Discovery of N-Alkyl Piperazine Side Chain Based CXCR4 Antagonists with Improved Drug-like Properties

Article abstract of DOI:10.1021/acsmedchemlett.8b00030

A novel series of CXCR4 antagonists with piperidinyl and piperazinyl alkylamine side chains designed as butyl amine replacements are described. Several of these compounds showed similar activity to the parent compound TIQ-15 (5) in a SDF-1 induced calcium flux assay. Preliminary structure-activity relationship investigations led us to identify a series containing N-propyl piperazine side chain analogs exemplified by 16 with improved off-target effects as measured in a muscarinic acetylcholine receptor (mAChR) calcium flux assay and in a limited drug safety panel screen. Further efforts to explore SAR and optimize drug properties led to the identification of the N′-ethyl-N-propyl-piperazine tetrahydroisoquinoline derivative 44 and the N-propyl-piperazine benzimidazole compound 37, which gave the best overall profiles with no mAChR or CYP450 inhibition, good permeability in PAMPA assays, and metabolic stability in human liver microsomes.

Full text of DOI:10.1021/acsmedchemlett.8b00030

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Discovery of N-Alkyl Piperazine Side Chain Based
CXCR4 Antagonists with Improved Drug-like Properties
Yesim Altas Tahirovic, Valarie M Truax, Robert James Wilson, Edgars Jecs, Huy
H. Nguyen, Eric J Miller, Michelle Bora Kim, Katie M. Kuo, Tao Wang, Chi S. Sum,
Mary Ellen Cvijic, Gretchen M Schroeder, Lawrence J Wilson, and Dennis C. Liotta
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00030 • Publication Date (Web): 09 Apr 2018
Downloaded from http://pubs.acs.org on April 10, 2018
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ACS Medicinal Chemistry Letters
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Discovery of N-Alkyl Piperazine Side Chain Based CXCR4 Antago-
nists with Improved Drug-like Properties
Yesim Altas Tahirovic†, Valarie M. Truax†, Robert J. Wilson†, Edgars Jecs†, Huy H. Nguyen†, Eric J. Miller†, Michelle B.
Kim†, Katie M. Kuo†, Tao Wang‡, Chi S. Sum‡, Mary E. Cvijic‡, Gretchen M. Schroeder‡, Lawrence J. Wilson†*, Dennis C.
Liotta†*
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†Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta GA 30322, USA
‡BristolꢀMyers Squibb R&D, US Route 206 and Province Line Road, Princeton, New Jersey 08543ꢀ4000, USA
*Corresponding authors
KEYWORDS: CXCR4 receptor, CXCR4 antagonists, piperazine, tetrahydroisoquinoline, Muscarinic receptor, calcium flux,
liver microsomes, PAMPA, CYP450
ABSTRACT: A novel series of CXCR4 antagonists with piperidinyl and piperazinyl alkylamine side chains designed as butyl
amine replacements are described. Several of these compounds showed similar activity to the parent compound TIQꢀ15 (5) in a
SDFꢀ1 induced calcium flux assay. Preliminary SAR investigations led us to identify a series containing Nꢀpropyl piperazine side
chain analogs exemplified by 16 with improved offꢀtarget effects as measured in a muscarinic acetylcholine receptor (mAChR)
calcium flux assay and in a limited drug safety panel screen. Further efforts to explore SAR and optimize drug properties led to the
identification of the N’ꢀethylꢀNꢀpropylꢀpiperazine tetrahydroisoquinoline derivative (44) which gave the best overall profile with no
mAChR or CYP450 inhibition, good permeability in PAMPA assays and metabolic stability in human liver microsomes.
The CꢀXꢀC chemokine receptor type 4 (CXCR4) is a seven
transmembrane Gꢀ protein coupled receptor (GPCR) and is
classified as a family I or rhodopsinꢀlike GPCR family.1ꢀ2 The
endogenous ligand CXCL12, or stromal cellꢀderived factorꢀ1
(SDFꢀ1), plays a vital role in the homing of hematopoietic
stem cells (HSCs) to the bone marrow and HSC quiescence.3ꢀ5
When SDFꢀ1 binds to CXCR4 it activates different signaling
pathways which lead to biological responses such as chemoꢀ
taxis, cell survival and proliferation, intracellular calcium flux
and gene transcription.6 Levels of CXCR4 expression are low
in healthy tissues, but this receptor is upregulated in ≥ 48 canꢀ
cer types, and has further been demonstrated as a prognostic
marker in breast, lung, ovarian, prostate and colorectal canꢀ
cers, as well as in hematological cancers like leukemia.7 Orꢀ
gans and tissues that express high levels of SDFꢀ1, such as the
liver, lung, bone marrow, and lymph nodes attract the migraꢀ
tion of CXCR4ꢀexpressing cancer cells in cancer metastasis.7
by modifying structural features of 2 resulted in the novel piꢀ
perazineꢀcontaining scaffold exemplified by 3 (GSK812397).
