mouse liver microsomes, and aqueous solubility
of 150 μM, alongside high mouse and human plasma stability
(100% compound remaining after 3 h). A pharmacokinetic
(PK) study was thus performed, dosed at 10 mg/kg po and 3
mg/kg iv. An oral dose of 10 mg/kg showed 4 to have blood
levels above EC90 for around 2 h (pEC90 5.7, 780 ng/mL),
which would translate to coverage above 6 h for a 50 mg/kg
dose, assuming a linear dose increase (see Supporting
Information). With the limited knowledge of the PK/PD
drivers for the treatment of VL, we considered this to be a
suitable profile for the progression of 4 into a previously
reported mouse efficacy model of VL,6,7,17,18 where it was
dosed twice daily at 50 mg/kg ip in order to further maximize
exposure and give the best chance of achieving proof-of-
concept.
After dosing twice daily for 5 days, a reduction in
parasitemia of 98% was observed (Table 1). This established
the proof-of-concept for this series in VL and, in fact, met our
criteria for a preclinical development candidate.7 Unfortu-
nately, despite reasonable kinetic aqueous solubility, 4 was
poorly soluble in FaSSIF (Fasted State Simulated Intestinal
Fluid)19,20 media and this, alongside a positive result in the
Ames assay,21−23 as reported previously,10 which indicated
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that changes to the pattern of hydrogen bond acceptors
(HBAs) could lead to similar improvements in solubility.
In order to define a set of alternative bi-cycles for synthesis,
we first looked at the SAR from our early hit discovery efforts,
on scaffolds with no 6-substituent (Table 3). On comparing
with initial hit 3, we noted that a scaffold which removed the
HBA in the 8-position (e.g., 14) showed no activity in the
INMAC assay. We therefore focused our scaffold-hopping
efforts on bi-cycles, which contained a HBA in the 8-position,
and then prioritized them based on cChromLogD (Chrom-
LogD24 is the chromatographically determined log D and
cChromLogD is the calculated version of this), cpKa
(calculated using ACD pKa predictor), calculated aqueous
solubility (using Gastroplus v9.7), and synthetic tractability
(based on literature precedent and in-house knowledge).
Without a robust model for activity in the Ames test, this was
not included in the design process, the intention being to
profile compounds in the assay when they demonstrated
suitable efficacy in the VL mouse model to warrant further
study.
A total of 28 alternative scaffolds were evaluated in silico, all
maintaining a HBA in the 8-position (for comparison
purposes, the numbering for the imidazo[1,2-a]pyrimidine
scaffold in Figure 2 is used in this section), plus the correct
orientation of the morpholinyl and phenyl substituents. We
explored HBDs and HBAs in various positions around the bi-
cycle and also examined the effect of reversing the 6,5-fused
system so that morpholine was attached to the five-membered
ring and phenyl to the six-membered ring. Finally, we also
profiled a fused 5,5 bi-cycle.
Table 1. In Vitro and In Vivo Profile of Compound 4
in vitro profile
a b
,
INMAC pEC50
THP-1 cells pEC50
6.2
a b
,
<4.3
150
15
c
aqueous solubility (μM)
a
FaSSIF solubility (μM)
ad
,
CLi mL/min/g
<0.5
e
in vivo profile
T1/2 (h)
iv PK (3 mg/kg)
0.5
135,000
AUC ng·min/mL
Clb mL/min/Kg
Cmax (ng/mL)
Tmax (h)
AUC ng·min/mL
Vdss L/kg
22
1300
po PK (10 mg/kg)
0.5
258,000
0.8
F %
57
98
f
% reduction in parasitemia
a
b
Data reported previously.10 INMAC is the intramacrophage assay
carried out in THP-1 cells with L. donovani amastigotes. Data are the
result of five independent replicates and standard deviations are ≤0.2.
c
d
Aqueous solubility is kinetic solubility measured by CLND. Cli is
e
the mouse liver microsomal intrinsic clearance. Mouse PK studies
dosed at 10 mg/kg po and 3 mg/kg iv. Infected mouse model of VL,
f
dosed at 50 mg/kg ip, b.i.d for 5 days.
potential genotoxicity issues, precluded further development of
this particular compound.
Having achieved the in vivo proof-of-concept, the series
moved into the lead-optimization phase, with an initial aim to
identify a compound with a suitable profile for rodent
toxicology studies. As this would require us to achieve an
exposure significantly higher than the minimum efficacious
dose, we continued to target compounds with pEC50 values >6
and now aimed for FaSSIF solubilities >100 μM and to
maintain Cli < 1 mg/mL/g. We carried out a systematic
exploration of the molecule, initially preparing further
analogues of 4 with variations to the pendant substituents of
the 2-phenylimidazo[1,2-a]pyrimidine core. However, this
failed to identify any better molecules (data not shown).
We next focused our attention on the core phenyl-
substituted bi-cycle. Initially, we investigated changes to the
phenyl ring and, as shown in Table 2, modifications to this part
of the scaffold led to analogues that maintained stability in
mouse liver microsomes and also showed improved solubility
in both aqueous (CLND) and FaSSIF media. Unfortunately,
these changes, such as moving or removing fluorine (5 and 6,
respectively), introducing alternative substituents (e.g., OMe
analogues 7 and 8), or replacing phenyl with six-membered
heterocycles (e.g., pyridine 9, pyrimidines 10 and 12, pyrazine
11, and pyridazine 13), all led to a >10-fold loss of potency.
We noted that similar findings were also reported by Tatipaka
et al.13 and Ferrins et al.14 on the related scaffolds. Analogue 4,
with the fluorine ortho to the bi-cyclic substituent, appeared to
be optimal and our subsequent knowledge of the binding
mode of these compounds allowed us to rationalize this (seen
later). We were interested to note that the changes made did
have a big impact on FaSSIF solubility, with methoxy analogue
8, pyrazine 11, pyridazine 13, and pyrimidines 10 and 12 all
having FaSSIF solubilities well above 100 μM. Also, 10−13, all
had intrinsic clearances of <0.5 mL/min/g. Encouraged by
this, we turned our attention to the core bi-cycle, surmising
As we reported for a previous series, we had focused on
reducing lipophilicity as a key strategy for improving
solubility.7 In that case, we targeted a PFI25 <7 (PFI =
property forecast index, ChromLogD + #Ar). Within the
current series which contained three aromatic rings, this would
equate to a cChromLogD below 4, so we utilized this as a key
property for the designed scaffolds. We also targeted scaffolds
which had a higher predicted solubility than 4 and scaffolds
which increased cpKa. The increased cpKa would indicate
potential for improved solubility, as well as allowing possible
salt formation. We then further prioritized based on the
potential synthetic tractability (based on literature precedent
and in-house synthetic expertise), as with each scaffold
requiring a bespoke synthesis, there would be a limited
chemistry resource to develop completely novel routes.
The initial scaffolds all examined the nature of the atom in
the 1-position of the bi-cycle (S-1 to S-16) (Table 4). Scaffolds
S-1−S-8 all examined the removal of the HBA, where the best
predicted profiles were obtained for S-1, S-4, S-5, and S-6, all
predicted to have cChromLogD below 4 and either higher
cpKa, improved solubility, or both when compared to 3 (Table
4). Scaffolds S-9 and S-10 would demonstrate whether a
hydrogen bond donor (HBD) was acceptable in position 1.
Although S-9 gave a more direct comparison to 3, S-10 proved
to be more synthetically tractable. On this basis, S-1 and S-10
were synthesized with the desired substituents, S-5 was
synthesized as the non-fluorinated analogue, and S-9 as the
phenyl analogue. Although S-4, S-6, and S-11 had good in silico
profiles, the syntheses of S-4 and S-6 were not completed
when it became apparent that the removal of HBA at the 1-
position would lead to an inactive compound, and for S-11,
there would be a possible tautomerization of the five-
membered ring, making any data generated difficult to
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Table 2. Profile of Analogues 4−13, with Substitutions and Nitrogen Insertions on the Central Phenyl Ring
a
INMAC is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes. Data are the result of at least three independent
b
c
replicates and standard deviations are ≤0.4. Aqueous solubility is the kinetic solubility measured by CLND. FaSSIF solubility is the fasted state
simulated intestinal fluid solubility. Cli is the mouse liver microsomal intrinsic clearance. Data reported previously.10 ND means not determined.
d
e
f
Table 3. Initial Modifications of the Bi-cyclic Core
Figure 2. Numbering for the imidazo[1,2-a]pyrimidine scaffold.
a
INMAC is the intramacrophage assay carried out in THP-1 cells
with L. donovani amastigotes. Data are the result of at least three
b
independent replicates and standard deviations are ≤0.4. Cli is the
Scaffolds S-17−S-21 all retained the HBA nitrogen in
mouse liver microsomal intrinsic clearance.
positions 1 and 8 and examined the effect of varying the
heteroatoms in the other positions around the rings. All of
interpret so this scaffold was not synthesized (see Figure 3). S-
12 would further probe the nature of the heteroatom in the 1-
position and had a very good in silico profile. Scaffolds S-13−S-
16 all examined the replacement of the nitrogen in the 1-
position with oxygen. All these compounds showed some
advantage over 3 and so were targeted for synthesis, although
S-15 was synthesized without the fluoro substituent and S-16
was not synthesized due to lack of a tractable route.
them showed some potential advantage over 3 and so were
selected for synthesis (although a tractable synthesis of S-21
was not successfully developed).
Scaffolds S-22−S-27 all examined the reversal of the 6,5-
fused scaffold to a 5,6-fused scaffold. S-22 had a cChromLogD
below 4 and higher predicted pKa compared to 3 and so was
selected for synthesis. While S-23−S-25 all had higher
predicted solubility, they proved to be synthetically challenging
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a
Table 4. Scaffolds Identified by the Medicinal Chemistry Team for In Silico Profiling
a
cChromLogD calculated using GSK predictive software. Colored as green <4 and amber >4. cpKa calculated using ACD labs pKa calculator for the
most basic nitrogen on the bi-cycle. Colored as green if >compound 4 (4.4) and amber if <compound 4. cAq. Sol (pH 7.4) mg/mL is GastroPlus
calculated aqueous solubility at pH 7.4, colored green if >compound 4 and amber if ≤compound 4. Synthesis color scheme: green = desired
compound synthesized, orange = alternative analogue synthesized (e.g., no F or phenyl instead of morpholinyl), red = not synthesized (no
successful route or abandoned due to SAR learnings).
Figure 3. Tautomerization of scaffold S-11.
and we only developed a tractable synthetic route to S-24 with
phenyl, rather than the morpholine substituent. S-26, which
replaced the nitrogen in the five-membered ring with oxygen,
had a poor in silico profile and so was not synthesized.
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Table 5. Effect of Scaffold Hops on Activity and Physicochemical Properties
a
b
Data reported previously for 1 and 24.10 INMAC is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes. Data are
c
the result of at least three independent replicates and standard deviations are ≤0.4. Aqueous solubility is the kinetic solubility measured by CLND.
d
e
f
FaSSIF solubility is the fasted state simulated intestinal fluid solubility. Cli is the mouse liver microsomal intrinsic clearance. ND means not
determined.
Although S-27 had predicted improved solubility, it was not
synthesized due to our understanding that the N-methyl group
would not be tolerated. Finally, we profiled some 5,5-bi-cycles,
as exemplified by S-28. The only advantage this might confer
would be a cChromLogD below 4, as the predicted solubility
was lower than 3, and the core did not contain the basic
nitrogen. Because of this, and also because the vectors of the
substituents would be very different to 3, as well as a lack of
literature precedent for synthesis, we elected not to synthesize
this analogue.
This led to a set of 10 scaffolds synthesized with the desired
substituents, plus a further 4 which were synthesized with
alternative substituents (Table 5). From this, we gained some
key SAR learnings. Scaffolds S-1 (compound 15) and S-5
(compound 16) showed that removing the HBA nitrogen from
position 1 led to inactive compounds in the INMAC assay.
Scaffolds S-9 and S-10 showed that a HBD in position 1 was
not tolerated (compounds 17 and 18, respectively). Scaffold S-
12 (compound 19), with N-Me in the 1-position, was inactive.
Scaffolds S-13−S-15 (compounds 20−22) which replaced the
HBD nitrogen with oxygen showed a range of potencies,
although all had poor aqueous solubility compared to 3, with
no improvement in FaSSIF solubility either. Scaffolds S-17−S-
20 (compounds 23−26), which retained the HBD nitrogens in
positions 1 and 8, all maintained good activity, with pEC50
values of 6 or above. Scaffolds S-17, S-18, and S-19
(compounds 23, 24, and 25 respectively) all showed a much
improved FaSSIF solubility compared to 3, with 24 and 25
having overall very similar profiles. The S-19 scaffold has
recently been published by Novartis.8,26 Although S-20
(compound 26) did have good potency, it showed no
improvement in FaSSIF solubility compared to 3. The
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Figure 4. Summary of the SAR around the core scaffold.
compounds with a “reversed” bi-cycle (S-22 and S-24,
compounds 1 and 27, respectively) proved to be very
interesting. Compound 27 was only synthesized as the phenyl
analogue; although this showed similar potency to 3, it had
very poor FaSSIF solubility. On the other hand, 1 was only
slightly less potent than 3 and had the highest FaSSIF
solubility of all the scaffold hop compounds. Based on these
findings, compounds 23 (S-17) and 1 (S-22) were investigated
further (23 was selected over 24 as it proved much more
straightforward to scale-up the synthesis to get suitable
quantities for in vivo studies). The progression of 23 was
subsequently stopped as the aniline resulting from the
hydrolysis of the urea was positive in the Ames assay,10
indicating a potential genotoxicity issue. This led us to focus
on 1 as the most promising scaffold for further development;
although it was slightly less potent than 23, it was the
compound with the best solubility profile and had high stability
in mouse liver microsomes.
To summarize the SAR, a HBD was essential in positions 8
(N) and 1 (N or O), whereas a HBD in the 7-position was
detrimental to activity (Figure 4). A 6-substituent, particularly
morpholine, was essential for activity and metabolic stability.10
In the other positions around the ring (e.g., 3, 4, 5, 7, and 9)
heteroatoms were tolerated but not essential to activity.
Regarding the central phenyl ring, 4-fluoro boosted activity
and 1-urea gave a better balance of potency, solubility, and
metabolic stability than the corresponding amides (data
reported previously).10 Finally, it proved possible to reverse
the central bi-cycle, a change which had a small detrimental
effect on potency that was compensated for by the improve-
ment in FaSSIF solubility.
A key aim of the scaffold-hopping exercise was to identify
compounds with improved solubility, in both aqueous and
FaSSIF media. Of the 10 scaffolds synthesized with the same
substituents as 4, only three showed an improvement in
solubility in both media (24, 25, and 1), with 23 showing an
improvement only in FaSSIF solubility (S-18, S-19, S-22, and
S-17, respectively). Compounds 23, 24, and 25 all had better
in silico predictions for solubility than for compound 4, whereas
1 was predicted to be similar. However, compound 1 had
much better solubility than compound 4, and of these four
compounds, it had the highest FaSSIF solubility. Interestingly,
scaffold S-22 was predicted to have a much higher pKa than S-
17 and S-18, indicating that for compound 1 a significantly
higher proportion is likely to be protonated at physiological
pH. Therefore, while the in silico profiling helped to triage the
number of scaffolds for synthesis, it was clear that a large
number of scaffolds needed to be synthesized in order to
identify the optimal scaffolds for progression.
Because reversing the imidazo[1,2-a]pyrimidine scaffold
(compounds 1 and 27) was liable to orient the pendant
amine and urea substituents slightly differently, we further
explored the SAR around the 3-position of the bi-cycle, where
morpholine was substituted (Table 6). The replacement of
morpholine of 4 with phenyl (28) gave an expected increase in
potency alongside a decrease in solubility, while switching to
pyridyl (29) maintained the improved potency alongside an
increase in FaSSIF solubility compared to 28. However, the Cli
was above our targeted level of <1 mL/min/g for a preclinical
candidate. We were interested in exploring saturated
substituents; isopropyl 30 showed a similar potency to
morpholine but with poorer FaSSIF solubility, and isobutyl
31 showed a small improvement in potency but with poorer
metabolic stability. In order to improve solubility, we explored
the introduction of a basic center with methylene-linked
morpholine 32 and directly linked morpholine 33. While this
was successful in delivering compounds with improved FaSSIF
solubility compared to 1, it unfortunately led to a loss of
potency.
We also further explored N-linked amines, where piperidine
34 showed good potency but with poor FaSSIF solubility.
