Pyrrolidine-5,5-trans-lactams as novel mechanism-based inhibitors of human cytomegalovirus protease. Part 3: Potency and plasma stability

Article abstract of DOI:10.1016/S0960-894X(02)00294-9

Mechanism-based inhibitors of HCMV protease, which are stable to human plasma (≥20 h) and have single-figure potency in the μM range against HCMV protease, have been developed based on the dansylproline α-methyl pyrrolidine-5,5-trans-lactam nucleus.

Full text of DOI:10.1016/S0960-894X(02)00294-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1719–1722
Pyrrolidine-5,5-trans-lactams as Novel Mechanism-Based
Inhibitors of Human Cytomegalovirus Protease.
Part 3:Potency and Plasma Stability
Alan D. Borthwick,* Anne M. Exall, Terry M. Haley, Deborah L. Jackson,
Andrew M. Mason and Gordon G. Weingarten
Department of Medicinal Chemistry CVU UK, GlaxoSmithKline Research and Development, Medicines Research Centre,
Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK
Received 20 February 2002; revised 15 April 2002; accepted 22 April 2002
Abstract—Mechanism-based inhibitors of HCMV protease, which are stable to human plasma (ꢀ20 h) and have single-figure
potency in the mM range against HCMV protease, have been developed based on the dansylproline a-methyl pyrrolidine-5,5-trans-
lactam nucleus. # 2002 Elsevier Science Ltd. All rights reserved.
We recently reported the design and synthesis of a novel
class of mechanism-based inhibitors (1, 2) of the human
cytomegalovirus protease (HCMV),1,2 based on the
a-methyl pyrrolidine-5,5-trans-lactam template (Fig. 1).
These inhibitors are novel, potent in the nanomolar
range and highly selective for the viral enzyme over the
mammalian enzymes elastase, thrombin and acetylcho-
line esterase.2 Mechanism of action studies showed that
these compounds acylate the nucleophile Ser 132 pre-
sent in the catalytic triad His, His, Ser at the active site
of this viral protease.
substituent on the lactam nitrogen less electron with-
drawing.
Initially a series of a-methyl pyrrolidine-5,5-trans-lac-
tams with Cbz on the pyrrolidine nitrogen and a range
of subsituents on the lactam nitrogen were tested for
stability in human plasma (Table 1). The stability range
was:
CONHMe >> COMe ꢀ CO2Me > SO2Me
The trend was similar to that seen with the mono-b-
lactam3 inhibitors of human leukocyte elastase.
Increasingsteric bulk adjacent to the lactam carbonyl at
the C-6 position by introducinga second methyl group
gave the gem-dimethyl derivative 3 which was >6 times
more stable than the a-methyl trans-lactam 4. Similarly
These compounds have reactive functionality so they
are potentially vulnerable to metabolism. The chemical
reactivity of the trans-lactam ringwill determine the
rates of both the non-specific hydrolysis due to the
hydrolytic plasma enzymes found in the blood as well as
the acylation of HCMV protease. Thus, the goal was to
find a compound that would react with the viral enzyme
after bindingto the active site, but would be sufficiently
stable to hydrolysis by the plasma enzymes. Two ways
were investigated to increase plasma stability and yet
retain potency against the viral protease. One way was
to sterically hinder the approach of the hydrolytic
plasma enzymes to the lactam carbonyl, the other was
to make the lactam carbonyl less reactive by makingthe
*Correspondingauthor. Fax: +44-1438-763616; e-mail: adb3028@
glaxowellcome.co.uk
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00294-9