Previous research of CXCR4 antagonists has resulted in severꢀ
al types of small molecule inhibitors such as AMD3100 (1,
Plerixafor), AMD11070 (2), GSK812397 (3), and IT1t (4)
(Figure 1). Of these the first FDA approved CXCR4 antagoꢀ
nist drug was Plerixafor (1, AMD3100), which was first deꢀ
veloped as an antiꢀHIV agent functioning as a potent antagoꢀ
nist, but drug toxicity prevented it from being administered
daily.8ꢀ9 Aside from antiꢀHIV activity, 1 also showed the moꢀ
bilization of hematopoietic stem cells (HSCs). Clinical invesꢀ
tigations of Plerixafor led to its FDAꢀapproval for hematopoiꢀ
etic stem cell mobilization in nonꢀHodgkin`s lymphoma and
multiple myeloma patients with autologous transplants.10 The
poor oral bioavailability displayed by 1 prompted the discovꢀ
ery of 2 as the first orally active CXCR4 antagonist, which is
currently in clinical trials.6, 11ꢀ13 Efforts to increase the potency
Figure 1. Selected small molecule CXCR4 antagonists.
Alternatively, the isothiourea series typified by 4 (IT1t) was
discovered through high throughput screening efforts.12ꢀ15 As
the benzimidazole ring of 2 was considered to be the most
likely source of reported offꢀtarget activity, much effort was
exerted to replace this motif. Our lab discovered that a tetraꢀ
hydroisoquinoline ring, exemplified by our parent compound
TIQꢀ15 (5), was a suitable replacement of the benzimidazole
core of 2. As previously reported, 5 is a potent and selective
CXCR4 antagonist with good drugꢀlike properties.16 Our efꢀ
forts to improve the ADMET properties of compound 5, mainꢀ
ly the CYP450 2D6 inhibition and poor cell permeability, by
structural changes to the nꢀbutyl amine side chain have recentꢀ
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ACS Medicinal Chemistry Letters
ly been reported.17,18 In this report, we describe synthetic efꢀ
Page 2 of 7
parent butylamine side chain, but with limited free rotation.
The protected ethyl piperidine intermediates 19a and 19b were
prepared via a reductive amination of 6 with (S)ꢀ18a and (R)ꢀ
18b, respectively. Protecting group removal from 19a and 19b
yielded the ethyl piperidine analogs 20 and 21.
1
2
3
4
5
6
7
8
forts focused on replacement of the nꢀbutylamine side chain
with various alkylꢀsubstituted heterocyclic side chains, such as
alkyl piperidines and piperazines, and replacement of the tetꢀ
rahydroisoquinoline ring with different heterocycles with the
same goals of improving CYP 2D6 inhibition and low permeꢀ
ability.
Table 1. Biological data of compounds from Scheme 1.
Scheme 1
CYP450
2D6
PAMPA
pH 7.4
CXCR4
Ca2+ Flux
IC50 (nM)
Microsomal
Stability
(H/R/M) a
Cpd.
No.
Boc
N
IC50 (µM)b Pc (nm/s)
9
OHC
X
n
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7-10
N
N
N
N
6
77/37/17
86/65/13
66/17/32
96/62/79
100/100/92
82/15/96
75/28/23
93/55/56
0.32
1.87
1.04
4.21
2.14
1.43
0.03
0.11
0
0
5
NH
N
i or ii
X
n
65
13
14
15
16
17
20
21
Boc
Boc
N
Boc
Br
N
150
553
17
19
10
0
N
11
Boc
6
12a (n=0, X=CH)
12b (n=1, X=CH)
12c (n=1, X=N)
12d (n=2, X=N)
12e (n=3, X=N)
Boc
66
14
0
*
OHC
N
iv or v
161
18a (*=S)
18b (*=R)
16
55
iii
a Liver microsomal stability measured as percent remaining by
LCꢀMS after ten minutes in Human (H), Rat (R) and Mouse
(M) microsomes. b All compounds have mAChR Ca2+ Flux
IC50 >14 ꢀM and CYP450 3A4 IC50 >12 ꢀM.