Piperazines 35−37 were inactive, although 36 and 37 did show
enhanced FaSSIF solubility. As we had seen success with
methylmorpholines in other series,6,7 we synthesized rac-2-
methyl morpholine 38 and the enantiomeric 3-methylmorpho-
lines 39 and 40. All of these compounds showed similar
potency to 1 and improved FaSSIF solubility, although all the
three were less metabolically stable. Likewise, dimethylmor-
pholines 41, 42, and 43 were synthesized, all showing similar
potency to 1 but with poorer metabolic stability, although 42
and 43 did show improved FaSSIF solubility. Spiro analogue
44 again was less metabolically stable than 1, and secondary
amines 45 and 46 showed a drop in potency compared to 1.
From all the in vitro profiling, none of the changes to the
morpholine ring led to compounds with an improved overall
profile; therefore, 1 was identified as the most suitable
compound for further in vitro and in vivo studies. As reported
previously,10 a PK study of 1, dosed at 25 mg/kg po, supported
its progression into the infected mouse model, where it showed
a candidate level efficacy against VL at this dose. When dosed
orally twice a day for 10 days, 1 had an ED90 of 16 mg/kg and
ED99 of 30 mg/kg. The compound had suitable PK for oral
delivery and a good predicted safety margin of at least 37-fold
when dosed in rats at 300 mg/kg. Critically, unlike compounds
4 and 23, both 1 and the aniline derivative potentially released
by hydrolysis of its urea were negative in the Ames assay, as
reported previously.10 Alongside the previously reported safety
profiling, 1 was also screened against a representative set of
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Table 6. Effect of Modifications or Replacements to the Morpholine Group on Activity and Physicochemical Properties
a
INMAC is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes. Data are the result of at least three independent
b
c
replicates and standard deviations are ≤0.4. Aq. solubility is the kinetic solubility measured by CLND. FaSSIF solubility is the fasted state
simulated intestinal fluid solubility. Cli is the mouse liver microsomal intrinsic clearance. Data reported previously.10 ND means not determined.
d
e
f
three kinases (LCK, PI3Kγ, and AuroraB) and was inactive
against them all (pIC50 <4.5). The compound is now being
advanced for the first time in human studies for VL.
Modeling. In parallel to these phenotypic optimization
studies, attempts to identify the target of these series were
carried out as previously reported.10 It was found that these
compounds act through the inhibition of the chymotrypsin-like
activity catalyzed by the β5 subunit of the L. donovani
proteasome, demonstrating good selectivity over the human
enzyme. A high-resolution cryo-EM structure of compound 1
bound to the Leishmania tarentolae proteasome10 revealed a
binding site that lies between the β4 and β5 subunits (Figure
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Figure 5. Compound 1 bound to the β4 (light blue) and β5 (white) subunits of the L. tarentolae 20S structure. PDB: 6QM7.
5) and exploits an induced cavity that is lined on one side by
β4 residues that are divergent between humans and
kinetoplastid protozoan. The cryo-EM structure of a related
compound in the complex with the Leishmania tarentolae
proteasome has also been recently published by Novartis.9
Description of the Binding Mode of Compound 1. A
homology model of compound 1 bound to the L. donovani 20S
proteasome β4 and β5 subunits was generated using the L.
tarentolae 20S proteasome cryo-EM structure as a template.
Compound 1 binds at the interface between the β4 and β5
subunits close to the catalytic Thr100 residue (Figure 6A),
which is the first residue of the β5 subunit. The pyrrolidine
carboxamide sits in the most buried part of the binding site, a
mainly hydrophobic cavity important for selectivity against the
human orthologue. The cavity is defined by Ile27, Ile29, and
Phe24 from the β4 subunit and by Phe225, Val227, and
Thr235 from the β5 subunit. The urea carbonyl oxygen is
hydrogen bonded to the Gly228 backbone nitrogen, and the
urea nitrogen is hydrogen bonded to the hydroxyl of the
Tyr212 side chain. The phenyl ring is placed on top of Gly197
of the terminal part of the β-strand 7 of the β5 subunit with the
fluorine substituent facing the Ser195 side chain hydroxyl. The
“top edge” (see Figure 2) of the imidazopyrimidine bi-cyclic
system is mainly solvent exposed with the six-membered ring
sitting on top of Gly146 and the sp2 nitrogen of the five-
membered ring interacting with the donor NH group of
Ser229. The nitrogen on the “bottom edge” of the pyrimidine
ring does not make any specific interactions; however, it
contributes to the charge delocalization, resulting in a favorable
charge interaction with Thr100 (see later). The morpholine
group is largely solvent exposed and is directed toward the β
hairpin motif critical for the recognition of bortezomib (β
strands 3 and 4 of the subunit β5), establishing a hydrogen
bond with the Gly122 backbone NH (Figure 6A).
(Bortezomib is a clinically used inhibitor of the human
proteasome.)
parameterized in molecular mechanics-based force fields
(e.g., CH−π, halogen−π, cation−π interactions, and non-
classical hydrogen bonds) resulting in a more accurate binding
energy. To overcome the high computational costs associated
with the QM calculation, the system is fragmented into smaller
parts and QM calculations are performed on each individual
fragment pair to derive pairwise interaction energy (PIE)
between each of the fragments and the ligand. By combining
the PIE of the ligand and the fragments, it is possible to derive
the total interaction energy of the ligands with the target. The
decomposition analysis of the PIE (PIEDA)28 allows the
derivation of four different energy terms (electrostatic, charge
transfer, dispersion, and exchange-repulsion) that provide a
deeper residue-by-residue insight into the nature of the ligand
interaction with the target. The electrostatic and charge
transfer terms are dominant in H-bonds, polar (favorable and
unfavorable) interactions, and salt bridges. The dispersion
term is more prominent in hydrophobic and van der Waals
interactions. The exchange-repulsion term describes the steric-
repulsion between electrons of different atoms accounting for
steric clashes.
The FMO-PIEDA analysis shows that, out of the 40 residues
in a 5 Å range from the ligand, there are 13 contributing
significantly (PIE < −3 kcal/mol) to the overall calculated
binding energy (Figure 6B,C). The initial work focused on
analyzing the H-bonding and hydrophobic interactions
revealed by the cryo-EM structure; the contribution to the
binding energy from the residues directly involved in hydrogen
bonding with the ligand was confirmed. The Gly228 hydrogen
bond to the urea carbonyl is the strongest H-bonding
interaction with a calculated electrostatic energy term of
−15.9 kcal/mol (PIE −14.3 kcal/mol).
In the cryo-EM structure of the L. tarentolae structure, a
CH2 group next to morpholine oxygen is adjacent to NH of
Gly 122, which is a part of a semi-flexible loop. However, in the
model of the L. donovani enzyme, the morpholine twists
slightly, allowing N−H of Gly 122 to establish a strong
hydrogen bond with the morpholino oxygen atom (PIE −9.0
kcal/mol, electrostatic term −9.0 kcal/mol). For Tyr212, the
PIE energy is −8.7 kcal/mol which is the result of both the
hydrogen bonding (electrostatic) with the urea NH and the
favorable hydrophobic interaction with the pyrrolidine ring
(dispersion). The FMO-PIEDA result also shows that the
Computational Analysis of Binding Interactions. The
fragment molecular orbital method (FMO-MP2)27 was used to
calculate the interaction energy between compound 1 and the
proteasome and to gain a deeper understanding of the
molecular recognition event. Being a quantum-mechanical
(QM)-based method, FMO-MP2 is capable of detecting and
accounting for nonclassical interactions that are poorly
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Figure 6. (A) View of compound 1 binding in the L. donovani 20S proteasome model β4 and β5 subunits. Shown in stick form are the 12 residues
that have a FMO-PIE <−3 kcal/mol; colored red if FMO-PIE <−30 kcal/mol, orange if between −30 and −10 kcal/mol, and yellow if FMO-PIE
between −10 and −3 kcal/mol. (B) FMO-PIE decomposition analysis results showing, for each residue within 5 Å, the contribution of the four
energy terms: electrostatic (yellow bar), exchange-repulsion (green bar), charge transfer (red bar), and dispersion (blue bar). (C) FMO-PIE
generated contribution to compound 1 binding energy per fragment residues within 5 Å from the ligand.
Ser229 interaction (PIE −6.26 kcal/mol) is mainly due to the
favorable interaction between the β carbon atom of the side
chain (dispersive interaction) with the heterocyclic scaffold of
the ligand rather than the hydrogen bonding of the nitrogen in
the 5-membered ring of imidazopyrimidine with the backbone
NH. This is consistent with the poor geometry observed for
the hydrogen bond present in both the L. tarentolae cryo-EM
structure and the L. donovani model. The favorable
contribution of hydrophobic contacts is also significant for
Gly197 (−4.2 kcal/mol) and Gly146 (−6.8 kcal/mol) as both
residues are in contact with the central phenyl ring. The
contact between the pyrrolidine carboxamide and the Phe24
side chain has a contribution of −9.2 kcal/mol.
Importantly, in addition to the interactions that could be
identified from a visual inspection of the cryo-EM structure,
the FMO-PIE analysis highlighted other residues that establish
nonintuitive interactions and are important in the molecular
recognition event. Thr100 (PIE −30.3 kcal/mol), Asp214, and
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Figure 7. Molecular ESP maps for some of the studied analogues. Protein surfaces were generated using the APBS plugin for PyMol and colored by
̈
calculated charges (red −5 kbT/ec, blue +5 kbT/ec). The ligand surfaces were generated using Jaguar in Schrodinger and colored by the
electrostatic potential (red −80 kcal/mol, blue +75 kcal/mol).
Figure 8. FMO-PIE generated contribution to binding energy in kcal/mol per fragment residues within 5 Å from the ligand calculated for
compound 1 analogues. Red indicates favorable, whereas green indicates unfavorable interaction energies.
Asp215 (PIE −16.4 and −16.0 kcal/mol, respectively) are
indicated as the residues with the strongest interaction with the
ligand. These are long-range electrostatic interactions between
the protein and the unevenly distributed electrons of the
ligand. The analysis of the electrostatic surface potential (ESP)
of the protein binding site highlighted a positively charged
patch consisting of the positively charged terminal Thr100 and
the backbone amide of Ser229. A charge−dipole interaction is
established between this positively charged patch of the protein
with the negatively charged sp2 nitrogen atoms of the
imidazopyrimidine scaffold in positions 1 and 8 on the bottom
edge (Figures 2 and 7). A similar effect is demonstrated by the
presence of the fluorine atom on the phenyl ring, which
increases potency by approximately 10-fold (see compounds 4
and 6). As previously discussed,10 the ESP of the ligand shows
that the presence of the fluorine atom on the phenyl ring
causes an accumulation of the positive charge on positions 3
and 4 of the fluorophenyl ring giving rise to another strong
dipole−charge interaction with the negatively charged Asp214
and Asp215. This effect of fluorine appears to be more
important to binding than its interaction with Ser195. Val227
also provides a significant contribution to binding energy (PIE
−12.7 kcal/mol) with the decomposition analysis indicating an
equal contribution from the dispersive term (related to the
hydrophobic side chain) and electrostatic term (related to the
positively charged patch localized on the pyrrolidine ringsee
Figure 7). Therefore, FMO-PIE analysis showed that the
binding of compound 1 to the proteasome is governed by a
complex mixture of specific hydrogen bonds, stacking
interactions, optimal electrostatic complementarity, and long-
range electrostatic interactions between the protein and the
ligand.
Computational Analysis of Structure Activity Rela-
tionships. To analyze the effect of the imidazopyrimidine
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scaffold replacement in the series related to compound 1, all
the synthesized scaffold hop compounds (Table 5) were
modeled in the L. donovani 20S proteasome model and studied
by FMO-PIEDA and ESP. The compounds tended to be either
active (pEC50 5.8−7.3) or inactive (pEC50 < 4.3). Derivatives
with a fluorine substituent on the phenyl ring ortho- to the bi-
cycle were consistently more active than the hydrogen
analogues. We and others have noted a correlation of activity
between the inhibition of proteasome and activity against
axenic Leishmania parasites.8,10 While there could be subtleties
in INMAC activity due to physicochemical properties of
compounds, this data allows an interpretation of the SAR.
The modeling data generated confirmed the important role
that the electrostatic complementarity between the ligands and
the proteasome binding site plays in the optimization of the
interaction. The ESP maps (Figure 7 and Supporting
Information), clearly show that even if the hydrogen acceptor
interacting with the Ser229 backbone nitrogen is conserved,
where there is a lack of the negative charge accumulation on
the bottom edge (positions 1 and 8) of the imidazopyrimidine
scaffold (as observed for compounds 15, 18, and 19), these
compounds are inactive (Table 5). This is further supported by
the FMO-PIEDA analysis results (Figure 8), indicating that
one of the strongest interactions that compound 1 establishes
is with Thr100, an interaction primarily electrostatic in nature,
which contributes with −30.3 kcal/mol. Compound 27 (Table
5) is also characterized by a strong interaction between Thr100
(FMO-PIE −30.3 kcal/mol) and the negatively charged
bottom face (positions 1 and 8) of the imidazotriazine
scaffold. The scaffold of compound 27 also seems to enhance
the partial positive charge on 3- and 4-positions of the phenyl
ring and strengthen the interaction with Asp214 and Asp215
(see the ESP, Supporting Information).
The “reversed” 5−6 bi-cyclic system compounds 1 and 27
not only are characterized by a particularly favorable
accumulation of the negative charge on position 1 of the bi-
cyclic scaffold but also by a slightly different placement of the
substituent in position 3 in relation to the 6−5 bi-cyclic
systems of the other analogues. An inspection of the bound
conformations shows that the vector of the substitution in the
3-position is similar between the 5−6 and 6−5 bi-cycles
(Figure 9), but in the 5−6 system, the substituent is about half
a bond longer than in the 6−5 system. This results in the
morpholine ring being pushed closer to Gly122 on the β
strands 3 and 4 of the subunit β5 and might explain the lower
potency of the compounds with a “reversed” scaffold. Docking
studies suggest that in order to avoid a steric clash with Gly122
and the beta-hairpin involved in bortezomib binding,
compound 24 appears to adopt a higher energy conformation
with the morpholine ring almost perpendicular to the bi-cyclic
scaffold. Due to this steric requirement, and together with an
unfavorable electrostatic distribution on the bi-cyclic scaffold,
resulting in the lack of a partial negative charge interacting with
Thr100 (Supporting Information), compounds 16 and 17,
where morpholine is replaced by a phenyl ring, failed to dock
and are reported as inactive.
Synthesis. To explore SAR around the central phenyl ring
in the 2-phenylimidazo[1,2-a]pyrimidin-6-morpholine sub-
series (e.g., compounds 5−13), the route shown in Scheme
1 is utilized. A suitably substituted 3-nitroacetophenone was
brominated to give 47a,c,d and then cyclized with 48 to give
49a,c,d. The nitro reduction gave 50a−i and subsequent urea
formation with pyrrolidine-1-carbonyl chloride yielded 5, 7,
and 8. Alternatively, a relevant aminoheterocycle was treated
with pyrrolidine-1-carbonyl chloride to give ureas 51b,e−i,
which were then brominated to give 52b,e−i. Again,
cyclization with 48 gave 6 and 9−13.
All the different cores required the bespoke synthesis, as
detailed in Schemes 2− 9 below (the synthesis of compounds
1, 3, 4, and 23 was described elsewhere10).
Compounds 15 and 27 are prepared, as outlined in Scheme
2. Compound 53 could be brominated with trimethyl(phenyl)-
ammonium tribromide to yield either monobromo 54 or
dibromo 55. The thermal cyclization of 54 with pyridazine 56
led to 15, whereas 55 could be cyclized with 1-aminoguanidine
to give 57 which was further cyclized with 2-bromo-1,1-
diethoxyethane to 58. Bromination to give 59 was followed by
Suzuki coupling with benzeneboronic acid to yield 27.
Compound 16 is synthesized according to Scheme 3, where
commercially available 3-(3-nitrophenyl)-1H-pyrazol-5-amine
was cyclized with 2-bromopropanedial to give 60, which with
subsequent Suzuki coupling with benzeneboronic acid gave 61.
The nitro group was then reduced, with subsequent urea
formation to give 16.
The synthesis of compounds 17 and 22 started from 3-
ethynylaniline, as shown in Scheme 4. The urea formation to
give 62 was followed by Sonagashira coupling with 3-bromo-5-
phenylpyridin-2-amine to give 63, which underwent base-
catalyzed cyclization to give 17. Alternatively, cyclization of 62
with 5-bromo-6-oxo-1,6-dihydropyridazin-3-yl triflate 64 gave
65, with subsequent thermal displacement of the triflate with
morpholine yielding 22.
The synthesis of compounds 18, 20, 21, and 26 all started
from 2-ethynyl-1-fluoro-4-nitrobenzene, as outlined in Scheme
5. Initially, 66 was synthesized from 2-ethynyl-1-fluoro-4-
nitrobenzene by nitroreduction and subsequent urea for-
mation. Compound 18 was then prepared via Sonagashira
coupling of 66 with 3,5-dibromopyrazin-2-amine to give 67,
followed by a base-catalyzed cyclization to give 6,5-bi-cycle 68.