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A. D. Borthwick et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1719–1722
increasingthe steric bulk of the acyl functionality on the
lactam nitrogen of the a-methyl trans-lactam template
with the tert-butyl acyl derivative 5 also increased sta-
bility to human plasma. However, both 3 and 5 lost in
vitro activity against HCMV protease (Table 1). A
similar situation has been observed in the mono-b-lac-
tam series4 of HCMV protease inhibitors. Interestingly
the cyclopropyl carbonyl compound 6 is more potent
and slightly more stable than the methyl carbonyl com-
pound 4 (Table 1). The amide 9 was stable to human
plasma as was the phosphonate 10 and the dini-
trophenyl derivative 11 (Table 1), but all were inactive
against HCMV protease.
Steric hindrance was achieved by substitution of
the cyclopropyl ringwith methyl groups. The stability
of these derivatives to plasma increased cyclopropyl
< methylcyclopropyl < cis-dimethyl cis-cyclopropyl
< tetramethylcyclopropyl. Methylation in the cyclo-
propyl ringgave 12 and 13 with increased stability to
plasma, but the dimethyl compound lost some potency,
and the tetramethyl analogue 14 although stable to
plasma lost all activity (Table 1). A similar increase in
stability to human plasma and decrease in potency was
seen in the dansylproline series: cyclopropyl > methyl-
cyclopropyl > cis-dimethyl cis-cyclopropyl > tetra-
methylcyclopropyl with the dimethyl analogue 19 in the
dansylproline series havingthe best profile ( Ki 1.1 mM
and t1/2=20 h in plasma).
To explore this plasma stability versus in vitro activity
against HCMV protease, the more active and slightly
more stable cyclopropyl acyl derivatives were investi-
gated in both the Cbz and the more active dansylproline
a-methyl pyrrolidine-5,5-trans-lactam templates.
In contrast, replacingthe cyclopropyl carbonyl sub-
stituent in the Cbz series with an amide, an electron-
withdrawingaryl or heterocyclic ringproduced stable
compounds but they were inactive against HCMV pro-
tease. However, the increase in potency in going from
the Cbz to the dansylproline series seen with the
cyclopropyl carbonyl derivatives was developed for
these plasma stable compounds (Table 2). Thus the
N-methylamide 9 and N-benzylamide 15 were inactive
(IC50 >500 mM) in the Cbz series, but in the dansyl-
proline series both the N-methylamide 21 and N-
benzylamide 22 had low mM activity against HCMV
protease in the order CH2Ph>Me.
Table 1.
Electronegative aromatic heterocycles have been used5
to activate peptidyl ketones towards nucleophilic addi-
tion by the active-site serine hydroxyl of human neu-
trophile elastase and prolyl endopeptidase. The potency
of peptidyl a-keto heterocyclic inhibitors of the mam-
malian serine protease HLE have been correlated5a,c
with the electon-withdrawingcharacter sI of the het-
erocyclic group. Although the plasma stable para-nitro-
phenyl analogue 16 in the Cbz series was inactive the
correspondingderivative 23 in the dansylproline series
was weakly active (IC50 41 mM). The stable thiazole was
Compd
R
R1
Human8
plasma stab
t1/2 min
HCMV
protease2 pNA
assay IC50 (mM)
3
4
5
6
7
8
9
10
11
COMe
COMe
COCMe3
CO-cyclopropyl
CO2Me
Me
H
H
H
H
H
H
H
H
6.5 h
54
3.7 h
1.4 h
50
>500
40
>500
9
148
ꢁ500
>500
>500
ꢁ500
SO2Me
<5
CONHMe
PO(OMe)2
Ph2,4-(NO2)
>24 h
>24 h
>24 h
2
Table 2.
Compd
R
Human8
plasma stab
t1/2 min
HCMV2
Protease
pNA assay
IC50 (mM)
Compd
R
Human8
plasma stab
t1/2 min
HCMV protease2
pNA assay (mM)
IC50
0.34
Ki
6
CO-cyclopropyl
1.4 h
2.6 h
9.8 h
9
16
72
2
CO-cyclopropyl
CO-cyclopropyl-Me
CO-(Z) diMecyclopropyl
CO-Me4cyclopropyl
CONHMe
5 h
2.5 h
20 h
>24 h
12 h
>33 h
12
13
14
9
15
16
17
CO-cyclopropyl-Me
CO-(Z) diMecyclopropyl
CO-Me4cyclopropyl
CONHMe
18
19
20
21
22
23
24
1.4
5.1
1.1
33 h
>500
>500
250
>100
22
6.6
41
2.1
>24 h
12 h
40 h
CONHCH2Ph
Ph-4-NO2
2-Thiazole
CONHCH2Ph
Ph-4-NO2
2-Thiazole
2.6
0.4
ꢁ500
44 h
ꢁ500
>50 h

A. D. Borthwick et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1719–1722
1721
Figure 2.
inactive in the Cbz series 17 but in the dansylproline
series 24 had a Ki of 0.4 mM (IC50 2.1 mM). In the dan-
sylproline series the greater potency of the thiazole over
the para-nitrophenyl derivative is reflected in the greater
electon-withdrawingcharacter of the thiazole rgoup
sI=3.4 over the para-nitrophenyl group sI=2.3.6
protease were investigated by ESI-MS to study their
mechanism of action and to see how quickly they were
turned over by the enzyme.
The ESI-MS study of these three compounds, incubated
with d-Ala protease, has shown three modes of action.
The N-benzylamide 22 inhibits without acylation, the
dimethylcyclopropyl carbonyl compound 19 acylates
within 30 min and the enzyme is restored after 24 h, and
the thiazole 24 also acylates within 30 min but the
enzyme remains almost completely acylated for 24 h
(Fig. 2). The thiazole 24 is thus the most potent
Examples of the three classes of compounds [the dime-
thylcyclopropyl carbonyl compound 19 (Ki 1.1 mM), the
amide 22 (Ki 2.6 mM ) and the thiazole 24 (Ki 0.4 mM )],
which were stable to human plasma (ꢀ20 h) and had
single figure potency in the mM range against HCMV
Scheme 1. (a) LiHMDS (1.5 equiv)/THF, ꢂ78ꢃ then RCOOCOCMe3 (2 equiv) ꢂ78 to 0 ꢃC, 2 h; (b) p-NO2-bromobenzene or 2-bromothiazole,
CuCl, K2CO3, TDA-1, xylene; (c) NaH (0.2–0.5 equiv)/THF, then RN=C¼O (1.3–2.5 equiv), 2 h; (d) H2, 10% Pd/C, IPA: HCl/Et2O; (e) 2-(4-
methoxybenzyloxycarbonyloxyimino)-2-phenylacetonitrile, Et3N, aq dioxan; (f) TFA/rt; (g) dansylproline, TBTU, HOBT, iPr2Net, MeCN, DMF.