N
N
Boc
*
N
N
Boc
N
N
X
n
NH
HN
Several in vitro pharmacological assays were used to screen
these derivatives of 5 and the results revealed a range of poꢀ
tencies and divergent structureꢀactivity relationships (SAR).
All compounds were tested as both agonists and antagonists in
CXCR4 calcium flux assays and none showed any agonistic
activity (Table 1). In addition, compounds were screened
against the muscarinic receptor to measure potential offꢀtarget
GPCR activity and all compounds were found to be inactive in
this assay. Replacing the butylamine side chain with 4ꢀmethyl
piperidine (13), 4ꢀethyl piperidine (14), (S)ꢀ3ꢀethyl piperidine
(20) or (R)ꢀ3ꢀethyl piperidine (21) resulted in robust CXCR4
inhibition, albeit with a slight loss in potency (2ꢀ3 fold) versus
5. We were encouraged to observe a decrease in CYP450 2D6
IC50 values for 13 and 14 relative to 5, however, 20 and 21
had slightly increased 2D6 activity. Also screened were the 2
through 4 carbon linked Nꢀpiperazines (15-17). Although all
three had reduced CYP450 2D6 activity with IC50 values up to
10ꢀfold higher than 5, propyl piperazine analog 16 proved to
be the best in terms of CXCR4 potency. Overall, these efforts
led to the discovery of compounds 21 and 16, which exhibited
the most potent CXCR4 antagonist activity of all compounds
screened in this initial series. In comparing piperidines to
piperazines, an increase in 2D6 for piperidine 21 and the lower
calcium flux value of piperidine 13 showed the limitations in
trying to optimize the piperidine series further. In contrast, we
found that the piperazine side chain analogs (15ꢀ17) had better
liver microsomal stability than the parent compound 5, as well
as piperidine analogs 13, 14, 20, and 21. In general, the piperiꢀ
dines were inferior compounds, having more problematic 2D6
potencies and liver microsomal stability across species. For
example, ethyl piperazine 15 was less potent than the ethyl
piperidine 14, but a slight improvement in metabolic stability
and CYP450 2D6 inhibition was observed. The propyl and
butyl piperazines (16, 17) had better onꢀtarget potency than
ethyl piperazine 15, and also maintained good microsomal
19a (*=S)
19b (*=R)
13 (n=0, X=CH)
14 (n=1, X=CH)
15 (n=1, X=N)
16 (n=2, X=N)
17 (n=3, X=N)
iii
N
N
*
NH
HN
20 (*=S)
21 (*=R)
Reagents: (i) 7 (n=0, X=CH), 8 (n=1, X=CH), 9 (n=1, X=N), 10
(n=3, X=N), NaBH(OAc)3, DCE, 75ꢀ95% yield; (ii) tertꢀbutylꢀ4ꢀ
(3ꢀbromopropyl)piperazineꢀ1ꢀcarboxylate (11), DIPEA, MeCN,
65oC, 73% yield; (iii) TFA, DCM, r.t. 35ꢀ48% yield; (iv) tertꢀ
(S)ꢀ3ꢀ(2ꢀoxoethyl)piperidineꢀ1ꢀcarboxylate
NaBH(OAc)3, DCE, 82% yield; (v) tertꢀbutyl (R)ꢀ3ꢀ(2ꢀoxoethyl)ꢀ
piperidineꢀ1ꢀcarboxylate (18b), NaBH(OAc)3, DCE, r.t.
butyl
(18a),
Synthetic modifications to the lead tetrahydroisoquinoline
scaffold exemplified by 5 began with key intermediate 6.16
We first synthesized several piperidine and piperazine side
chain analogs with varying carbon chain lengths, similar to the
optimization efforts of the butylamine side chain of 2.11 Reꢀ
ductive amination reaction of 6 with Boc protected piperidinyl
aldehydes (7, 8) and Boc protected piperazinyl aldehydes (9,
10) gave compounds 12a-c and 12e, respectively (Scheme 1).