Buchwald−Hartwig cross-coupling with morpholine then led
to 18. Alternatively, acetylene 66 could be cyclized with 5-
bromo-3-iodopyridin-2-one to give 69, which was converted to
20 via Buchwald−Hartwig cross-coupling. To synthesize 21,
Sonagashira coupling of 66 with 1-PMB protected 3,5-
dichloropyrazin-2(1H)-one yielded 70, which could be
cyclized to 71 in the presence of silver nitrate and TFA.
Again, Buchwald−Hartwig coupling facilitated the conversion
of 71 to 21. Finally, to synthesize 26, 2-ethynyl-1-fluoro-4-
nitrobenzene was treated with LDA then ethyl chloroformate
to give 72, with base-catalyzed cyclization with 1-amino-4-
Figure 9. Superimposition of a 6,5-bi-cyclic system (compound 1,
shown in green) with a 5,6-bi-cyclic system (compound 24, shown in
orange).
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a
Scheme 1. Synthesis of Compounds 5−13
a
Reagents and Conditions: (a) trimethyl(phenyl)ammonium tribromide, THF, RT, 18 h; (b) MeCN, 60 °C, 4 d, 43% over two steps; (c) SnCl2,
EtOH, reflux, 2 d, quant.; and (d) pyrrolidine-1-carbonyl chloride, DMAP, pyridine, 40 °C, 2 d, 9−36%.
a
Scheme 2. Synthesis of Compounds 15 and 27
a
Reagents and Conditions: (a) trimethyl(phenyl)ammonium tribromide (1 equiv.), THF, RT, 18 h, 73%; (b) MeCN, 60 °C, 2 d, 43%; (c)
trimethyl(phenyl)ammonium tribromide (1 equiv), THF, 60 °C, 18 h, 46%; (d) morpholine and THF, 35 °C, 18 h, then aminoguanidine
bicarbonate, acetic acid, MeOH, 60 °C, 18 h, 26%; (e) 2-bromo-1,1-diethoxy-ethane, HBr, water, 90 °C, 30 min, 36%; (f) Br2, NaOAc, acetic acid,
RT, 1 h, 50%; (g) Pd(PPh3)4, sodium carbonate, DMF, 80 °C, 18 h, 38%.
a
Scheme 3. Synthesis of Compound 16
a
Reagents and Conditions: (a) 2-bromopropanedial, AcOH, EtOH, 75 °C, 30 min, 66%; (b) benzeneboronic acid, Pd(PPh3)4, KOAc, dioxane, 3 h,
64%; (c) Fe, NH4Cl, EtOH, water, 75 °C, 2 h; (d) pyrrolidine-1-carbonyl chloride, DMAP, pyridine, DCM, 70 °C, 48 h, 62% over two steps.
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a
Scheme 4. Synthesis of Compounds 17 and 22
a
Reagents and Conditions: (a) pyrrolidine-1-carbonyl chloride, DMAP, pyridine, DCM, 50 °C, 18 h, 79%; (b) PdCl2(PPh3)2, CuI, NEt3, DMF, 80
°C, 18 h, 46%; (c) KOtBu, NMP, 75 °C, 18 h, 57%; (d) PdCl2(PPh3)2, CuI, NEt3, MeCN, RT, 6 h; (e) morpholine, 50 °C, 18 h, 12% over two
steps.
morpholinopyridazin-1-ium iodide 73 leading to 74. Subse-
quent decarboxylation to give 75 was followed by nitro
reduction and urea formation, to yield 26.
The preparation of 19 is reported in Scheme 6. First, 5-
bromo-1H-pyrrolo[2,3-b]pyridine was methylated on the
pyrrole nitrogen to give 76, with Buchwald−Hartwig coupling
leading to 77. Alongside this, 4-fluoro-3-iodoaniline was
treated with pyrrolidine-1-carbonyl chloride to give 78,
which underwent a palladium-catalyzed direct C-2 arylation
with 77 to give 19.
Alternatively, to synthesize C-linked morpholine analogues
32 and 33, the routes shown in Scheme 10 were utilized.
Intermediate 90 could undergo a Mannich reaction to give 91,
with nitro reduction and subsequent urea formation giving 32.
A Vilsmeier−Haack reaction on 90 led to formylated 92. Its
treatment with a SnAP reagent30 led to the carbon-linked
morpholine substituent 93, which could be Boc-protected to
give 94, the nitro group reduced, and converted to pyrrolidinyl
urea 95, with Boc deprotection and subsequent methylation
giving 33.
The 3-amino analogues were synthesized according to
Schemes 11 and 12. Where applicable, the previously
published route for compound 1 was utilized (Scheme 11),
whereby the relevant amine was condensed with glyoxal and
benzotriazole to give 1,2-bis electrophiles 96a-i, which were
subsequently cyclized with 87 under Lewis acid-promoted
conditions to give compounds 1, 34−38, and 41−43. In cases
where this was unsuccessful, due to the relevant 96 not
forming, the alternative route31 in Scheme 12 was utilized,
whereby 2-chloropyrimidine analogue 97 was treated with
relevant glycinamide 98a−e to give 99a−e which was cyclized
to 100a−e. Cbz-Deprotection, followed by generation of the
urea thus led to compounds 39, 40, and 44−46.
Compound 24 (Scheme 7) was prepared starting from 3-
amino-5-morpholinopyridazine which was aminated on the 2-
position to give diamino compound 79. Alongside this, ethyl 5-
amino-2-fluorobenzoate was treated with pyrrolidine-1-carbon-
yl chloride, with subsequent ester hydrolysis giving 80, which
was converted to acid chloride 81 by treatment with sulfonyl
chloride. Compounds 79 and 81 were then cyclized to give 24.
Compound 25 was synthesized according to Scheme 8,
whereby 2-chloro-5-bromopyrimidine was treated with hydra-
zine to give 82, which was condensed with 2-fluoro-5-
nitrobenzaldehyde to give 83 and then cyclized to 84. Thermal
rearrangement led to 85, with nitroreduction giving 86. This
was cross-coupled with morpholine and then treated with
pyrrolidine-1-carbonyl chloride to give 25.
To explore the SAR around the 3-position of the 6-
phenylimidazo[1,2-a]pyrimidine scaffold, two approaches were
taken; either an initial cyclization, followed by functionalization
of the 3-position of the bi-cycle or cyclization of a precursor
with the 3-substituent in place. The synthesis of 28 was
previously described,29 and a similar route was used to
synthesize the pyridyl analogue 29, as shown in Scheme 9,
where the previously reported 2-aminopyrimidine 87 was
cyclized with 2-bromo-1,1-dimethoxyethane to give 88, which
could be brominated to 89.10 The Suzuki coupling of 89 with
2-pyridineboronic acid then led to 29. Alternatively, to access
alkyl substitutions, 87 could be cyclized directly with a suitable
α-bromoaldehyde to give 30 and 31.
CONCLUSIONS
■
2-Phenylimidazo[1,2-a]pyrimidine 3 was a suitable starting
point for a phenotypic lead-optimization program for VL.
Changes to the central phenyl ring demonstrated that it was
possible to significantly improve FaSSIF solubility but failed to
yield compounds with a suitable balance of potency, metabolic
stability, and FaSSIF solubility; we, therefore, embarked on a
scaffold-hopping exercise. A round of design and synthesis led
to a set of 14 compounds with different cores, giving us an
understanding of the requirements for potency, where HBA’s
at positions 1 and 8 of the bi-cycle were critical for activity.
While the scaffolds, which fulfilled these requirements, notably
20−26 and the “reversed” scaffolds 1 and 27, all met our target
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a
Scheme 5. Synthesis of Compounds 18, 20, 21, and 26
a
Reagents and Conditions: (a) (i) Fe, NH4Cl, EtOH, water, 75 °C, 2 h. (ii) pyrrolidine-1-carbonyl chloride, DMAP, pyridine, DCM, 50 °C, 18 h,
78%; (b) PdCl2(PPh3)2, CuI, NEt3, DMF, RT, 30 min, 23%; (c) KOtBu, NMP, 75 °C, 2 h, 86%; (d) morpholine, Pd2dba3, Xphos, NaOtBu, DMF,
110 °C, 2 h, 26%; (e) CuI, PdCl2(PPh3)4, NEt3, DMF, 50 °C, 16 h, 69%; (f) morpholine, RuPhos, Pd2(dba)3, KHMDS, 1,3-dioxane, 90 °C, 15%;
(g) CuI, PdCl2(PPh3)2, NEt3, DMF, 80 °C, 2 h, 81%; (h) Silver nitrate, TFA, DCM, RT, 0.5 h, 89%; (i) morpholine, Pd2dba3, Xphos, NaOtBu,
DMF, 100 °C, 1 h, 33%; (j) LDA, ethyl chloroformate, THF, −78 °C, 2 h, 91%; (k) DBU, MeCN, RT, 18 h, 26%; (l) HBr, 120 °C, 1 h, 26%; (m)
(i) iron, NH4Cl, EtOH, water, 80 °C, 4 h. (ii) Pyrrolidine-1-carbonyl chloride, DMAP, pyridine, DCM, 50 °C, 18 h, 24% over three steps.
potency in the INMAC assay, the solubilities, in both aqueous
and FaSSIF media, were highly variable in ways that were
difficult to predict.
was selected as a preclinical candidate for VL, as previously
reported.10
Modeling studies were carried out using the cryoEM L.
tarentolae proteasome structure that we had previously
reported. This was used to generate a model of the L.
donovani proteasome. The structure was not available until
after the chemistry program had finished. By performing a pair
interaction energy decomposition analysis (PIEDA) using the
fragment molecular orbital method (FMO), we were able to
predict which are the important interactions between the
protein and ligand on a residue by residue basis. Important
interactions were with Thr100, Gly122, Asp214, Asp215,
Val227, and Gly228. The presence of a negative charge on the
From this, compounds 1 and 23 were identified as the most
promising for progression, although the development of 23 was
halted due to a genotoxicity issue. Attempts were made to
optimize the morpholine substituent in compound 1 with a
variety of different analogues and replacements. While some of
these showed greater potency, this came at the cost of
solubility and/or microsomal stability. Compound 1 was
selected for further profiling, showing that neither the parent,
nor the aniline potentially released by the hydrolysis of the
urea were positive in the Ames assay. After safety profiling, 1
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a
Scheme 6. Synthesis of Compound 19
a
Reagents and conditions: (a) NaH, MeI, THF, 0 °C, 5 h, 70%; (b) morpholine, NaOtBu, Pd2(dba)3, xantphos, toluene, 80 °C, 18 h, 48%; (c)
DMAP, pyridine, DCM, 50 °C, 18 h, 82%; (d) Pd(OAc)2, 2-nitrobenzoic acid, Ag2O, DMF, 90 °C, 18 h, 2%.
a
Scheme 7. Synthesis of Compound 24
a
Reagents and conditions: (a) O-(mesitylsulfonyl)hydroxylamine, DCM, MeOH, 0 °C, 10 min; (b) (i) pyrrolidine-1-carbonyl chloride, DMAP,
pyridine, DCM, 60 °C, 3 h; (ii) NaOH, water, MeOH, RT, 18 h, 64% over two steps; (c) SOCl2, DCM, 50 °C, 1 h; (d) DIPEA, MeCN, 80 °C, 24
h, 13% over two steps.
a
Scheme 8. Synthesis of Compound 25
a
Reagents and conditions: (a) hydrazine hydrate, MeOH, 80 °C, 18 h, 99%; (b) 2-fluoro-5-nitrobenzaldehyde, EtOH, RT, 18 h, 93%; (c)
iodobenzene diacetate, DCM, RT, 18 h, 87%; (d) formic acid, reflux, 5 h, 90%; (e) iron, NH4Cl, EtOH, THF, water, 75 °C, 18 h, 79%; (f) (i)
morpholine, 100 °C, 1 h. (ii) pyrrolidine-1-carbonyl chloride, DMAP, pyridine, 50 °C, 18 h, 29%.
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a
Scheme 9. Synthesis of Compound 29−31
a
Reagents and conditions: (a) HBr, EtOH, 80 °C, 9 h; (b) Br2, NaOAc, MeOH, RT, 0.5 h, 34% over two steps; (c) 2-pyridineboronic acid,
Pd(dppf)Cl2·CH2Cl2, K2CO3, 1,4-dioxane, water, 100 °C, 10 h, 10%; (d) EtOH, reflux, 4 h, 25−59%.
a
Scheme 10. Synthesis of Compounds 32 and 33
a
Reagents and conditions: (a) paraformaldehyde, morpholine, acetic acid, 50 °C, 18 h; (b) (i) iron, NH4Cl, EtOH, water, 80 °C, 18 h; (ii)
pyrrolidine-1-carbonyl chloride, DMAP, pyridine, DCM, 50 °C, 18 h, 7% over three steps; (c) POCl3, DMF, 80 °C, 20 h; (d) 2-
[(tributylstannyl)methoxy]ethanamine (SnAP-M), DCM, then 2,6-lutidine, hexafluoro-2-propanol, Cu(OTf)2, 50 °C, 3 h, 50% over two steps; (e)
Boc2O, MeOH, 50 °C, 3 h, 62%; (f) (i) iron, NH4Cl, EtOH, water, 75 °C, 3 h (ii) pyrrolidine-1-carbonyl chloride, DMAP, pyridine, DCM, 50 °C,
20 h, 36%; (g) (i) TFA DCM, RT, 1 h; (ii) paraformaldehyde, acetic acid, THF, RT, 4 h, then sodium triacetoxyborohydride, RT, 24 h, 28%.
bottom edge of the bi-cycle (positions 1 and 8, Figure 2) was
important for the interaction with Thr100 and positive charge
on positions 3 and 4 for interaction with Asp214 and Asp215.
The relative importance of the protein−ligand interactions
would not have been identified unless such a sophisticated
analysis had been carried out. We also analyzed the ESP of the
ligands. When combining this with the FMO analysis, we were
able to rationalize the SAR that we have seen. We suggest that
this approach could be used going forward in predicting
whether modifications to compounds are likely to improve
binding.
This project demonstrates the utility of Cryo-EM co-
structures for rationalizing protein−ligand interactions. In this
case, we were able to obtain structures of sufficient resolution
to be able to understand in a detailed manner the protein−
ligand interactions. As well as being able to rationalize the
SAR, as we have previously reported, we were able to use the
structural information to rationalize the selectivity of our
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a
Scheme 11. Synthesis of Compounds 1, 34−38, and 41−43
a
Reagents and conditions: (a) EtOH, RT, 12 h, 83%; (b) ZnBr2, DCM, reflux, 12 h, 5−46%.
a
Scheme 12. Synthesis of Compounds 39, 40, and 44−46
a
Reagents and conditions: (a) DIPEA, 1,4-dioxane, 120 °C, 18 h, 56%; (b) POCl3, 80 °C, 1 h, 67%; (c) (i) Pd/C, H2, MeOH, RT, 18 h; (ii) CDI,
DIPEA, DCM, 18 h, then pyrrolidine, RT, 4.5 h, 22−46%.
DPX 500 spectrometer (1H at 500.1 MHz, 13C at 125.8 MHz).
compounds for the parasite proteasome compared to the
human proteasome, as reported previously.10
The proteasome represents an exciting new drug target for
VL and our lead compound, 1, is being progressed toward
human studies.
Chemical shifts (δ) are expressed in ppm recorded using the residual
solvent as the internal reference in all cases. Signal splitting patterns
are described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), broad (b), or a combination thereof. Coupling
constants (J) are quoted to the nearest 0.1 Hz. LC−MS analyses were
performed with either an Agilent HPLC 1100 series connected to a
EXPERIMENTAL SECTION
■
Bruker Daltonics MicrOTOF or an Agilent Technologies 1200 series
Chemistry. Chemicals and solvents were purchased from the
HPLC connected to an Agilent Technologies 6130 quadrupole LC/
Aldrich Chemical Company, Fluka, ABCR, VWR, Acros, Fluorochem,
MS, where both instruments were connected to an Agilent diode array
and Alfa Aesar and were used as received. Air- and moisture-sensitive
detector. The mobile phase was water/acetonitrile + 0.1% HCOOH
reactions were carried out under an inert atmosphere of argon in
or water/acetonitrile + 0.1% NH3; linear gradient 80:20 to 5:95 over
oven-dried glassware. Analytical thin-layer chromatography (TLC)
3.5 min, and then held for 1.5 min; flow rate 0.5 mL min−1. All
was performed on precoated TLC plates (layer 0.20 mm silica gel 60
intermediates had a measured purity ≥90% and all assay compounds
had a measured purity of ≥95% as determined using this analytical
LC−MS system (TIC and UV). High-resolution electrospray
measurements were performed on a Bruker Daltonics MicrOTOF
mass spectrometer. Microwave-assisted chemistry was performed
using a Biotage initiator microwave synthesizer. The synthesis of all
with fluorescent indicator UV254, from Merck). Developed plates
were air-dried and analyzed under a UV lamp (UV254/365 nm).