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A. D. Borthwick et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1719–1722
(Ki 0.4 mM) and stable (t1/2 >50 h in plasma) acylating
inhibitor of HCMV protease and is essentially stable to
turnover by the viral enzyme during24 h.
References and Notes
1. Borthwick, A. D.; Angier, S. J.; Crame, A. J.; Exall, A. M.;
Haley, T. M.; Hart, G. J.; Mason, A. M.; Pennell, A. M. K.;
Weingarten, G. W. J. Med. Chem. 2000, 43, 4452.
2. Borthwick, A. D.; Crame, A. J.; Ertl, P. F.; Exall, A. M.;
Haley, T. M.; Hart, G. J.; Mason, A. M.; Pennell, A. M. K.;
Singh, O. M. P.; Weingarten, G. W.; Woolven, J. M. J. Med.
Chem. 2002, 45, 1.
3. Shah, K. S.; Dorn, C. P.; Finke, P. E.; Hale, J. J.; Hag-
mann, W. K.; Brause, K. A.; Chandler, G. O.; Kissenger,
A. L.; Ashe, B. M.; Weston, H.; Knight, W. B.; Maycock,
A. L.; Dellea, P. S.; Flechter, D. S.; Hand, K. M.; Mumford,
R. A.; Underwood, D. J.; Doherty, J. B. J. Med. Chem. 1992,
35, 3745.
4. Borthwick, A. D.; Weingarten, G. G.; Haley, T. M.;
Tomaszewski, M.; Wang, W.; Hu, Z.; Bedard, J.; Jin, H.;
Yeun, L.; Mansour, T. S. Bioorg. Med. Chem. Lett. 1998, 8,
365.
5. (a) Edwards, P. D.; Wolanin, D. J.; Andisik, D. W.; Davis,
M. W. J. Med. Chem. 1995, 38, 76. (b) Tsutsumi, S.; Okonogi,
T.; Shibahara, S.; Ohuchi, S.; Hatsushiba, E.; Patchett, A. A.;
Christensen, B. G. J. Med. Chem. 1994, 37, 3492. (c) Chan,
A. W. E.; Golec, J. M. C. Bioorg. Med. Chem. 1996, 4, 1673.
6. Taylor, P. J.; Wait, A. R. J. Chem. Soc., Perkin Trans. 2
1986, 11 1765.
Chemistry
The cyclopropyl acyl derivatives 2, 6, 12–14, 18–20 were
prepared by acylatingthe anion of the required lactam
25 or 26, with the correspondingmixed anhydride or
acid chloride,1,2 while the amides 9, 15, 21, 22 were
prepared by reactingthe lactam nitrogen with the cor-
respondingisocyanate under base-catalysed conditions
(Scheme 1). The aryl 16, 23, or heterocyclic ring 17, 24,
derivatives were prepared by reactingthe lactam nitro-
gen with the corresponding aryl or heterocyclic bromide
under Cu catalysis usingmodified Goldbergcondi-
tions.7 The plasma stable thiazole derivative 17 was
prepared in 67% yield, however when the modified
Goldbergreaction was carried out on the dansylproline
translactam 26 only a 3% yield of thiazole 24 could be
obtained, possibly due to co-ordination of Cu by the
dansylproline moiety. The Cbz group could not be
removed by hydrogenolysis from the more readily pre-
pared Cbz analogue 17, hence a more labile protecting
group was used. The Cbz group was removed from the
lactam 25 and the pyrrolidine nitrogen protected by a
p-methoxybenzyloxy group to give 27. The thiazole was
then added in the usual way,7 in 53% yield, and the
protecting group removed with TFA to give the proline
28. Couplingwith dansylproline gave 24 (Scheme 1).
7. Greiner, A. Synthesis 1989, 312.
8. Each compound was incubated in fresh human plasma at a
concentration of 10 mM and aliquots deproteinated with
acetonitile at the followingtimes 0, 15, 30, 60, 120, 240 min,
and 22 h. The samples were then assayed individually by LC–
MS on an API-300 usingan APCI source and sinlge-ion
monitoring.