Subsequent removal of the Boc group with trifluoroacetic acid
gave desired products 13-15, and 17. Alternatively, an alkylaꢀ
tion reaction of 6 was necessary for the synthesis of propyl
piperazine intermediate 12d. The secondary amine 6 was
treated with alkyl bromide 11 to give 12d, followed by deproꢀ
tection of the Boc groups to yield 16. A series of enantiomeriꢀ
cally pure piperidines containing an ethyl linker (20 and 21)
was also explored. These side chains closely resembled the
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ACS Medicinal Chemistry Letters
Reagents: (i) 11, DIPEA, MeCN, r.t., 75% yield; (ii) 24 or 27,
stability. Overall, propyl piperazine 16 was the best compound
NaBH(OAc)3, DCE, r.t., 16ꢀ26% yield; (iii) TFA, DCM, r.t., 85ꢀ
98% yield.
1
2
3
from these first generation results.
Table 2. Offꢀtarget panel of compounds 5, 16 and 21.
After finding that the propyl piperazine side chain delivered
good CXCR4 activity, improved metabolic stability, and reꢀ
duced off target activity, we focused our SAR on substituting
different heterocycles for the bottom THIQ ring system. This
effort was also designed to compare the different heterocycles
used in CXCR4 medicinal chemistry with the novel propyl
piperazine substitution versus the butyl amine. These included
pyridine for phenyl swaps and various aromatic heterocyclic
substitutions and the previously reported benzimidazole (as
that found in 2) and isoquinoline rings.11, 19 The synthesis of
the THIQ and alternate heterocyclic scaffolds began with the
alkylation of compound 22 with the side chain alkyl bromide
11 to yield the intermediate 23 (Scheme 2). Compound 23 was
then either used in a reductive amination reaction with its corꢀ
responding aldehyde to give compounds 25, 28, 30, and 34 or
alkylated with the corresponding chloride to give compounds
32, 36 or 38 (Schemes 2-3). Diastereomers 28 and 30 were
readily separable by column chromatography. A final deproꢀ
tection of the Boc group furnished the analogs 26, 29, 31, 33,
35, 37 and 39.
4
5
6
7
8
9
Offꢀtarget Assay
IC50 (µM)
16
5
21
αꢀAdrenergic 2A
ꢀꢀOpioid
3.7
>30
5.9
14.9
>30
>30
>30
11.3
11.3
10.7
>30
nAChR α1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Acetylcholine esterase
>30
In order to further distinguish between the piperazine and piꢀ
peridine sideꢀchain series, we chose two compounds with the
best profiles for each heterocycle type (16, 21) as well as TIQꢀ
15 (5) for comparison to test for additional nonꢀCXCR4 pharꢀ
macology beyond the CYP450 2D6 and Muscarinic receptor
(mAChR) assays. A limited panel of four assays (Table 2)
were selected based on proteins which are known to interact
with many nitrogenꢀbased chemoꢀtypes (such as phenethyl
amines, pyridines, piperazines and piperidines) similar to
those found in our compounds. As there are a vast number of
possibilities for crossꢀpharmacology and our goal was not
New Chemical Entity (NCE) candidate selection at this stage,
this panel was very limited to convey potential liabilities.
First, no responses were observed lower than 3 ꢀM for all
compounds indicating a therapeutic index of at least 100ꢀfold
compared to the CXCR4 Ca2+ responses. In the two class A
GPCRs (adrenergic, opioid) both 5 and 21 had similar reꢀ
sponses in the single digit ꢀM ranges but still with therapeutic
indices at 100 to 1,000 fold. Furthermore, compound 21 had
similar responses in the nicotinic receptor and acetyl choline
esterase with IC50 values in the 10 ꢀM range while both 5 and
16 were devoid of this activity up to 30 ꢀM. In comparison,
the propyl piperazine 16 had the least off target effects with all
four assays showing no responses up to 30 ꢀM. Although piꢀ
peridine 21 had slightly improved PAMPA permeability, all
four offꢀtarget effects in the 6ꢀ11 ꢀM range indicating a less
favorable profile even compared to TIQꢀ15. Since the piperaꢀ
zine based side chain had the best profile, we selected comꢀ
pound 16 as a more selective agent for continued SAR exploꢀ
ration.
Table 3. Biological data of compounds from Schemes 2 and 3.
CYP450
2D6
IC50 (ꢀM)b,c
PAMPA
pH 7.4
CXCR4
Ca2+ Flux
IC50 (nM)
Microsomal
Stability
(H/R/M) a
Cpd.
No.