Flash column chromatography was performed using prepacked silica
gel cartridges (230−400 mesh, 40−63 mm, from SiliCycle) using a
Teledyne ISCO CombiFlash Companion, or CombiFlash Retrieve.
1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE
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intermediates, and spectral data for final compounds, is included in
the Supporting Information.
The organic phases were combined, dried over anhydrous Na2SO4,
filtered, and concentrated to give a brown pale solid, which was dried
under vacuum to yield 8 (0.011 g, 0.03 mmol, 9%). 1H NMR
(DMSO-d6): δ 8.50 (d, J = 2.9 Hz, 1H), 8.39 (d, J = 2.1 Hz, 1H), 8.29
(d, J = 2.9 Hz, 1H), 7.96 (s, 1H), 7.49 (dd, J = 8.4, 2.2 Hz, 1H), 7.12
(s, 1H), 6.99 (d, J = 8.6 Hz, 1H), 3.80 (s, 3H), 3.76−3.65 (m, 4H),
3.35−3.26 (m, 4H), 3.06−2.97 (m, 4H), 1.81 (t, J = 6.5 Hz, 4H); 13C
NMR (DMSO-d6): δ 153.9, 149.3, 146.3, 145.9, 145.8, 136.5, 129.5,
126.6, 120.2, 119.0, 118.2, 111.3, 107.0, 66.3, 56.5, 50.1, 45.9, 25.6;
HRMS (ES+): m/z [M + H]+ calcd for C22H27N6O3, 423.2145; found,
423.2139.
N-(6-(6-Morpholinoimidazo[1,2-a]pyrimidin-2-yl)pyridin-2-
yl)pyrrolidine-1-carboxamide (9). 9 was synthesized by an
analogous method to 6 from 49 (0.21 g, 1.17 mmol) and 50e
(0.364 g, 1.17 mmol) with purification done by flash chromatography
(2−10% MeOH/DCM) to give 9 (0.1 g, 0.25 mmol, 22%). 1H NMR
(DMSO-d6): δ 8.69−8.67 (m, 1H), 8.50−8.48 (m, 1H), 8.45 (s, 1H),
8.21 (s, 1H), 7.83−7.69 (m, 3H), 3.82−3.78 (m, 4H), 3.48−3.42 (m,
4H), 3.14−3.10 (m, 4H), 1.90 1.84 (m, 4H); 13C NMR (DMSO-d6):
δ 153.8, 153.5, 151.2, 147.4, 145.7, 145.3, 138.8, 136.7, 119.2, 114.4,
112.9, 110.1, 66.3, 49.9, 46.2, 25.4; HRMS (ES+): m/z [M + H]+
calcd for C20H24N7O2, 394.1986; found, 394.1984.
N-(6-(6-Morpholinoimidazo[1,2-a]pyrimidin-2-yl)pyrimidin-
4-yl)pyrrolidine-1-carboxamide (10). 10 was synthesized by an
analogous method to 6 from 49 (0.306 g, 1.7 mmol) and 52f (0.682
g, 1.7 mmol) with purification by flash chromatography (0.5−2%
MeOH/DCM) to give 10 (0.322 g, 0.82 mmol, 48%). 1H NMR
(DMSO-d6): δ 9.32 (s, 1H), 8.76−8.74 (m, 2H), 8.58 (s, 1H), 8.47−
8.45 (m, 1H), 8.37 (s, 1H), 3.82−3.78 (m, 4H), 3.51−3.42 (m, 4H),
3.14−3.10 (m, 4H), 1.89−1.82 (m, 4H); 13C NMR (DMSO-d6): δ
160.6, 159.7, 158.4, 153.2, 148.6, 145.9, 143.7, 137.0, 66.3, 50.0, 46.4,
31.0; HRMS (ES+): m/z [M + H]+ calcd for C19H23N8O2, 395.1938;
found, 395.1939.
N-(6-(6-Morpholinoimidazo[1,2-a]pyrimidin-2-yl)pyrazin-2-
yl)pyrrolidine-1-carboxamide (11). 11 was synthesized by an
analogous method to 6 from 49 (0.208 g, 1.16 mmol) and 52g (0.452
g, 1.45 mmol), with purification by prep. HPLC to give 11 (0.071 g,
0.18 mmol, 12%). 1H NMR (DMSO-d6): δ 9.06−9.04 (m, 2H), 8.86
(s, 1H), 8.75−8.73 (m, 1H), 8.53−8.51 (m, 1H), 8.26 (s, 1H), 3.82−
3.79 (m, 4H), 3.50−3.43 (m, 4H), 3.15−3.11 (m, 4H), 1.92−1.84
(m, 4H); 13C NMR (DMSO-d6): δ 153.5, 149.9, 148.2, 145.9, 145.7,
142.7, 136.9, 135.8, 134.6, 119.2, 111.1, 66.2, 49.8, 46.4, 30.5; HRMS
(ES+): m/z [M + H]+ calcd for C19H23N8O2, 395.1938; found,
395.1946.
N-(2-Fluoro-5-(6-morpholinoimidazo[1,2-a]pyrimidin-2-yl)-
phenyl)pyrrolidine-1-carboxamide (5). To a suspension of 50a
(0.32 g, 1.02 mmol) in pyridine (19 mL) was added 4-
dimethylaminopyridine (DMAP) (0.0062 g, 0.051 mmol) and
pyrrolidine-1-carbonyl chloride (0.169 mL, 1.532 mmol), and the
resulting suspension stirred at RT for 40 h then at 40 °C for 24 h.
Further pyrrolidine-1-carbonyl chloride (0.169 mL, 1.532 mmol) was
added, and the resulting suspension stirred at 40 °C for 2 days.
Further pyrrolidine-1-carbonyl chloride (0.113 mL, 1.021 mmol) was
then added and the reaction mixture stirred at 40 °C for a further 24
h. The temperature was then increased to 55 °C and the mixture was
stirred for another 48 h. The reaction mixture was then heated at 65
°C before adding more pyrrolidine-1-carbonyl chloride (0.205 g,
1.532 mmol). The resulting mixture was heated at 55 °C for 2 days.
Solvents were evaporated, and the residue purified by flash
chromatography (0−5% MeOH/DCM). Brown oil was obtained
which was further purified by prep. HPLC to yield 5 as a yellow solid
(0.05 g, 0.12 mmol, 12% yield). 1H NMR (DMSO-d6): δ 8.54 (d, J =
2.9 Hz, 1H), 8.30 (d, J = 3.0 Hz, 1H), 8.06 (d, J = 7.2 Hz, 2H), 7.82
(s, 1H), 7.58 (ddd, J = 8.5, 4.6, 2.3 Hz, 1H), 7.17 (dd, J = 10.6, 8.5
Hz, 1H), 3.77−3.65 (m, 4H), 3.36−3.28 (m, 4H), 3.05−2.98 (m,
4H), 1.86−1.69 (m, 4H); 13C NMR (DMSO-d6): δ 156.3, 154.4,
154.2, 147.0, 145.8, 136.7, 130.2, 128.4 (d, JCF = 12.1 Hz), 123.3,
122.1 (d, JCF = 7.5 Hz), 119.1, 116.2 (d, JCF = 20.1 Hz), 107.8, 66.3,
50.0, 46.2, 25.5; HRMS (ES+): m/z [M + H]+ calcd for
C21H24FN6O2, 411.1939; found, 411.1939.
N-(3-(6-Morpholinoimidazo[1,2-a]pyrimidin-2-yl)phenyl)-
pyrrolidine-1-carboxamide (6). A mixture of 49 (0.347 g, 1.92
mmol) and 52b (0.6 g, 1.92 mmol) in dimethylformamide (DMF) (5
mL) was stirred at 90 °C for 18 h. After cooling, the solvent was
evaporated and the crude material was purified by flash chromatog-
raphy (0−5% MeOH/DCM) to give 6 (0.035 g, 0.085 mmol, 5%).
1H NMR (DMSO-d6): δ 8.64−8.62 (m, 1H), 8.42−8.40 (m, 1H),
8.25 (s, 1H), 8.14−8.13 (m, 1H), 8.11 (s, 1H), 7.56 (d, J = 8.0 Hz,
1H), 7.49 (d, J = 7.6 Hz, 1H), 7.29 (dd, J = 7.9 and 7.3 Hz, 1H),
3.81−3.78 (m, 4H), 3.42−3.38 (m, 4H), 3.12−3.09 (m, 4H), 1.89−
1.85 (m, 4H); 13C NMR (DMSO-d6): δ 154.4, 146.8, 145.9, 145.6,
141.6, 136.6, 134.2, 129.1, 119.4, 119.2, 119.1, 117.0, 107.8, 66.4,
50.2, 46.2, 25.6; HRMS (ES+): m/z [M + H]+ calcd for C21H25N6O2,
393.2039; found, 393.2054.
N-(4-Methoxy-3-(6-morpholinoimidazo[1,2-a]pyrimidin-2-
yl)phenyl)pyrrolidine-1-carboxamide (7). To a suspension of 50c
(0.158 g, 0.486 mmol) in pyridine (9.2 mL), DMAP (0.003 g, 0.024
mmol) and pyrrolidine-1-carbonyl chloride (0.080 mL, 0.728 mmol)
were added, and the resulting suspension was stirred at RT for 40 h.
Solvents were evaporated and the crude product was purified by flash
chromatography (0−10% MeOH/DCM). Brown oil was obtained
which was triturated with acetone and further purified by preparative
HPLC to yield 7 (0.074 g, 0.17 mmol, 36% yield). 1H NMR (DMSO-
d6): δ 8.53 (d, J = 2.9 Hz, 1H), 8.38 (d, J = 2.9 Hz, 1H), 8.23 (d, J =
2.8 Hz, 1H), 8.14 (s, 1H), 8.05 (s, 1H), 7.49 (dd, J = 8.9, 2.8 Hz,
1H), 6.93 (d, J = 9.0 Hz, 1H), 3.83 (s, 3H), 3.71 (dd, J = 5.9, 3.5 Hz,
4H), 3.34−3.26 (m, 4H), 3.04−2.97 (m, 4H), 1.81−1.74 (m, 4H);
13C NMR (DMSO-d6): δ 154.7, 152.2, 147.0, 144.9, 141.4, 136.3,
N-(4-(6-Morpholinoimidazo[1,2-a]pyrimidin-2-yl)pyrimidin-
2-yl)pyrrolidine-1-carboxamide (12). 12 was synthesized by an
analogous method to 6 from 49 (0.206 g, 1.14 mmol) and crude 52h
(1 equiv), with purification by flash chromatography (0.5−3%
1
MeOH/DCM) giving 12 (0.293 g, 0.73 mmol). H NMR (DMSO-
d6): δ 9.20 (s, 1H), 8.77−8.74 (m, 1H), 8.60 (d, J = 5.0 Hz, 1H),
8.53−8.50 (m, 1H), 8.33 (s, 1H), 7.60 (d, J = 5.0 Hz, 1H), 3.82−3.78
(m, 4H), 3.47−3.40 (m, 1H), 3.15−3.10 (m, 4H), 1.89−1.84 (m,
4H); 13C NMR (DMSO-d6): δ 160.6, 159.8, 159.4, 153.1, 148.6,
145.9, 143.1, 136.9, 119.1, 112.3, 110.3, 66.2, 49.7, 46.5, 25.6; HRMS
(ES+): m/z [M + H]+ calcd for C19H23N8O2, 395.1938; found,
395.1954.
N-(6-(6-Morpholinoimidazo[1,2-a]pyrimidin-2-yl)pyridazin-
4-yl)pyrrolidine-1-carboxamide (13). 13 was synthesized by an
analogous method to 6 from 49 (0.208 g, 1.15 mmol) and 52i (0.355
g, 1.43 mmol), with purification by flash chromatography (0−5%
MeOH/DCM) giving 13 (0.013 g, 0.03 mmol, 2%). 1H NMR
(DMSO-d6): δ 9.40−9.38 (m, 1H), 8.96 (s, 1H), 8.73−8.71 (m, 1H),
8.56−8.54 (m, 1H), 8.51−8.49 (m, 1H), 8.43 (s, 1H), 3.82−3.79 (m,
4H), 3.47−3.41 (m, 4H), 3.14−3.11 (m, 4H), 1.92−1.87 (m, 4H);
13C NMR (DMSO-d6): δ 155.2, 153.3, 147.9, 145.8, 142.8, 142.6,
134.4, 121.9, 120.8, 120.5, 119.2, 111.8, 111.7, 66.3, 56.2, 50.1, 46.1,
25.6; HRMS (ES+): m/z [M + H]+ calcd for C22H27N6O3, 423.2139;
found, 423.2138.
N-(2-Methoxy-5-(6-morpholinoimidazo[1,2-a]pyrimidin-2-
yl)phenyl)pyrrolidine-1-carboxamide (8). To a suspension of
50d (0.094 g, 0.289 mmol) in pyridine (5.45 mL) were added DMAP
(1.8 mg, 0.014 mmol) and pyrrolidine-1-carbonyl chloride (0.048 mL,
0.433 mmol), and the resulting suspension was stirred at RT for 40 h.
More pyrrolidine-1-carbonyl chloride (0.048 mL, 0.433 mmol) was
added, and the resulting solution was stirred at RT over 3 days.
Solvents were evaporated and the crude material was purified by flash
chromatography (0−10% MeOH/DCM). The resulting yellow solid
was dissolved in DCM/MeOH (5 mL) and washed with water (15
mL). The aqueous layer was further extracted with DCM (2 × 5 mL).
141.0, 136.9, 119.2, 110.5, 109.9, 66.8, 49.9, 46.4, 25.3; HRMS (ES+):
m/z [M + H]+ calcd for C19H23N8O2, 395.1944; found, 395.1961.
N-[4-Fluoro-3-(7-morpholinoimidazo[1,2-b]pyridazin-2-yl)-
phenyl]pyrrolidine-1-carboxamide (15). A solution of 5-
morpholinopyridazin-3-amine32 (56, 0.12 g, 0.67 mmol) and 54
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(0.241 g, 0.73 mmol) in MeCN (10 mL) was heated to 60 °C for 2
days. After cooling, the resulting precipitate was collected by filtration,
loaded onto a column, and purified by flash chromatography (10%
MeOH/EtOAc). Clean fractions were combined and concentrated,
and the resulting yellow solid was triturated and collected by filtration
1H), 7.97 (dd, J = 2.7, 6.9 Hz, 1H), 7.50 (ddd, J = 2.7, 4.3 and 8.9 Hz,
1H), 7.24 (dd, J = 8.9, 10.8 Hz, 1H), 6.65 (dd, J = 2.2, 2.2 Hz, 1H),
3.77 (t, J = 4.8 Hz, 4H), 3.47 (t, J = 4.8 Hz, 4H), 3.41−3.37 (m, 4H),
1.88 (t, J = 6.6 Hz, 4H); 13C NMR (DMSO-d6): 155.9, 154.5, 153.9,
153.1, 137.7 (d, JCF = 30.2 Hz), 136.2 (d, JCF = 44.9 Hz), 126.6, 122.0
(d, JCF = 8.1 Hz), 120.6, 119.6 (d, JCF = 13.1 Hz), 116.4 (d, JCF = 23.0
Hz), 100.1 (d, JCF = 7.4 Hz), 66.5, 46.8, 46.1, 25.5; m/z [M + H]+
calcd for C21H24N6O2F, 411.1945; found, 411.1957.
1
to give 15 (0.121 g, 0.29 mmol, 43%). H NMR (DMSO-d6): δ 8.66
(d, J = 1.8 Hz, 1H), 8.36 (d, J = 4.4 Hz, 1H), 8.30 (s, 1H), 8.21 (d, J =
3.7 Hz, 1H), 7.56−7.54 (m, 1H), 7.20−7.14 (m, 2H), 3.80 (s, 4H),
3.39 (s, 4H), 3.30 (s, 4H), 1.87 (s, 4H); 13C NMR (DMSO-d6): δ
156.0, 154.5, 154.0, 143.0, 140.3, 138.9, 137.7 (d, JCF = 19.9 Hz),
121.2 (d, JCF = 13.5 Hz), 120.5 (d, JCF = 7.9 Hz), 119.4, 115.7 (d, JCF
= 10.1 Hz), 113.8 (d, JCF = 13.1 Hz), 102.4, 66.1, 48.1, 46.1, 25.5; [M
+ H]+ calcd for C21H24FN6O2, 411.1945; found 411.1948.
N-[4-Fluoro-3-(1-methyl-5-morpholino-pyrrolo[2,3-b]-
pyridin-2-yl)phenyl]pyrrolidine-1-carboxamide (19). In a sealed
vial, 78 (0.4614 g, 1.38 mmol), Pd(OAc)2 (0.0077 g, 0.03 mmol), 2-
nitrobenzoic acid (0.1731 g, 1.04 mmol), and Ag2O (0.120 g, 0.52
mmol) were flushed with nitrogen. A solution of 77 (0.150 g, 0.69
mmol) in DMF (1 mL) was added, and the reaction mixture stirred at
90 °C overnight. The RM was allowed to cool to RT, filtered through
a pad of celite, and eluted with DCM and then MeOH to give a
brown solution. The solvent was evaporated, and the crude material
was purified by flash chromatography (0−100% EtOAc/heptane then
0−10% MeOH/EtOAc). Fractions containing the product were
combined and purified by prep. HPLC (20−95% MeCN) to afford 19
N-(3-(6-Phenylpyrazolo[1,5-a]pyrimidin-2-yl)phenyl)-
pyrrolidine-1-carboxamide (16). To a suspension of crude 61
(0.350 g, 1.11 mmol) in EtOH (20 mL) was added a solution of
ammonium chloride (0.237 g, 4.43 mmol) in water (3 mL). The
stirred mixture was heated to 75 °C and then treated in a single
portion with finely divided iron (0.495 g, 8.85 mmol). The reaction
mixture was stirred at this temperature for 2 h, cooled to RT, and
filtered through a pad of celite, further eluting with MeOH. The
resulting solution was concentrated under reduced pressure, and the
residue partitioned between DCM and water. The aqueous layer was
further extracted with DCM, and the combined organics were washed
with brine, dried over MgSO4, and concentrated to give crude 3-(6-
phenylpyrazolo[1,5-a]pyrimidin-2-yl)aniline (0.272 g, 0.95 mmol),
which was used without purification. The crude material (0.250 g,
0.87 mmol) was taken up in pyridine (1 mL)/DCM (10 mL), treated
dropwise with pyrrolidine-1-carbonyl chloride (0.175 g, 1.31 mmol),
and stirred at 70 °C for 48 h. After cooling, the reaction was diluted
with DCM (20 mL), washed with sat. NaHCO3, dried (MgSO4), and
the solvent was evaporated. The resulting solid was triturated with
EtOAc, collected by filtration, and chromatographed (100% EtOAc)
1
(0.005 g, 0.01 mmol, 2%). H NMR (CDCl3): δ 8.22 (d, J = 2.6 Hz,
1H), 7.53 (dd, J = 2.7, 6.3 Hz, 1H), 7.51−7.47 (m, 2H), 7.16−7.13
(m, 1H), 6.48 (s, 1H), 6.24 (br s, 1H), 3.96−3.93 (m, 4H), 3.79 (d, J
= 1.5 Hz, 3H), 3.50−3.48 (m, 4H), 3.17−3.15 (m, 4H), 2.01−1.99
(m, 4H). 13C NMR (DMSO-d6): δ 155.7, 154.4, 153.8, 143.9 (d, JCF
= 175 Hz), 137.7 (d, JCF = 2.5 Hz), 136.3, 136.1, 122.7, 122.0 (d, JCF
= 7.7 Hz), 119.9 (d, JCF = 15.9 Hz), 116.1 (d, JCF = 22.9 Hz), 115.3,
100.5, 66.7, 51.3, 46.2, 29.7, 25.5; HRMS (m/z): [M + H]+ calcd for
C23H27FN5O2, 424.2143; found 424.2167.
N-(4-Fluoro-3-(5-morpholinofuro[2,3-b]pyridin-2-yl)-
phenyl)pyrrolidine-1-carboxamide (20). To a degassed suspen-
sion of 69 (0.462 g, 1.14 mmol) in 1,4-dioxane (11 mL) were added
morpholine (300 μl, 3.43 mmol) and potassium bis(trimethylsilyl)-
amide (KHMDS) (4.6 ml, 2.29 mmol, 0.5 M in toluene). The
resulting mixture was purged with nitrogen and then 2-dicyclohex-
ylphosphino-2′,6′-diisopropoxybiphenyl (RuPhos) (0.107 g, 0.23
mmol) and (Pd2dba3) (0.105 g, 0.11 mmol) were added. The RM
was stirred at 90 °C for 16 h, cooled, filtered through celite, and
washed with DCM. The solvent was evaporated, and the crude
material was purified by flash chromatography (0−40% EtOH/EtOAc
(1:3)/cyclohexane) to give the crude product, which was further
purified by flash chromatography (0−2% MeOH/DCM) to give 20
(0.070 g, 0.17 mmol, 15%). 1H NMR (CDCl3): δ 8.08−8.04 (m, 1H),
7.87−7.80 (m, 1H), 7.75−7.71 (m, 1H), 7.46 (s, 1H), 7.16−7.09 (m,
2H), 6.31 (s, 1H), 3.95−3.88 (m, 4H), 3.54−3.45 (m, 4H), 3.23−
3.14 (m, 4H), 2.03−1.96 (m, 4H); m/z 410.6 [M + H]+; 13C NMR
(DMSO-d6): δ 156.4, 155.5, 154.4, 153.6, 150.0 (d, JCF = 3.8 Hz),
145.9, 138.1, 135.5, 121.9 (d, JCF = 7.9 Hz), 121.2, 117.7, 117.1, 116.6
(d, JCF = 22.3 Hz), 105.9 (d, JCF = 12.7 Hz), 66.6, 50.3, 46.2, 25.5; m/
z [M + H]+ calcd for C22H24N4O3F, 411.1832; found, 411.1840.
N-[4-Fluoro-3-(2-morpholinofuro[2,3-b]pyrazin-6-yl)-
phenyl]pyrrolidine-1-carboxamide (21). Pd2dba3 (0.0013 g,
0.0014 mmol), sodium tert-butoxide (0.027 g, 0.28 mmol), and
Xphos (0.002 g, 0.0042 mmol) in a sealed vial were purged with
nitrogen, and a suspension of 71 (0.05 g, 0.14 mmol) in DMF (1 mL)
was added, followed by morpholine (0.06 g, 0.69 mmol). The RM was
stirred at 100 °C for 1 h, cooled to RT and partitioned between water
and EtOAc. The aqueous layer was further extracted with EtOAc, and
the combined organics washed with brine, dried over MgSO4, and
concentrated. The crude mixture was purified by flash chromatog-
raphy (0−5% EtOAc/heptane) and fractions containing the product
were combined and the solvent was removed. The residue was further
purified by prep. HPLC (20−95% MeCN, 0.1% NH4OH) to give 21
(0.02 g, 0.046 mmol, 33%). 1H NMR (CDCl3): δ 7.84−7.81 (m, 3H),
7.26 (d, J = 3.2 Hz, 1H), 7.16 (dd, J = 9.0, 10.7 Hz, 1H), 6.29 (s, 1H),
3.93−3.90 (m, 4H), 3.61−3.58 (m, 4H), 3.52 (dd, J = 6.6, 6.6 Hz,
4H), 2.05−2.02 (m, 4H); 13C NMR (DMSO-d6): δ 155.6, 155.2,
154.3, 153.6, 153.0 (d, JC−F = 3.8 Hz), 149.8, 138.2, 137.8, 126.2,
122.4 (d, JC−F = 7.8 Hz), 117.2, 116.7 (d, JC−F = 20.1 Hz), 106.0 (d,
1
to give 16 (0.234 g, 0.55 mmol, 62%). H NMR (DMSO-d6): δ 9.47
(s, 1H), 8.97−8.92 (m, 1H), 8.29 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 7.6
Hz, 2H), 7.64−7.60 (m, 2H), 7.57−7.53 (m, 2H), 7.49−7.44 (m,
1H), 7.39−7.34 (m, 1H), 7.17 (s, 1H), 3.44−3.40 (m, 4H), 1.92−
1.86 (m, 4H). 13C NMR (DMSO-d6): 156.4, 154.4, 149.9, 148.7,
141.7, 134.2, 132.9, 132.7, 129.7, 129.3, 128.7, 127.3, 121.9, 120.6,
120.2, 117.5, 93.3, 46.2, 25.5; m/z [M + H]+ calcd for C23H22N5O,
384.1836; found, 384.1824.
N-(3-(5-Phenyl-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-
pyrrolidine-1-carboxamide (17). To 63 (0.075 g, 0.20 mmol) in
N-methyl-2-pyrrolidone (NMP) (1 mL) was added potassium tert-
butoxide (0.066 g, 0.59 mmol) and stirred at 75 °C overnight. After
cooling to RT, the RM was partitioned between satd. NH4Cl and
DCM. The aqueous layer was further extracted with DCM, and the
combined organics were washed with brine, dried over MgSO4, and
the solvent was evaporated. The crude material was chromatographed
1
(0−1% MeOH/EtOAc) to give 17 (0.045 g, 0.11 mmol, 57%). H
NMR (CDCl3): δ 9.77 (br s, 1H), 8.57 (d, J = 2.1 Hz, 1H), 8.10 (d, J
= 1.6 Hz, 1H), 8.01−7.99 (m, 1H), 7.69−7.66 (m, 2H), 7.53−7.49
(m, 2H), 7.45−7.39 (m, 4H), 6.86 (d, J = 2.1 Hz, 1H), 6.28 (s, 1H),
3.52−3.48 (m, 4H), 2.02−1.99 (m, 4H); 13C NMR (DMSO-d6):
154.4, 149.7, 142.2, 141.6, 140.2, 139.5, 132.1, 129.4, 129.3, 129.1,
127.3, 126.2, 121.5, 120.1, 119.3, 117.4, 97.5, 46.2, 25.5; m/z [M +
H]+ calcd for C24H23N4O, 383.1872; found, 383.1882.
N-(4-Fluoro-3-(2-morpholino-5H-pyrrolo[2,3-b]pyrazin-6-
yl)phenyl)pyrrolidine-1-carboxamide (18). To 68 (0.225 g, 0.56
mmol), morpholine (0. 242 g, 2. 78 mmol), tris-
(dibenzylideneacetone)dipalladium(0) (Pd2dba3) (0.0255 g, 0.028
mmol), and 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl
(Xphos) (0.265 g, 0.56 mmol) in DMF (5 mL) was added sodium
tert-butoxide (0.0053 g, 0.056 mmol), sealed, evacuated, flushed with
N2, and stirred at 110 °C for 2 h. After cooling, RM was partitioned
between water/EtOAc, and the organics was evaporated and
chromatographed (0−6% MeOH/EtOAc). Fractions containing the
product were combined, evaporated, and the resulting solid washed
with MeOH and dried under vacuum to give 18 (0.063 g, 0.15 mmol,
1
26%). H NMR (DMSO-d6): δ 11.95 (s, 1H), 8.26 (s, 1H), 8.07 (s,
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JC−F = 15.0 Hz), 66.3, 46.2, 46.1, 25.5; m/z [M + H]+ calcd for
C21H23N5O3F, 412.1785; found, 412.1797.
EtOH/EtOAc (1:3)/cyclohexane). The fractions containing the
product were evaporated, EtOAc (100 mL) was added, and washed
with water (250 mL)/HCl (2 M, 20 mL). The phases were separated
and the organic layer was dried over Na2SO4, filtered, and
concentrated to give 25 (0.57 g, 1.4 mmol, 29%). 1H NMR
(DMSO-d6): δ 9.01 (d, J = 2.8 Hz, 1H), 8.92 (d, J = 2.8 Hz, 1H),
8.43−8.31 (m, 2H), 7.77−7.67 (m, 1H), 7.29−7.20 (m, 1H), 3.87−
3.74 (m, 4H), 3.44−3.35 (m, 4H), 3.26−3.18 (m, 4H), 1.91−1.82
(m, 4H); 13C NMR (DMSO-d6): δ 161.1 (d, JCF = 5.3 Hz), 156.3,
N-(4-Fluoro-3-(3-morpholinofuro[2,3-c]pyridazin-6-yl)-
phenyl)pyrrolidine-1-carboxamide (22). To a stirred solution of
4-bromo-1,2-dihydropyridazine-3,6-dione (3 g, 15.7 mmol) in
pyridine (27 mL) under Ar at 5 °C was added triflic anhydride
(2.64 mmol, 15.7 mmol) and stirred for 2h. The solvent was
evaporated and RM was diluted with EtOAc, washed with sat.
aqueous NaHCO3 and brine, and the combined organic layers was
dried over Na2SO4, filtered, and concentrated to give 64 (3.7 g, 11.49
mmol, 73%), which was used without purification.
A suspension of 64 (0.590 g, 1.82 mmol), PdCl2(PPh3)2 (0.038 g,
0.05 mmol), copper (I) iodide (0.034 g, 0.18 mmol), and 62 (0.506 g,
2.37 mmol) in a mixture of MeCN/NEt3 (3 mL, 10:1) was stirred at
RT for 6 h. DCM was added and the mixture was washed with water.
The organic phase was dried and evaporated to obtain crude 65 which
was used in the next step without further purification (0.892 g, 1.9
mmol). 65 was dissolved in dry morpholine (30 mL) and stirred at 50
°C overnight. After cooling, EtOAc was added followed by water. The
organic phase was separated, the solvent was evaporated, and the
crude material purified by flash chromatography (EtOAc/cyclohexane
0−100%). The resulting product was further purified by a SCX
column, eluting with 2 M ammonia in MeOH. A second purification
by SCX column, eluting with MeOH and then with 2 M ammonia in
MeOH yielded 22 (0.093 g, 0.24 mmol, 12%). 1H NMR (DMSO-d6):
δ 8.40 (s, 1H), 8.24−8.22 (m, 1H), 7.76−7.73 (m, 1H), 7.63−7.61
(m, 1H), 7.52 (s, 1H), 7.44−7.40 (m, 1H), 7.31 (s, 1H), 3.79−3.76
(m, 4H), 3.54−3.50 (m, 4H), 3.42−3.39 (m, 4H), 1.90−1.87 (m,
4H); 13C NMR (DMSO-d6): δ 162.1, 160.4, 159.7, 154.2, 142.0,
129.8, 128.5, 127.9, 121.8, 119.7, 116.6, 106.0, 100.2, 66.4, 46.9, 46.2,
25.5; m/z [M + H]+ calcd for C21H24N5O3, 394.1879; found,
394.1895.
154.4, 154.3, 151.4, 150.8, 138.6, (d, JCF = 2.8 Hz), 123.0 (d, JCF
=
18.7 Hz), 121.3 (d, JCF = 4.8 Hz), 118.4 (d, JCF = 12.0 Hz), 116.7 (d,
JCF = 22.4 Hz), 66.2, 49.5, 46.2, 25.5; m/z [M + H]+ calcd for
C20H23N7O3F, 412.1897; found, 412.1903.
N-(4-Fluoro-3-(5-morpholinopyrazolo[1,5-b]pyridazin-2-yl)-
phenyl)pyrrolidine-1-carboxamide (26). 74 (0.035 g, 0.08 mmol)
in HBr (2 mL, 48% aqueous) was heated in a microwave (120 °C, 1
h). The solvent was evaporated, water was added, and the solution
was neutralized by the addition of sat. NaHCO3. The resulting solid
was collected, washed with water, and dried to give crude 75 which
was used directly in the next step (0.028 g, 0.08 mmol, 97%, m/z [M
+ H]+ 344.1). To crude 75 in EtOH (4 mL) was added iron (0.0325
g, 0.58 mmol) and ammonium chloride (0.0156 g, 0.29 mmol, in
water (1 mL)) and stirred at 80 °C for 4 h. RM was filtered through
celite, the solvent was evaporated and partitioned between water/
EtOAc, and the organics was evaporated to dryness. This crude
material was taken up in DCM (4 mL)/Pyridine (1 mL), and DMAP
(0.001 g, 0.007 mmol) and pyrrolidine-1-carbonyl chloride (0.0146 g,
0.11 mmol) added. After stirring overnight at 50 °C, further
pyrrolidine-1-carbonyl chloride (0.0146 g, 0.11 mmol) was added
and stirred for a further night at 50 °C. After cooling, further DCM
(10 ml) was added and washed with water, 1 M HCl, brine and the
solvent evaporated. Crude material was purified by prep. HPLC to
give 26 (0.008 g, 0.018 mmol, 24% over three steps). 1H NMR
(DMSO-d6): δ 8.56 (d, J = 3.0 Hz, 1H), 8.31 (s, 1H), 8.24 (dd, J =
2.9, 6.8 Hz, 1H), 7.66 (ddd, J = 2.8, 4.4, 9.0 Hz, 1H), 7.33 (d, J = 3.1
Hz, 1H), 7.23−7.17 (m, 1H), 6.76 (d, J = 4.0 Hz, 1H), 3.84−3.77 (m,
4H), 3.41−3.36 (m, 4H), 3.27 (t, J = 4.8 Hz, 4H), 1.89−1.84 (m,
4H); 13C NMR (DMSO-d6): δ 156.7, 154.4, 154.3, 146.0, 141.0,
137.9, 137.7, 136.1, 121.3 (d, JCF = 7.7 Hz), 120.2 (d, JCF = 12.3 Hz),
119.5, 116.3 (d, JCF = 22.9 Hz), 104.8, 95.0 (d, JCF = 11.2 Hz), 66.1,
48.1, 46.2, 25.5; m/z [M + H]+ calcd for C21H24N6O2F, 411.1945;
found, 411.1955.