Pc (nm/s)
5
63/<1/<1
99/88/88
79/79/78
100/87/63
100/68/78
66/59/74
63/29/68
50/21/45
1.64
1.40
>20
>20
>20
2.96
>20
>20
86
16
9
2
1154
171
2639
4316
17
26
29
31
33
35
37
39
0
31
243
146
ndd
45
1199
a Liver microsomal stability measured as percent remaining by
LCꢀMS after ten minutes in Human (H), Rat (R) and Mouse
(M). bAll compounds have mAChR Ca2+ Flux IC50 >17 µM
except 35 (mAChR IC50 = 3.2 µM). cAll CYP450 3A4 values
IC50 >20 µM except 2 (>6.67 µM), 37 (12.7 µM) and 39 (9.4
µM). dnd=not determined.
Scheme 2
i
Scheme 3
Boc
N
11
N
N
NH2
HN
N
22
23
X
CHO
Boc
*
N
ii
24 (X=CH,*=S)
27 (X=N,*=rac)
R
N
R
N
N
N
N
N
N
N
N
+
R
R
N
N
X
28 (R=Boc)
29 (R=H)
R=Boc [25 (X=CH)/30 (X=N)]
R=H [26 (X=CH)/31 (X=N)]
iii
iii
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R
R
Cbz
N
N
N
1
2
3
4
5
6
7
8
N
N
N
N
N
N
N
i
N
N
N
N
N
N
N
N
NH
Boc
Boc
N
R
N
N
Br
Cbz
32 (R=Boc)
33 (R=H)
40
iii
36 (R=Boc)
37 (R=H)
iii
6
41
N
ii
Cl
Cl
iiꢁiii
N
Boc
N
N
ii
ii
9
Boc
R1
R1
N
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
N
N
N
N
HN
N
N
N
N
N
iv
Cl
S
R2
23
Boc
N
N
N
i
CHO
43 (R1=CH3, R2=H)
44 (R1=C2H5, R2=H)
45 (R1=i-Pr, R2=H)
42a (R1=CH3)
42b (R1=C2H5)
42c (R1=iPr)
R
R
v
46 (R1=C(O)NH2, R2=H)
N
N
N
42d (R1=C(O)NH2)
N
N
N
47 (R1=
, R2=H)
42e (R1=
)
N
N
N
S
N
48 (R1=R2=CH3)
Reagents (i) benzylꢀ4ꢀ(3ꢀbromopropyl)piperazineꢀ1ꢀcarboxylate)
(40), DIPEA, MeCN, 65oC, 78% yield; (ii) Pd(OH)2, HCOONH4,
EtOH, reflux; (iii) HCHO (for 42a), CH3ꢀCHO (for 42b), acetone
(for 42c), NaBH(OAc)3, DCE, r.t. or TMSNCO, DIPEA, THF
(for 42d) or (1ꢀethoxycyclopropoxy)trimethylsilane, AcOH,
NaBH3CN, MeOH (for 42e), r.t.; (iv) TFA, DCM, 40ꢀ61% yield
over 3 steps (v) HꢀCHO, NaBH(OAc)3, DCE, r.t.
34 (R=Boc)
35 (R=H)
38 (R=Boc)
39 (R=H)
iii
iii
Reagents: (i) isoquinolineꢀ3ꢀcarbaldehyde, NaBH(OAc)3, DCE,
r.t.; (ii) NꢀBocꢀ2ꢀchloromethylbenzimidazole11, 2ꢀ(chloromethꢀ
yl)benzo[d]thiazole, or 2ꢀ(chloromethyl)pyrimidine hydroꢀ
chloride, DIPEA, MeCN, 65oC; (iii) TFA, DCM, r.t., 22ꢀ52%
yield over two steps.
Since compound 16 had low permeability observed in the
PAMPA assay, we decided to continue to alter the structure of
the propyl piperazine side chain of this compound by focusing
on the terminal piperazine nitrogen. Altering the basicity of
the piperazine nitrogen and reducing Hꢀbond donor count have
been shown to improve permeability in other efforts.20 A seꢀ
ries of Nꢀsubstituted propyl piperazines 43-48 were syntheꢀ
sized to probe how substitution at the terminal piperazine
might alter CXCR4 activity, metabolic stability and permeaꢀ
bility. The side chain benzyl 4ꢀ(3ꢀbromopropyl)piperazineꢀ1ꢀ
carboxylate 40 was synthesized using Cbz protected piperaꢀ
zine and 1,3ꢀdibromopiperazine (see Supporting Information).