N-(4-Fluoro-3-(2-morpholinoimidazo[1,2-b][1,2,4]triazin-6-
yl)phenyl)pyrrolidine-1-carboxamide (23). Previously described
in ref 10.
N-(4-Fluoro-3-(7-morpholino-[1,2,4]triazolo[1,5-b]-
pyridazin-2-yl)phenyl)pyrrolidine-1-carboxamide (24). To a
mixture of 80 (0.2 g, 0.80 mmol) in DCM (5 mL) was added thionyl
chloride (0.174 mL, 2.379 mmol) and stirred at 50 °C for 1 h. The
solvent was evaporated, further DCM was added, and evaporated (2 ×
10 mL) to give crude 81, which was used without purification. To 79
and N,N-diisopropylethylamine (DIPEA) (0.174 mL, 0.994 mmol) in
MeCN (3 mL) was added crude 81 (0.13 g, 0.66 mmol), and the
mixture stirred at 80 °C for 24 h. After cooling, the mixture was
filtered to remove the solid, and the filtrate concentrated and purified
by flash chromatography (0−40% EtOH/EtOAc(1:3)/cyclohexane)
to give 24 (0.035 g, 0.17 mmol, 13%). 1H NMR (DMSO-d6): δ 8.83−
8.71 (m, 1H), 8.42−8.30 (m, 2H), 7.79−7.62 (m, 1H), 7.46−7.35
(m, 1H), 7.30−7.16 (m, 1H), 3.86−3.72 (m, 4H), 3.48−3.30 (m,
N-(3-(7-Phenylimidazo[1,2-b][1,2,4]triazin-3-yl)phenyl)-
pyrrolidine-1-carboxamide (27). A mixture of 59 (0.030 g, 0.074
mmol), 4,4,5,5-tetramethyl-2-phenyl-1,3,2-dioxaborolane (0.030 g,
0.15 mmol), sodium carbonate (0.023 g, 0.22 mmol), and
tetrakis(triphenylphosphine)palladium(0) (0.0011 g, 0.0015 mmol)
in DMF (1.5 mL)/water (0.5000 mL) was heated to 80 °C in a sealed
tube overnight. After cooling, water (10 mL) and EtOAc (10 mL)
were added, the layers were separated, and the organic layer was
evaporated. The crude product was purified by prep. HPLC to yield
8H), 1.93−1.80 (m, 4H); 13C NMR (DMSO-d6): δ 159.0 (d, JCF
=
5.3 Hz), 156.4, 154.5, 146.7, 145.7, 137.7, 137.6 (d, JCF = 2.7 Hz),
123.0 (d, JCF = 7.7 Hz), 121.4, 118.5 (d, JCF = 12.0 Hz), 116.7 (d, JCF
= 22.5 Hz), 110.9, 66.0, 47.4, 46.1, 25.5; m/z 412.1 [M + H]+. m/z
[M + H]+ calcd for C20H23N7O2F, 412.1897; found, 412.1901.
N-(4-Fluoro-3-(6-morpholino-[1,2,4]triazolo[1,5-a]-
pyrimidin-2-yl)phenyl)pyrrolidine-1-carboxamide (25). A mix-
ture of 85 (9 g, 29.2 mmol) and morpholine (115 mL, 1314 mmol)
was stirred at 100 °C for 1 h. The mixture was concentrated to
dryness and the crude solid washed with MeOH (2 × 50 mL),
collected, and dried to give crude 4-fluoro-3-(6-morpholino-[1,2,4]-
triazolo[1,5-a]pyrimidin-2-yl)aniline (86, 6.4 g, 20.4 mmol, 70%
1
27 (0.012 g, 0.028 mmol, 38%). H NMR (DMSO-d6): δ 9.16 (s,
1H), 8.61 (s, 1H), 8.49 (s, 1H), 8.35−8.31 (m, 1H), 8.20 (d, J = 7.5
Hz, 2H), 7.89−7.84 (m, 1H), 7.61−7.55 (m, 2H), 7.48−7.43 (m,
1H), 7.37−7.32 (m, 1H), 3.42−3.38 (m, 4H), 1.91−1.84 (m, 4H);
m/z 403.2 [M + H]+.
N-(4-Fluoro-3-(3-phenylimidazo[1,2-a]pyrimidin-7-yl)-
phenyl)pyrrolidine-1-carboxamide (28). As described in ref 29.
N-(4-Fluoro-3-(3-(pyridin-2-yl)imidazo[1,2-a]pyrimidin-7-
yl)phenyl)pyrrolidine-1-carboxamide (29). A mixture of 89 (0.19
g, 0.47 mmol), 2-pyridineboronic acid (0.195 g, 0.94 mmol),
Pd(dppf)Cl2·CH2Cl2 (0.077 g, 0.094 mmol), and potassium
carbonate (0.195 g, 1.41 mmol) in 1,4-dioxane (30 mL)/water (2
mL) was stirred under nitrogen at 100 °C for 10 h. The mixture was
cooled to RT, evaporated, diluted with water (20 mL), and extracted
with EtOAc (5 × 30 mL). The combined organic layers were dried
over Na2SO4, filtered, and the solvent was evaporated. The crude
material was purified by flash chromatography (1−5% MeOH/DCM)
1
crude yield), which was used without further purification. H NMR
(DMSO-d6): δ 8.98 (d, J = 3.0 Hz, 1H), 8.90 (d, J = 2.8 Hz, 1H), 7.37
(dd, J = 6.2 and 2.9 Hz, 1H), 7.02 (dd, J = 10.9 and 8.8 Hz, 1H), 6.68
(dt, J = 8.5 and 3.6 Hz, 1H), 5.18 (s, 2H), 3.84−3.73 (m, 4H), 3.27−
3.15 (m, 4H). To crude 86 (1.5 g, 4.77 mmol) and DMAP (0.029 g,
0.239 mmol) in pyridine (40 mL) was added pyrrolidine-1-carbonyl
chloride (0.790 mL, 7.16 mmol), and the solution was stirred at 50 °C
overnight. The resulting suspension was concentrated to dryness, and
the crude residue was purified by flash chromatography (0−100%
1
to give 29 as a yellow solid (0.02 g, 0.048 mmol, 10%). H NMR
(MeOD): δ 10.42 (d, J = 7.5 Hz, 1H), 8.91 (br s, 1H), 8.70 (d, J = 4.6
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Hz, 1H), 8.32−8.27 (m, 1H), 7.89−7.76 (m, 3H), 7.43−7.38 (m,
1H), 7.33 (dd, J = 5.4, 6.7 Hz, 1H), 7.11 (dd, J = 11.2, 9.3 Hz, 1H),
3.30−3.26 (m, 4H), 1.94−1.89 (m, 4H). m/z 403.2 [M + H]+.
N-(4-Fluoro-3-(3-isopropylimidazo[1,2-a]pyrimidin-7-yl)-
phenyl)pyrrolidine-1-carboxamide (30). A mixture of 87 (0.15 g,
0.5 mmol) and 2-bromo-3-methylbutanal (0.123 g, 0.75 mmol) in
EtOH (2 mL) was heated to reflux for 4 h. The reaction mixture was
cooled to RT, and the solvent was evaporated. The crude material was
purified by prep. HPLC to obtain 30, which was then diluted in DCM
(5 mL) and treated dropwise with 2 N HCl in Et2O (0.191 mL, 0.381
mmol). The resulting solution was stirred at RT for 2 h, the solvent
was evaporated, and the resulting solid was dried at 50 °C to give 30·
HCl (0.12 g, 0.297 mmol, 59%). 1H NMR (DMSO-d6): δ 9.39 (d, J =
7.2 Hz, 1H), 8.51 (s, 1H), 8.42 (dd, J = 6.8 and 2.8 Hz, 1H), 8.19 (s,
1H), 7.97 (dd, J = 7.2 and 1.6 Hz, 1H), 7.75 (ddd, J = 8.8, 7.2 and 2.8
Hz, 1H), 7.37 (dd, J = 11.2 and 8.8 Hz, 1H), 3.51−3.41 (m, 1H),
3.39 (t, J = 6.8 Hz, 4H), 1.87 (t, J = 6.8 Hz, 4H), 1.37 (d, J = 7.2 Hz,
6H). 13C NMR (DMSO-d6): δ 158.8, 155.2, 154.3, 144.0, 138.4,
136.5, 131.9, 125.3 (JCF = 7.3 Hz), 123.3 (JCF = 10.9 Hz), 121.2,
117.2 (JCF = 24.4 Hz), 113.4 (JCF = 7.8 Hz), 101.4, 46.2, 25.9, 23.4,
21.0; m/z 368.1 [M + H]+. HRMS (ES+): m/z [M + H]+ calcd for
C20H23FN5O, 368.1887; found, 368.1899.
N-(4-Fluoro-3-(3-isobutylimidazo[1,2-a]pyrimidin-7-yl)-
phenyl)pyrrolidine-1-carboxamide (31). A mixture of 87 (0.15 g,
0.5 mmol) and 2-bromo-4-methylpentanal (0.18 g, 1 mmol) in EtOH
was heated to reflux for 24 h. The reaction mixture was cooled to RT
and partitioned between 10% aq NaHCO3 and EtOAc. The organic
layer was dried over anhydrous Na2SO4, filtered, and the solvent
evaporated. The crude material was purified by prep. HPLC to afford
31 (0.047 g, 0.123 mmol, 25%). 1H NMR (DMSO-d6): δ 9.36 (d, J =
7.2 Hz, 1H), 8.48 (s, 1H), 8.42 (dd, J = 6.8 and 2.4 Hz, 1H), 8.15 (s,
1H), 7.93 (d, J = 6.4 Hz, 1H), 7.73 (ddd, J = 8.8, 7.6 and 2.8 Hz, 1H),
7.37 (dd, J = 11.2 and 9.2 Hz, 1H), 3.38 (t, J = 6.8 Hz, 4H), 2.88 (d, J
= 6.8 Hz, 2H), 2.11−2.01 (m, 1H), 1.87 (t, J = 6.4 Hz, 4H), 0.99 (d, J
= 6.8 Hz, 6H); 13C NMR (DMSO-d6): δ 158.4 (d, JCF = 33.6 Hz),
157.0, 155.0, 154.3, 138.4, 136.1, 125.1, 125.0, 123.6, 121.2, 117.2 (d,
JCF = 24.4 Hz), 115.7, 113.1, 46.2, 31.5, 26.9, 25.5, 22.5; HRMS
(ES+): m/z [M + H]+ calcd for C21H25FN5O2, 382.2038; found,
382.2040.
for 1 h and evaporated to dryness. To the crude compound was added
acetic acid (0.6 mL, 10.5 mmol) and paraformaldehyde (0.045 g, 1.5
mmol) in tetrahydrofuran (THF) (3 mL), and the mixture stirred at
RT for 4 h. Sodium triacetoxyborohydride (0.127 g, 0.6 mmol) was
added, and the RM was stirred at RT for 24 h, evaporated to dryness,
and purified by prep. HPLC to afford 33 (0.018 g, 0.042 mmol, 28%).
1H NMR (DMSO-d6): δ 9.29 (d, J = 7.3 Hz, 1H), 8.43 (s, 1H), 8.24−
8.22 (m, 1H), 7.80−7.77 (m, 2H), 7.47 (d, J = 7.3 and 2.2 Hz, 1H),
7.27 (dd, J = 11.4 and 8.9 Hz, 1H), 3.88−3.85 (m, 1H), 3.77−3.69
(m, 4H), 3.41−3.37 (m, 4H), 2.91−2.88 (d, J = 11.7 Hz, 1H), 2.35−
2.33 (m, 1H), 2.02 (s, 3H), 1.89−1.85 (m, 4H); m/z 425.19 [M +
H]+. HRMS (ES+): m/z [M + H]+ calcd for C22H26FN6O2, 425.2101;
found, 425.2088.
N-(4-Fluoro-3-(3-(piperidin-1-yl)imidazo[1,2-a]pyrimidin-7-
yl)phenyl)pyrrolidine-1-carboxamide (34). 34 was synthesized
by an analogous method to 1, from 87 (0.2 g, 0.66 mmol), 96b (0.567
g, 1.32 mmol), and zinc bromide (0.148 g, 0.66 mmol) with
purification by prep. HPLC to yield 34 (0.021 g, 0.061 mmol, 9%).
1H NMR (DMSO-d6): δ 8.64 (d, J = 7.2 Hz, 1H), 8.43 (s, 1H), 8.23−
8.20 (m, 1H), 7.80−7.76 (m, 1H), 7.45 (s, 1H), 7.41 (d, J = 6.9 Hz,
1H), 7.26 (dd, J = 9.5 and 10.7 Hz, 1H), 3.41−3.37 (m, 4H), 3.01−
2.98 (m, 4H), 1.88−1.84 (m, 4H), 1.76−1.71 (m, 4H), 1.62−1.58
(m, 2H); 13C NMR (DMSO-d6): δ 156.7, 154.8, 154.4, 151.1 (d, JCF
= 2.7 Hz), 144.3, 138.0, 135.0, 131.7, 125.2 (d, JCF = 11.6 Hz),
123.11, 121.1, 116.7 (d, JCF = 23.8 Hz), 108.8 (d, JCF = 11.4 Hz),
52.6, 46.2, 25.9, 25.5, 24.1; HRMS (ES+): m/z [M + H]+ calcd for
C22H26FN6O, 409.2147; found, 409.2154.
N-(4-Fluoro-3-(3-(piperazin-1-yl)imidazo[1,2-a]pyrimidin-7-
yl)phenyl)pyrrolidine-1-carboxamide (35). 35 was synthesized
by an analogous method to 1, from 87 (0.3 g, 1 mmol), 96c (0.693 g,
1.1 mmol), and zinc bromide (0.045 g, 0.19 mmol) to give crude Boc-
35. This was treated with TFA (3 mL)/DCM (6 mL), the solvent was
evaporated, and crude material was purified by prep. HPLC to give 35
1
as the TFA salt (0.092 g, 0.23 mmol, 23% over two steps). H NMR
(DMSO-d6): δ 9.06−8.98 (m, 3H), 8.45 (s, 1H), 8.34−8.31 (m, 1H),
7.91 (s, 1H), 7.78−7.74 (m, 1H), 7.73−7.70 (m, 1H), 7.35−7.30 (m,
1H), 3.42−3.34 (m, 8H), 3.30−3.27 (m, 4H), 1.89−1.85 (m, 4H);
13C NMR (DMSO-d6): δ 158.7, 158.4, 156.9, 154.9, 154.3, 138.2 (d,
JCF = 2.3 Hz), 133.6, 133.2, 124.3, 121.2, 117.0 (d, JCF = 23.8 Hz),
48.4, 46.2, 43.4, 25.5; m/z 409.9 [M + H]+. m/z [M + H]+ calcd for
C21H25N7OF, 410.2105; found, 410.2119.
N-(4-Fluoro-3-(3-(morpholinomethyl)imidazo[1,2-a]-
pyrimidin-7-yl)phenyl)pyrrolidine-1-carboxamide (32). A mix-
ture of 90 (0.774 g, 3 mmol), paraformaldehyde (0.087 g, 3 mmol),
and morpholine (0.261 g, 3 mmol) in glacial acetic acid (10 mL) was
stirred at 50 °C overnight. After cooling, the RM was basified with 2
N NaOH to pH approx. 8 and extracted with DCM (3 × 30 mL). The
combined organics were washed with brine (2 × 50 mL), dried over
Na2SO4, filtered, and the solvent was evaporated to give crude 91.
This was taken up in EtOH (15 mL) to which iron (0.753 g, 13.4
mmol), and ammonium chloride (0.758 g, 6.7 mmol, in 4 mL water)
were added, and the RM was heated to 80 °C for 1 h, cooled to RT,
filtered through celite, and the solvent was evaporated. Water was
added and extracted with EtOAc (3 × 50 mL), and the combined
organics were dried over anh. Na2SO4, filtered, and concentrated to
give a crude residue. This was taken up in pyridine (2 mL)/DCM (10
mL), DMAP (0.0056 g, 0.046 mmol), and pyrrolidine-1-carbonyl
chloride (0.122 g, 0.92 mmol), and the RM was heated at 50 °C
overnight. The solvent was evaporated and the crude material was
purified by prep. HPLC to give 32 (0.083 g, 0.196 mmol, 7% over
N-(3-(3-(4-Acetylpiperazin-1-yl)imidazo[1,2-a]pyrimidin-7-
yl)-4-fluorophenyl)pyrrolidine-1-carboxamide (36). To a mix-
ture of N-(3-(2-aminopyrimidin-4-yl)-4-fluorophenyl)pyrrolidine-1-
carboxamide (87, 0.5 g, 1.66 mmol) in 1,2-DCE (11 mL) were added
96d (1.29 g, 2.49 mmol) and zinc bromide (0.187 g, 0.83 mmol) and
stirred at 80 °C overnight. The solvent was evaporated and the
residue was purified by column chromatography (0−10% MeOH/
DCM), followed by prep. HPLC. The resulting solid was triturated
with MeOH (5 mL) and dried to yield 36 (0.039 g, 0.08 mmol, 5%).
1H NMR (DMSO-d6): δ 8.78 (d, J = 7.2 Hz, 1H), 8.41 (s, 1H), 8.22
(dd, J = 2.8 and 7.1 Hz, 1H), 7.80−7.75 (m, 1H), 7.53 (s, 1H), 7.47−
7.43 (m, 1H), 7.29−7.23 (m, 1H), 3.70−3.65 (m, 4H), 3.41−3.36
(m, 4H), 3.08−2.97 (m, 4H), 2.09 (s, 3H), 1.89−1.85 (m, 4H); 13C
NMR (DMSO-d6): δ 168.9, 157.0, 154.6, 154.4, 151.7, 144.5, 138.1,
113.7, 131.9, 125.2 (d, JCF = 11.8 Hz), 123.8, 123.2 (d, JCF = 8.3 Hz),
121.2, 116.7 (d, JCF = 23.8 Hz), 109.0 (d, JCF = 11.1 Hz), 51.7, 51.4,
46.2, 25.5, 21.7; m/z 452.1 [M + H]+. m/z [M + H]+ calcd for
C21H23N5O3F, 452.2210; found, 452.2229.
1
three steps). H NMR (CDCl3): δ 8.80 (d, J = 7.2 Hz, 1H), 8.15−
7.74 (m, 3H), 7.59 (d, J = 6.8 Hz, 1H), 7.12 (dd, J = 10.8 and 9.2 Hz,
1H), 6.71 (broad s, 1H), 3.88 (br s, 2H), 3.74−3.67 (m, 4H), 3.53−
3.46 (m, 4H), 2.55−2.44 (m, 4H), 2.05−1.92 (m, 4H); 13C NMR
(DMSO-d6): δ 154.8, 154.4, 152.5, 148.8, 138.1, 135.8, 134.4, 125.2
(d, J = 11.7 Hz), 123.3 (d, J = 8.0 Hz), 121.3, 119.7, 116.7 (d, J = 23.8
Hz), 109.0 (d, J = 10.9 Hz), 66.6, 53.3, 51.6, 46.2, 25.5. HRMS (ES+):
m/z [M + H]+ calcd for C22H26FN6O2, 425.2096; found, 425.2104.
N-(4-Fluoro-3-(3-(4-methylmorpholin-3-yl)imidazo[1,2-a]-
pyrimidin-7-yl)phenyl)pyrrolidine-1-carboxamide (33). To a
solution of 95 (0.08 g, 0.16 mmol) in DCM (1 mL), trifluoroacetic
acid (0.5 mL, 6.53 mmol) was added, and the resulting mixture stirred
N-(4-Fluoro-3-(3-(3-oxopiperazin-1-yl)imidazo[1,2-a]-
pyrimidin-7-yl)phenyl)pyrrolidine-1-carboxamide (37). 37 was
synthesized by an analogous method to 1, from 87 (0.5 g, 1.66
mmol), 96e (1.15 g, 2.49 mmol), and zinc bromide (0.187 g, 0.83
mmol) with purification by flash chromatography (0−10% MeOH/
DCM), followed by trituration with MeOH to give 37 (0.06 g, 0.14
1
mmol, 8%). H NMR (DMSO-d6): δ 8.76 (d, J = Hz, 1H), 7.2 Hz,
8.41 (s, 1H), 8.21 (dd, J = 7.2 and 2.8 Hz, 1H), 8.03 (s, 1H), 7.81−
7.76 (m, 1H), 7.55 (s, 1H), 7.42 (dd, J = 2.1 and 7.1 Hz, 1H), 7.25
(dd, J = 9.0 and 11.3 Hz, 1H), 3.69 (s, 2H), 3.42−3.36 (m, 6H),
3.24−3.20 (m, 2H), 1.89−1.84 (m, 4H); 13C NMR (DMSO-d6): δ
5926
https://doi.org/10.1021/acs.jmedchem.1c00047
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Journal of Medicinal Chemistry
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Article
167.3, 156.8, 154.8, 154.4, 151.7, 144.7, 138.1 (d, JCF = 2.1 Hz),
132.3, 132.1, 125.1 (d, JCF = 11.6 Hz), 124.4, 123.3 (d, JCF = 8.1 Hz),
121.2, 116.6 (d, JCF = 23.9 Hz), 109.0 (d, JCF = 11.1 Hz), 54.7, 48.2,
46.2, 40.8, 25.5; m/z 424.0 [M + H]+. m/z [M + H]+ calcd for
C21H23N7O2F, 424.1897; found, 424.1902.
14.5; HRMS (ES+): m/z [M + H]+ calcd for C22H26FN6O2, 425.2101;
found, 425.2113.
N-(3-(3-((2S,6R)-2,6-Dimethylmorpholino)imidazo[1,2-a]-
pyrimidin-7-yl)-4-fluorophenyl)pyrrolidine-1-carboxamide
(41). To a mixture of 87 (3.5 g, 11.62 mmol) in 1,2-DCE (77 mL),
96g (8.55 g, 17.42 mmol) and zinc bromide (1.308 g, 5.81 mmol)
were added and then stirred at 80 °C under a N2 atmosphere for 2 h,
cooled to RT, and stirred overnight. The solvent was evaporated, and
the residue purified by flash chromatography (0−10% MeOH/
DCM). The resulting solid was triturated with MeOH (20 mL), and
the solids were collected by filtration to give 41 (1.0 g, 2.28 mmol,
20%). 1H NMR (DMSO-d6): δ 9.16 (d, J = 7.2 Hz, 1H), 8.49 (s, 1H),
8.39 (dd, J = 7.1, 2.8 Hz, 1H), 8.01 (s, 1H), 7.87 (dd, J = 7.3, 1.7 Hz,
1H), 7.74 (ddd, J = 9.0, 4.4, 2.8 Hz, 1H), 7.35 (dd, J = 11.3, 9.0 Hz,
1H), 3.93−3.80 (m, 2H), 3.43−3.32 (m, 4H), 3.22 (d, J = 2.1 Hz,
2H), 2.55 (dd, J = 11.8, 10.2 Hz, 2H), 1.91−1.80 (m, 4H), 1.14 (d, J
= 6.3 Hz, 6H); 13C NMR (DMSO-d6): δ 157.0, 155.1, 154.3, 141.3,
138.4, 134.9, 134.4, 125.2, 123.5 (d, JCF = 11.1 Hz), 121.2, 117.2 (d,
N-(4-Fluoro-3-(3-(2-methylmorpholino)imidazo[1,2-a]-
pyrimidin-7-yl)phenyl)pyrrolidine-1-carboxamide (38). A mix-
ture of 87 (0.15 g, 0.5 mmol), 96f (0.345 g, 0.75 mmol), and zinc
bromide (0.056 g, 0.25 mmol) 1,2-DCE (5 mL) was heated to reflux
for 6 h. The reaction was cooled to RT, solvent was evaporated, and
the residue was purified by prep. HPLC to give 38 (0.036 g, 0.13
mmol, 17%). 1H NMR (DMSO-d6): δ 8.76 (d, J = 7.2 Hz, 1H), 8.43
(s, 1H), 8.23−8.19 (m, 1H), 7.80−7.75 (m, 1H), 7.50 (s, 1H), 7.42
(d, J = 6.8 Hz, 1H), 7.28 (dd, J = 9.5 and 10.7 Hz, 1H), 3.93−3.89
(m, 1H), 3.83−3.76 (m, 2H), 3.41−3.37 (m, 4H), 3.20 (d, J = 11.3
Hz, 1H), 3.13 (d, J = 11.4 Hz, 1H), 2.91−2.84 (m, 1H), 2.59 (dd, J =
10.5 and 10.9 Hz, 1H), 1.88−1.85 (m, 4H), 1.14 (d, J = 6.2 Hz, 3H);
13C NMR (DMSO-d6): δ 156.7, 154.7, 154.4, 151.5, 144.5, 138.0,
J
CF = 24.0 Hz), 113.2, 71.3, 56.5, 46.2, 25.5, 19.1; HRMS (ES+): m/z
133.8, 131.9, 125.2 (d, JCF = 11.6 Hz), 123.4, 121.2, 116.7 (d, JCF
=
[M + H]+ calcd for C23H28FN6O2, 429.2252; found, 439.2227.
N-(3-(3-(2,2-Dimethylmorpholino)imidazo[1,2-a]pyrimidin-
7-yl)-4-fluorophenyl)pyrrolidine-1-carboxamide (42). A mix-
ture of 87 (0.2 g, 0.66 mmol), 96h (0.486 g, 0.99 mmol), and zinc
bromide (0.223 g, 0.99 mmol) in MeCN (4 mL) was stirred at 40 °C
for 18 h. The solvent was evaporated, and the crude product purified
24.0 Hz), 108.9 (d, JCF = 11.5 Hz), 71.7, 66.3, 57.5, 51.0, 46.2, 25.5,
19.1; HRMS (ES+): m/z [M + H]+ calcd for C22H26FN6O2, 425.2096;
found, 425.2100.
(R)-N-(4-Fluoro-3-(3-(3-methylmorpholino)imidazo[1,2-a]-
pyrimidin-7-yl)phenyl)pyrrolidine-1-carboxamide (39). A mix-
ture of 100a (0.343 g, 0.743 mmol) and 10% activated palladium on
carbon (0.040 g, 0.037 mmol) was cooled to −78 °C and MeOH
(7.43 mL) was added. The flask was purged with N2 and H2, and the
resulting mixture stirred at RT under a H2 atmosphere overnight. The
RM was filtered through a celite pad, washed with DCM (5 × 20
mL), and the eluent was concentrated to dryness and dried under
high vacuum to afford (R)-4-fluoro-3-(3-(3-methylmorpholino)-
imidazo[1,2-a]pyrimidin-7-yl)aniline which was used without purifi-
cation. The crude material was taken up in DCM (10 mL) to which
CDI (0.213 g, 1.314 mmol) and DIPEA (0.229 mL, 1.314 mmol)
were added and stirred overnight. Pyrrolidine (0.110 mL, 1.314
mmol) was added dropwise and the RM was stirred at RT for 4.5 h.
Further pyrrolidine (0.02 mL) was added and the reaction was stirred
at RT for a further 30 min, then poured into 2 M NaOH (50 mL),
and extracted with DCM (5 × 20 mL). The organic layers were
combined, dried over Na2SO4, filtered, concentrated, and the crude
material was purified by flash chromatography (0−50% EtOAc/EtOH
(3:1)/cyclohexane). Fractions containing the product were further
purified by flash chromatography (0−50% EtOAc/EtOH (3:1)/
cyclohexane) to give 39 (0.127 g, 0.34 mmol, 46%). 1H NMR
(DMSO-d6): δ 8.79 (d, J = 7.2 Hz, 1H), 8.41 (s, 1H), 8.21 (dd, J =
7.1, 2.8 Hz, 1H), 7.77 (ddd, J = 9.0, 4.4, 2.8 Hz, 1H), 7.68 (s, 1H),
7.44 (dd, J = 7.2, 2.1 Hz, 1H), 7.25 (dd, J = 11.4, 9.0 Hz, 1H), 3.91−
3.80 (m, 2H), 3.72 (ddd, J = 11.3, 9.5, 2.9 Hz, 1H), 3.45−3.33 (m,
5H), 3.21 (dqd, J = 9.1, 6.2, 2.8 Hz, 1H), 3.04 (dddd, J = 21.5, 12.0,
9.2, 3.0 Hz, 2H), 1.92−1.80 (m, 4H), 0.78 (d, J = 6.3 Hz, 3H); 13C
NMR (DMSO-d6): δ 156.7, 154.8, 154.4, 152.0, 144.7, 138.0, 131.9
(d, JCF 9.1 Hz), 127.5, 125.2 (d, JCF 11.8 Hz), 123.2 (d, JCF 8.1 Hz),
121.2, 116.7 (d, JCF 23.9 Hz), 109.1 (d, JCF 11.1 Hz), 72.6, 67.3, 55.5,
52.4, 46.2, 25.5, 14.6; HRMS (ES+): m/z [M + H]+ calcd for
C22H26FN6O2, 425.2096; found, 425.2093.
(S)-N-(4-Fluoro-3-(3-(3-methylmorpholino)imidazo[1,2-a]-
pyrimidin-7-yl)phenyl)pyrrolidine-1-carboxamide (40). 40 was
prepared by an analogous method to 39, from 100b (0.37 g, 0.802
mmol) using 10% activated palladium on carbon (0.0427 g, 0.04
mmol), then CDI (0.155 g, 0.953 mmol), DIPEA (0.166 mL, 0.953
mmol), and pyrrolidine (0.080 mL, 0.953 mmol), purifying by prep.
HPLC to yield 42 (0.12 g, 0.28 mmol, 35% over two steps). 1H NMR
(DMSO-d6): δ 8.80 (d, J = 7.2 Hz, 1H), 8.43 (s, 1H), 8.24−8.20 (m,
1H), 7.80−7.76 (m, 1H), 7.69 (s, 1H), 7.44 (d, J = 6.7 Hz, 1H), 7.26
(dd, J = 9.5 and 10.8 Hz, 1H), 3.90−3.83 (m, 2H), 3.75−3.69 (m,
1H), 3.42−3.35 (m, 5H), 3.24−3.19 (m, 1H), 3.11−2.98 (m, 2H),
1.90−1.83 (m, 4H), 0.79 (d, J = 6.2 Hz, 3H); 13C NMR (DMSO-d6):
δ 156.7, 154.8, 154.4, 152.0, 144.7, 138.0, 131.9 (d, JCF 9.5 Hz),
127.5, 125.2 (d, JCF 11.9 Hz), 123.3 (d, JCF 8.0 Hz), 121.2, 116.7 (d,
1
by prep. HPLC to give 42 (0.134 g, 46% yield). H NMR (DMSO-
d6): δ 8.98 (d, J = 7.0 Hz, 1H), 8.48 (s, 1H), 8.38−8.35 (m, 1H), 7.90
(s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.77−7.73 (m, 1H), 7.35 (dd, J =
7.4 and 10.6 Hz, 1H), 3.89−3.85 (m, 4H), 3.42−3.37 (m, 4H), 3.00−
2.97 (m, 2H), 2.91−2.88 (m, 2H), 1.89−1.85 (m, 4H), 1.33 (s, 6H);
13C NMR (DMSO-d6): 157.3, 154.9, 154.3, 141.6, 138.4, 134.8,
134.4, 125.1 (d, JCF = 8.4 Hz), 123.5 (d, JCF = 11.1 Hz), 121.2, 117.1
(d, JCF = 23.7 Hz), 114.0, 113.1 (d, JCF = 11.6 Hz), 71.2, 60.5, 60.4,
51.7, 46.2, 25.5, 24.9; HRMS (ES+): m/z [M + H]+ calcd for
C23H28FN6O2, 439.2258; found, 439.2252.
N-(3-(3-(3,3-Dimethylmorpholino)imidazo[1,2-a]pyrimidin-
7-yl)-4-fluorophenyl)pyrrolidine-1-carboxamide (43). A mix-
ture of 87 (0.676 g, 2.243 mmol), 96i (1.642 g, 3.347 mmol), and
zinc bromide (0.777 g, 3.45 mmol) in MeCN (12 mL) was stirred at
60 °C for 4 h and evaporated to dryness. The crude material was
purified by flash chromatography (2% MeOH/DCM) and then by
prep. HPLC to obtain 43 (0.11 g, 0.25 mmol, 12%). 1H NMR
(CD3OD): δ 9.26 (d, J = 7.2 Hz, 1H), 8.46 (dd, J = 6.8 and 2.8 Hz,
1H), 8.15 (s, 1H), 8.10 (d, J = 6.8 Hz, 1H), 7.63 (ddd, J = 8.8, 7.2 and
2.8 Hz, 1H), 7.29 (dd, J = 11.2 and 9.2 Hz, 1H), 4.01−3.78 (m, 2H),
3.65 (s, 2H), 3.49 (t, J = 6.8 Hz, 4H), 3.06−2.74 (m, 2H), 2.00 (t, J =
6.4 Hz, 4H), 1.54−0.80 (m, 6H); 13C NMR (DMSO-d6): δ 158.7,
158.4, 157.0, 155.1, 154.3, 141.9, 138.4, 135.0, 130.8, 125.1 (d, JCF
=
8.6 Hz), 123.6 (d, JCF = 11.2 Hz), 121.2, 117.2 d, (JCF = 23.3 Hz),
76.9, 67.7, 55.8, 48.3, 46.2, 25.5, 17.4; HRMS (ES+): m/z [M + H]+
calcd for C23H28FN6O2, 439.2258; found, 439.2265.