Alkylation of 6 with 40 gave intermediate 41 (Scheme 4), and
deprotection of the Cbz group with Pd/C followed by reducꢀ
tive amination with either formaldehyde, acetaldehyde, aceꢀ
tone or (1ꢀethoxycyclopropoxy)trimethylsilane furnished
compounds 42a-c and 42e respectively whereas reaction with
(trimethylsilyl)isocyanate resulted in compound 42d. Boc
deprotection of compounds 42a-e provided compounds 43-47.
Reductive methylation of 43 gave compound 48.
First, the TIQ substitutions yielded further SAR. Pharmacoꢀ
logical results of 26, the epimer of 16, demonstrated a nearly
100ꢀfold reduction in CXCR4 activity, illustrating a preference
for the Rꢀisomer (Table 3) which was similarly observed durꢀ
ing the discovery of TIQꢀ15.16 The pyridine for phenyl swap
(29, 31) resulted in a compound (29) with 10ꢀfold lower
CXCR4 activity but a marked improvement in CYP 2D6. For
the heteroaromatic substitution SAR, the pyrimidine (33),
isoquinoline (35), benzimidazole (37), and benzothiazole (39)
analogs were tested. The isoquinoline 35, synthesized as a
replacement for the TIQ ring on 16, had similar CXCR4 and
CYP450 2D6 potencies but with more potent muscarinic activꢀ
ity (3 µM versus >17 ꢀM). The muscarinic activity, although
not reported for the analogous nꢀbutyl amine compound, are
likely attributed solely to the isoquinoline ring.19 Benzimidazꢀ
ole (37) had 3ꢀfold lower CXCR4 activity compared to 35, but
showed no CYP450 2D6 or muscarinic activities. Benzothiaꢀ
zole (39) and pyrimidine (33) were much less active against
CXCR4 with both compounds having single digit micromolar
activities in the calcium flux assay. One advantage to the aroꢀ
matic heterocycle compounds was an improvement in
PAMPA permeability, especially 35 and 37 showing >100
nm/s Pc values. In this effort, the benzimidazole compound
(37), had the best overall properties with slightly higher
CXCR4 activity than 16 (2 to 3ꢀfold) but no CYP450 2D6
inhibition and acceptable PAMPA permeability. Also,
when comparing this nꢀpropyl piperazine side chain comꢀ
pound (37) to AMD11070 (2), improvements in CYP450 2D6,
liver microsomal stabilities and PAMPA are observed with a
10ꢀfold drop in CXCR4 activity. The majority of these trends
were now consistent for both THIQ and benzimidazole substiꢀ
tutions (16 and 37).
Compounds (43-48) were subsequently assessed for CXCR4
and mAChR inhibition, microsomal stability, CYP450 2D6
inhibition and PAMPA permeability. A methyl substitution on
piperazine 43 resulted in an almost 10ꢀfold loss in CXCR4
potency, increased muscarinic activity, and a reduction in miꢀ
crosomal stability In contrast, the CYP450 2D6 liability saw a
slight improvement. The biggest change was the large enꢀ
hancement in PAMPA permeability, which increased 100ꢀfold
versus 16. The compound with both the piperazine nitrogen
and THIQ nitrogen methylated (48), was less active in the
CXCR4 calcium flux assay by 20ꢀfold, but again saw an imꢀ
provement in PAMPA permeability as compared to 16. The
effect on PAMPA permeability in reduction of Hꢀbond donor
count, from two on 16 to one on 43 and then none on 48,
Scheme 4
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ACS Medicinal Chemistry Letters
showed the biggest improvement came from capping the piꢀ
15 and 2 (~10ꢀfold). In contrast, substituting the distal piperaꢀ
zine nitrogen of 16 in order to improve permeability was merꢀ
ited, providing a greater than 100ꢀfold increase in Pc values in
the PAMPA assay. This led to identification of the N’ꢀethylꢀ
substituted Nꢀpropyl piperazine compound 44, which provided
the best overall combination of properties. In comparison to
the starting butyl amine compound 5 (TIQꢀ15), compound 44
was equivalent or superior in all respects with the exception of
CXCR4 activity being 5ꢀfold less potent. However, with good
CXCR4 potency, reduced offꢀtarget effects, good human liver
microsomal stability, PAMPA permeability and greatly reꢀ
duced CYP450 2D6 activity, compounds 37 and 44 offer imꢀ
proved selections to progress into lead optimization.