N-(3-(3-(2-Oxa-7-azaspiro[4.4]nonan-7-yl)imidazo[1,2-a]-
pyrimidin-7-yl)-4-fluorophenyl)pyrrolidine-1-carboxamide
(44). 44 was prepared by an analogous method to 39, from 100c
(0.283 g, 0.580 mmol) using 10% activated palladium on carbon
(0.0309 g, 0.029 mmol), then CDI (0.125 g, 0.770 mmol), DIPEA
(0.134 mL, 0.770 mmol), and pyrrolidine (0.064 mL, 0.770 mmol),
purifying by flash chromatography (0−4% EtOAc/EtOH (3:1)/
cyclohexanes) to yield 44 (0.07 g, 0.155 mmol, 27% over two steps).
1H NMR (DMSO-d6): δ 8.73 (d, J = 7.6 Hz, 1H), 8.40 (s, 1H), 8.20
(dd, J = 7.2 and 2.8 Hz, 1H), 7.76 (ddd, J = 8.8, 7.2 and 2.8 Hz, 1H),
7.40 (s, 1H), 7.36 (dd, J = 7.6 and 2.4 Hz, 1H), 7.24 (dd, J = 11.2 and
8.8 Hz, 1H), 3.84−3.77 (m, 2H), 3.71 (t, J = 8 Hz, 1H), 3.61 (t, J =
8.4 Hz, 1H), 3.42−3.34 (m, 6H), 3.26 (d, J = 9.2 Hz, 6H), 3.22 (d, J
= 9.2 Hz, 6H), 2.06−1.95 (m, 4H), 1.86 (t, J = 6.8 Hz, 4H); 13C
NMR (DMSO-d6): δ 156.7, 154.8, 154.4, 150.4, 144.0, 138.0, 133.5,
132.1, 125.3 (d, JCF = 11.8 Hz), 123.0 (d, JCF = 7.9 Hz), 121.1 (d, JCF
= 15.3 Hz), 116.8 (d, JCF = 24.0 Hz), 108.7 (d, JCF = 11.4 Hz), 77.1,
67.4, 60.8, 50.9, 49.7, 46.2, 37.7, 35.2, 25.5; HRMS (ES+): m/z [M +
H]+ calcd for C24H28FN6O2, 451.2252; found, 451.2234.
JCF 23.9 Hz), 109.1 (d, JCF 11.2 Hz), 72.6, 67.2, 55.5, 52.4, 46.2, 25.5,
5927
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Article
N-(4-Fluoro-3-(3-(isopropylamino)imidazo[1,2-a]pyrimidin-
7-yl)phenyl)pyrrolidine-1-carboxamide (45). 45 was synthesized
by an analogous method to 39, from 100d (0.27 m, 0.64 mmol) and
10% activated palladium on carbon (0.034 g, 0.03 mmol), followed by
DIPEA (233 μL, 1.33 mmol), CDI (0.216 g, 1.33 mmol), and
pyrrolidine (111 μL, 1.33 mmol), purifying by prep. HPLC to give 45
(0.056 g, 0.15 mmol, 22%). 1H NMR (DMSO-d6): δ 8.61 (d, J = 7.3
Hz, 1H), 8.41 (s, 1H), 8.20−8.16 (m, 1H), 7.78−7.73 (m, 1H), 7.35
(d, J = 7.0 Hz, 1H), 7.23 (dd, J = 9.5 and 10.9 Hz, 1H), 7.16 (s, 1H),
5.37 (d, J = 6.3 Hz, 1H), 3.56−3.46 (m, 1H), 3.42−3.36 (m, 1H),
1.91−1.84 (m, 4H), 1.22 (d, J = 6.3 Hz, 6H); 13C NMR (DMSO-d6):
δ 154.7, 154.4, 148.8, 142.9, 138.0, 131.3, 130.6, 125.5 (d, JCF = 12.1
Hz), 122.7 (d, JCF = 8.1 Hz), 121.1, 118.8, 116.5 (d, JCF = 23.9 Hz),
108.0 (d, JCF = 11.3 Hz), 46.6, 46.2, 25.5, 23.2; HRMS (ES+): m/z
[M + H]+ calcd for C20H24FN6O, 383.1996; found, 383.1999.
N-(4-Fluoro-3-(3-((4-methoxybutan-2-yl)amino)imidazo-
[1,2-a]pyrimidin-7-yl)phenyl)pyrrolidine-1-carboxamide (46).
46 was prepared by an analogous method to 39 from 100e (0.24 g,
0.53 mmol) and 10% activated palladium on carbon (0.028 g, 0.03
mmol), followed by DIPEA (0.188 mL, 1.08 mmol), CDI (0.175 g,
1.08 mmol), and pyrrolidine (0.090 mL, 1.08 mmol), purifying by
prep. HPLC to yield 46 (0.098 g, 0.24 mmol, 44%). 1H NMR
(DMSO-d6): δ 8.64 (d, J = 7.2 Hz, 1H), 8.41 (s, 1H), 8.20−8.17 (m,
1H), 7.79−7.74 (m, 1H), 7.38−7.35 (m, 1H), 7.27−7.20 (m, 2H),
5.39 (d, J = 6.5 Hz, 1H), 3.57−3.50 (m, 1H), 3.44−3.32 (m, 5H,
under solvent signal), 3.31−3.28 (m, 4H), 1.90−1.83 (m, 4H), 1.19
(d, J = 6.4 Hz, 3H); 13C NMR (DMSO-d6): δ 156.7, 154.7, 154.4,
149.1, 143.0, 137.9, 130.9 (d, JCF = 11.1 Hz), 125.5 (d, JCF = 11.8
Hz), 122.8 (d, JCF = 7.4 Hz), 121.1, 119.3, 116.7 (d, JCF = 23.8 Hz),
108.1 (d, JCF = 11.3 Hz), 76.2, 58.8, 50.7, 46.2, 25.5, 18.4; HRMS
(ES+): m/z [M + H]+ calcd for C21H26FN6O2, 413.2101; found,
413.2083.
University of Dundee, Dundee DD1 5EH, U.K.;
orcid.org/0000-0002-3171-3519
Fabio Zuccotto − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.;
orcid.org/0000-0002-3888-7423
Iva Lukac − Drug Discovery Unit, Wellcome Centre for Anti-
Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Peter G. Dodd − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Eun-Jung Ko − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.;
orcid.org/0000-0003-1222-1938
Sujatha Manthri − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Kate McGonagle − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.;
orcid.org/0000-0001-8485-7180
Maria Osuna-Cabello − Drug Discovery Unit, Wellcome
Centre for Anti-Infectives Research, Division of Biological
Chemistry, University of Dundee, Dundee DD1 5EH, U.K.
Jennifer Riley − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Caterina Pont − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Frederick Simeons − Drug Discovery Unit, Wellcome Centre
for Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Laste Stojanovski − Drug Discovery Unit, Wellcome Centre
for Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
John Thomas − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Stephen Thompson − Drug Discovery Unit, Wellcome Centre
for Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Elisabet Viayna − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00047.
■
sı
Experimental information, synthesis of intermediates,
methods for parasitology and modeling, molecular ESP
maps and HPLC traces of key compounds, in vitro and
in vivo assays, and in vivo pharmacokinetics (PDF)
Homology model of L. donovani protease subunits β4
and β5 (PDB)
Molecular formula strings for key compounds (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Maria Marco − Global Health R&D, GlaxoSmithKline, Tres
Cantos 28760, Spain; orcid.org/0000-0002-2442-0021;
Phone: +34 650220972; Email: maria.m.marco@gsk.com
Ian H. Gilbert − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.;
orcid.org/0000-0002-5238-1314; Phone: +44 1382
386240; Email: i.h.gilbert@dundee.ac.uk
Jose M. Fiandor − Global Health R&D, GlaxoSmithKline,
Tres Cantos 28760, Spain
Julio Martin − Global Health R&D, GlaxoSmithKline, Tres
Cantos 28760, Spain; orcid.org/0000-0003-1887-0859
Paul G. Wyatt − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.;
orcid.org/0000-0002-0397-245X
Timothy J. Miles − Global Health R&D, GlaxoSmithKline,
Tres Cantos 28760, Spain; orcid.org/0000-0001-7407-
7404
Kevin D. Read − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.;
orcid.org/0000-0002-8536-0130
Authors
Michael Thomas − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.;
orcid.org/0000-0003-0377-0281
Stephen Brand − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
University of Dundee, Dundee DD1 5EH, U.K.
Manu De Rycker − Drug Discovery Unit, Wellcome Centre for
Anti-Infectives Research, Division of Biological Chemistry,
5928
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J. Med. Chem. 2021, 64, 5905−5930

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Simeons, F. R. C.; Manthri, S.; MacLean, L. M.; Zuccotto, F.;
Homeyer, N.; Pflaumer, H.; Boesche, M.; Sastry, L.; Connolly, P.;
Albrecht, S.; Berriman, M.; Drewes, G.; Gray, D. W.; Ghidelli-Disse,
S.; Dixon, S.; Fiandor, J. M.; Wyatt, P. G.; Ferguson, M. A. J.;
Fairlamb, A. H.; Miles, T. J.; Read, K. D.; Gilbert, I. H. Cyclin-
dependent kinase 12 is a drug target for visceral leishmaniasis. Nature
2018, 560, 192−197.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00047
Author Contributions
§M.T. and S.B. contributed equally to the project.
Notes
The authors declare the following competing financial
interest(s): Several authors have shares in GlaxoSmithKline.
All regulated procedures on living animals in Dundee were
carried out under the authority of a project license issued by
the Home Office under the Animals (Scientific Procedures)
Act 1986, as amended in 2012 (and in compliance with EU
Directive EU/2010/63). License applications have been
approved by the University’s Ethical Review Committee
(ERC) before submission to the Home Office. The ERC has
a general remit to develop and oversee policy on all aspects of
the use of animals on University premises and is a
subcommittee of the University Court, its highest governing
body. All animal studies were reviewed by GlaxoSmithKline’s
(GSK) internal ethical review committee and performed in
accordance with Animals (Scientific Procedures) Act 1986 and
the GSK Policy on the Care, Welfare, and Treatment of
Laboratory Animals (UK 1986).
(7) Thomas, M. G.; De Rycker, M.; Ajakane, M.; Albrecht, S.;
́
Alvarez-Pedraglio, A. I.; Boesche, M.; Brand, S.; Campbell, L.;
Cantizani-Perez, J.; Cleghorn, L. A. T.; Copley, R. C. B.; Crouch, S.
D.; Daugan, A.; Drewes, G.; Ferrer, S.; Ghidelli-Disse, S.; Gonzalez,
S.; Gresham, S. L.; Hill, A. P.; Hindley, S. J.; Lowe, R. M.; MacKenzie,
C. J.; MacLean, L.; Manthri, S.; Martin, F.; Miguel-Siles, J.; Nguyen,
V. L.; Norval, S.; Osuna-Cabello, M.; Woodland, A.; Patterson, S.;
Pena, I.; Quesada-Campos, M. T.; Reid, I. H.; Revill, C.; Riley, J.;
Ruiz-Gomez, J. R.; Shishikura, Y.; Simeons, F. R. C.; Smith, A.; Smith,
V. C.; Spinks, D.; Stojanovski, L.; Thomas, J.; Thompson, S.;
Underwood, T.; Gray, D. W.; Fiandor, J. M.; Gilbert, I. H.; Wyatt, P.
G.; Read, K. D.; Miles, T. J. Identification of GSK3186899/
DDD853651 as a Preclinical Development Candidate for the
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Yeh, V.; Groessl, T.; Federe, G.; Koh, H. X. Y.; Venable, J. D.;
Bursulaya, B.; Shapiro, M.; Mishra, P. K.; Spraggon, G.; Brock, A.;
Mottram, J. C.; Buckner, F. S.; Rao, S. P. S.; Wen, B. G.; Walker, J. R.;
Tuntland, T.; Molteni, V.; Glynne, R. J.; Supek, F. Proteasome
inhibition for treatment of leishmaniasis, Chagas disease and sleeping
sickness. Nature 2016, 537, 229−233.
(9) Nagle, A.; Biggart, A.; Be, C.; Srinivas, H.; Hein, A.; Caridha, D.;
Sciotti, R. J.; Pybus, B.; Kreishman-Deitrick, M.; Bursulaya, B.; Lai, Y.
H.; Gao, M.-Y.; Liang, F.; Mathison, C. J. N.; Liu, X.; Yeh, V.; Smith,
J.; Lerario, I.; Xie, Y.; Chianelli, D.; Gibney, M.; Berman, A.; Chen, Y.-
L.; Jiricek, J.; Davis, L. C.; Liu, X.; Ballard, J.; Khare, S.; Eggimann, F.
K.; Luneau, A.; Groessl, T.; Shapiro, M.; Richmond, W.; Johnson, K.;
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ACKNOWLEDGMENTS
■
We would like to acknowledge the Wellcome Trust for the
support (092340, 100476, 105021, 203134, and 204672). We
thank Gina MacKay, Daniel Fletcher, Darren Edwards, and
Denise Barrett for performing HRMS analyses and for
assistance with performing other NMR and MS analyses, the
Galchimia chemistry team for synthesizing key compounds,
Susan Wyllie and team for elucidating the MoA, and Alastair
Pate, Francesco Gastaldello, James Burkinshaw, and Kashish
Sharma for data management.
ABBREVIATIONS
■
CLND, chemiluminescent nitrogen detection; ESP, electro-
static surface potential; FaSSIF, fasted state simulated intestinal
fluid; FMO, Frontier molecular orbital method; INMAC,
intracellular L. donovani assay; MoA, mechanism of Action;
PFI, property forecast index; PIE, PIEDA, pair interaction
energy decomposition analysis; VL, visceral leishmaniasis
(10) Wyllie, S.; Brand, S.; Thomas, M.; De Rycker, M.; Chung, C.-
w.; Pena, I.; Bingham, R. P.; Bueren-Calabuig, J. A.; Cantizani, J.;
Cebrian, D.; Craggs, P. D.; Ferguson, L.; Goswami, P.; Hobrath, J.;
Howe, J.; Jeacock, L.; Ko, E.-J.; Korczynska, J.; MacLean, L.; Manthri,
S.; Martinez, M. S.; Mata-Cantero, L.; Moniz, S.; Nuhs, A.; Osuna-
̈
Cabello, M.; Pinto, E.; Riley, J.; Robinson, S.; Rowland, P.; Simeons,
F. R. C.; Shishikura, Y.; Spinks, D.; Stojanovski, L.; Thomas, J.;
Thompson, S.; Viayna Gaza, E.; Wall, R. J.; Zuccotto, F.; Horn, D.;
Ferguson, M. A. J.; Fairlamb, A. H.; Fiandor, J. M.; Martin, J.; Gray,
D. W.; Miles, T. J.; Gilbert, I. H.; Read, K. D.; Marco, M.; Wyatt, P.
G. Preclinical candidate for the treatment of visceral leishmaniasis that
acts through proteasome inhibition. Proc. Natl. Acad. Sci. U.S.A. 2019,
116, 9318−9323.
(11) Sykes, M. L.; Baell, J. B.; Kaiser, M.; Chatelain, E.; Moawad, S.
R.; Ganame, D.; Ioset, J.-R.; Avery, V. M. Identification of compounds
with anti-proliferative activity against Trypanosoma brucei brucei
strain 427 by a whole cell viability based HTS campaign. PLoS
Neglected Trop. Dis. 2012, 6, No. e1896.
(12) Hwang, J. Y.; Smithson, D. C.; Holbrook, G.; Zhu, F.;
Connelly, M. C.; Kaiser, M.; Brun, R.; Kiplin Guy, R. Optimization of
the electrophile of chloronitrobenzamide leads active against
Trypanosoma brucei. Bioorg. Med. Chem. Lett. 2013, 23, 4127−4131.
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McQueen, J.; Duster, N. A.; Nagle, A.; Supek, F.; Molteni, V.;
Wenzler, T.; Brun, R.; Glynne, R.; Buckner, F. S.; Gelb, M. H.
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