1
2
3
4
5
6
7
8
perazine NꢀH. The Pc values for 43 and 48 were not substanꢀ
tially different (Table 4, 360 versus 513 nm/s) but over 100ꢀ
fold better than 16. Showing great improvement in the
PAMPA permeability, we continued our focus on the terminal
piperazine nitrogen. The ethylꢀsubstituted piperazine 44 had
better CXCR4 potency than 43, but a decrease in microsomal
stability across all three species was observed. However, this
compound was devoid of CYP450 2D6 inhibition, and addiꢀ
tionally retained the improvement in PAMPA permeability
(Table 4). Further alkyl group substitutions, including isoproꢀ
pyl (45) and cyclopropyl (47) motifs had similar results to 43,
exhibiting less potent CXCR4 inhibition and no improvement
in CYP450 2D6 or liver microsomal stability. Urea substituted
piperazine 46 demonstrated a 50ꢀfold potency loss, and also
introduced moderate muscarinic activity. Surprisingly, this
compound (46) also showed lower permeability by 10ꢀfold
versus the alkylated analogs (43-45, 47, 48) despite the nonꢀ
basic terminal piperazine nitrogen. Overall, the ethyl analog
44 was the best compound in this series, having similar potenꢀ
cy to 16 and greatly improved CYP450 2D6 and PAMPA
properties.
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16
17
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21
22
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43
44
45
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48
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When comparing the newly discovered nꢀpropylꢀpiperazine
side chain to our other nꢀbutyl amine structural changes, we
can directly determine that this current effort provides the best
overall compound identified in 44. Previously, one effort
found a cycloꢀhexyl amine compound with similar CXCR4
potency but lower 2D6 activity and PAMPA permeability than
44.17 Our other work found that substituting the butyl chain
with methyl groups and unsaturation coupled with nitrogen
substitutions provided compounds similar in CXCR4 activity
and PAMPA permeability to 44 but lower 2D6 activity.18 In
the later case, it was the nitrogen substitutions not structural
changes on the butyl amine chain that led to the improvements
over TIQꢀ15. Many of these previously described structural
variations (gemꢀdimethyls, unsaturation, nitrogen pyranyl
groups) were not attempted in the current series due to the
quick identification of compounds 37 and 44, however our
interest in these modifications may lead to further improveꢀ
ments. Future efforts, including pharmacokinetic studies and
evaluation in models of immunoꢀoncology will be reported in
due course.
Table 4. Biological data of compounds from Scheme 4.
CXCR4
Ca2+ Flux
IC50 (nM)
Microsomal
Stability
(H/R/M) a
CYP450
2D6
IC50 (ꢀM)b
PAMPA
pH 7.4
Cpd.
No.
Pc (nm/s)
125
33
72/10/53
59/1/19
46/4/58
79/4/42
21/2/3
5.60
>20
6.65
4.14
6.04
5.14
360
317
344
67
43
44
45
46
47
48
62
959
50
505
513
ASSOCIATED CONTENT
Supporting Information
536
60/2/43
a
Liver microsomal stability measured as percent remaining by
Experimental and characterization data for all new compounds
and all biological data. The Supporting Information is available
free of charge on the ACS Publications website.
LCꢀMS after ten minutes in Human (H), Rat (R) and Mouse (M).
bAll compounds have mAChR Ca2+ Flux IC50 > 17 µM except 43
(6.56 µM) and 46 (4.98 µM). All compounds have CYP450 3A4
IC50 >20 µM.
AUTHOR INFORMATION
In conclusion, a novel series of highly potent and selective
CXCR4 antagonists featuring Nꢀalkylꢀsubstituted heterocycles
as replacements of the butylamine side chain of lead comꢀ
pound TIQꢀ15 (5) was presented. The SAR around these side
chain replacements led to the Nꢀpropyl piperazine compound
16, which not only retained CXCR4 activity and improved
metabolic stability in human, rat and mouse liver microsomes,
but also reduced CYP450 2D6 activity, as compared to 5. In
addition, compound 16 had fewer offꢀtarget liabilities than the
best piperidine compound 21 or 5 (TIQꢀ15; Table 2). We
were also able to replace the THIQ bottom ring with various
heterocycles using the newly discovered nꢀpropyl piperazine
side chain with mixed results showing both the THIQ and
benzimidazole rings having the best overall properties. In fact,
the benzimidazole compound 37 had eliminated CYP450 2D6
activity with only a 3ꢀfold drop in CXCR4 potency, but did
increase CYP450 23A4 activity. Also, compound 37 had
slightly improved PAMPA permeability compared 16 likely
due to the lower basicity of the benzimidazole versus THIQ
rings. The Nꢀalkylations of the piperazine nitrogen on 37
were not carried out as it already had optimal properties and
the CXCR4 activity was already significantly higher than TIQꢀ
Corresponding Author
*Eꢀmail: ljwilso@emory.edu, dliotta@emory.edu
Author Contributions
The manuscript was written by YAT and through contributions of
all authors. All authors have given approval to the final version of
the manuscript.
Funding Sources
The authors acknowledge the use of shared instrumentation proꢀ
vided by grants from NSF (CHE1531620).
Notes
DCL is the principle investigator on a research grant from Bristolꢀ
Myers Squibb Research and Development to Emory Universiꢀ
ty. DCL, LJW, YAT, EJM, EJ, HHN, RJW, VTT, and MBK are
coꢀinventors on Emoryꢀowned Intellectual Property that includes
CXCR4 antagonists.
ACKNOWLEDGMENT
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The authors would like to acknowledge Dr. Shaoxiang Wu of
13.
Wilson, L. J.; Liotta, D. C., Emergence of Small-Molecule
Emory NMR facility, and Dr. Fred Strobel for mass spectrometry.
1
CXCR4 Antagonists as Novel Immune and Hematopoietic System
Regulatory Agents. Drug Dev. Res. 2011, 72 (7), 598-602.
2
3
4
5
6
7
8
9
ABBREVIATIONS
CXCR4, CXC chemokine receptor type 4; GPCR, G proteinꢀ
coupled receptor; CXCL12, CXC chemokine ligand type 12;
14.
Jenkinson, S.; Thomson, M.; McCoy, D.; Edelstein, M.;
Danehower, S.; Lawrence, W.; Wheelan, P.; Spaltenstein, A.;
Gudmundsson, K., Blockade of X4-Tropic HIV-1 Cellular Entry by
THQ,
5,6,7,8ꢀtetrahydroquinoline;
THIQ,
1,2,3,4ꢀ
GSK812397,
a
Potent Noncompetitive CXCR4 Receptor
tetrahydroisoquinoline; HLM, human liver microsomes; RLM, rat
liver microsomes; MLM, mouse liver microsomes; TFA, triꢀ
fluoroacetic acid; DCE, 1,2ꢀdichloroethane; mAChR, muscarinic
acetylcholine receptor; STABꢀH, sodium triacetoxyborohydride;
PAMPA, parallel artificial membrane permeability assay.
Antagonist. Antimicrob. Agents Chemother. 2010, 54 (2), 817-
824.
15.
Thoma, G.; Streiff, M. B.; Kovarik, J.; Glickman, F.;
Wagner, T.; Beerli, C.; Zerwes, H.-G., Orally Bioavailable
Isothioureas Block Function of the Chemokine Receptor CXCR4 in
Vitro and in Vivo. J. Med. Chem. 2008, 51 (24), 7915-7920.
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37
38
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43
44
45
46
47
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N
NH
N
N
N
R
N
N
N
N
N
N
N
N
NH2
N
X
HN
HN
HN
Improve:
1. CYP450 2D6
2. PAMPA
n
Het
9
Permeability
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16
17
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X= N or CH
n= 1ꢁ4
TIQ-15
44
CXCR4 Ca2+ Flux IC50= 33 nM
mAChR Ca2+ Flux IC50 > 17 ꢀM mAChR Ca2+ Flux IC50 > 17 ꢀM
37
Het= Various
N heterocycles
R= H or alkyl
CXCR4 Ca2+ Flux IC50= 6 nM
mAChR Ca2+ Flux IC50 > 17 ꢀM
CYP 450 2D6 = 0.32 ꢀM
CXCR4 Ca2+ Flux IC50= 45 nM
CYP 450 2D6 > 20 ꢀM
PAMPA (pH 7.4): Pc= 317 nm/s
CYP 450 2D6 > 20 ꢀM
PAMPA (pH 7.4): Pc= 146 nm/s
PAMPA (pH 7.4): Pc= 0 nm/s
7
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